Molecular Robotics: An Introduction 9811939861, 9789811939860

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Satoshi Murata   Editor

Molecular Robotics An Introduction

Molecular Robotics

Satoshi Murata Editor

Molecular Robotics An Introduction

Editor Satoshi Murata Department of Robotics Tohoku University Sendai, Japan

ISBN 978-981-19-3986-0 ISBN 978-981-19-3987-7 (eBook) https://doi.org/10.1007/978-981-19-3987-7 Translation from the Japanese language edition: ブンシロボティクスガイロン by Satoshi Murata, © Intelligent Molecular Robotics Research Group, S&I division, SICE 2019. Published by CBI Press. All Rights Reserved © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2022 This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part of the material is concerned, specifically the rights of reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Singapore Pte Ltd. The registered company address is: 152 Beach Road, #21-01/04 Gateway East, Singapore 189721, Singapore

Organization of this Book

Molecular robotics is related to many fields, such as systems engineering, control engineering, computer science, biochemistry, biophysics, polymer chemistry, nucleic acid chemistry, molecular biology, and ethics; only by taking a bird’s-eye view, we can grasp the whole picture of molecular robotics. We believe that it is essential for students and beginners to understand the ongoing expansion of molecular robotics and, therefore, we planned this book. This book consists of eight chapters: the design theory of molecular robots, systematization technology, molecular nanotechnology, molecular actuators, molecular materials, medical applications, and social acceptance. Each chapter provides a general overview of the theory, underlying technologies, medical applications, and social acceptance. A list of references to the representative papers is included in each chapter. The Introduction, "Welcome to Molecular Robotics," provides an introduction to molecular robotics, including a definition of molecular robotics, the academic status of molecular robotics, and its near-future applications. In Chap. 2, “Design Theory of Molecular Robots,” important techniques for molecular robotics design are explained for each level of design, such as logic circuits, dynamic circuits, and large-scale simulations. Because many methods have been proposed even for a logic circuit, it is impossible to cover them all, but particularly useful and interesting ones are introduced. Chapter 3, “Systematization Technology for Molecular Robots” describes systematization of molecular robots. There are several stages in the systematization of molecular robots, and this chapter describes the working principle of amoebatype (artificial cell-type) molecular robots. It also explains molecular computational models on gels (gellular automat), focusing on basic research results. Chapter 4, “Molecular Nanotechnology for Molecular Robots,” focuses on recent research trends in DNA nanotechnology, which can be regarded as fundamental technology for molecular robotics. Initially, DNA was used exclusively as a material; however, in recent years, RNA, peptides, and other biopolymers with higher functionality have become available, and it is expected that an increasing number of DNA, RNA, and peptide complexes will be designed and used in the future. v

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In Chap. 5, “Molecular Actuator for Molecular Robots,” we introduce the varieties of biomolecular motor systems: microtubule and kinesin systems, artificial peptide polymerization propulsion systems, light-driven actin systems, and F1-ATPase-based rotary molecular motors. Belousov–Zhabotinsky (BZ) gel actuators, inorganic BZ gel actuators, and inorganic nanosheet actuators have also been introduced as inorganic molecular motors. In Chap. 6, “Molecular Material for Molecular Robots,” we explain the trend in new technologies from the viewpoint of materials for molecular robots. In this section, we first describe the mass synthesis of DNA, which is important when considering DNA gels as a material for molecular robots. In addition, various artificial nucleic acids that are different from those derived from living organisms have been developed, and these have various useful functions that are not found in natural nucleic acids. Nucleic acids that bind and disassociate in response to the wavelength of light (UV or visible light) and orthogonal nucleic acids that do not interact with natural nucleic acids will expand the degree of freedom in the design of molecular robots as systems or circuits. Chapter 7, “Medical Application of Molecular Robots,” introduces molecular robotics research in the field of medicine, in which molecular robots or molecular devices are expected to be applied in the near future. This explains how nucleic acid medicine and induced pluripotent stem cells are connected to molecular robotics and how they will develop. Chapter 8, “Social Acceptance of Molecular Robots,” explains how research and development of molecular robotics should be conducted from the perspective of ethics, law, and society, considering the differences between molecular robotics and conventional engineering technology. New technologies, such as molecular robotics, are expected to have various ripple effects, and it is important to consider how they will be accepted by society as well as to promote research and development. In this section, we explain the framework for specific considerations such as technology assessment and technology development guidelines.

Foreword

Accelerating the evolution of science through the intersection of academic disciplines that were previously thought to be independent and completely unrelated is at the heart of Kurzweil’s singularity, at which point machine intelligence outperforms all human intelligence, and the future after that point becomes unpredictable by humans. The idea of singularity may have been thought of as a big prophecy of a suspicious future predictor, but it has been widely accepted until now. The law of accelerating returns, saying that technologies produce technologies, is at least qualitatively correct. The accelerated evolution by fusions of various academic disciplines is now frequently observed here and there in science. It seems getting more and more frequent recently. Molecular robotics, which aims to design and implement autonomous molecular systems, is not only a typical example of a fusion of academic disciplines but also highly universal in the following sense. Most of the natural phenomena on the earth, including life, are caused by the reaction of molecules with each other. Therefore, it is no exaggeration to say that all systems on the earth are molecular systems. In particular, life is the culmination of molecular systems, and its main feature is autonomy. Autonomous molecular systems that constitute life could only be realized by the process of evolution on Earth. Therefore, it is no doubt that trying to artificially design and implement such systems is extremely challenging and fundamental, comparable to the challenge of exploring the origin of the universe, among other challenges in science. Of course, it is not possible to face such challenges only by a few academic disciplines. It is required to include almost all academic disciplines. This is only possible in the era (age) of singularity, where academic disciplines influence each other and accelerate each other’s progress. Indeed, the research field of molecular robotics can only emerge in the era of singularity, and at the same time, it should emerge in the development of science due to the universality of the research theme of autonomous molecular systems. In fact, what I have written so far is clearly visualized in Fig. 3 at the beginning of this book. Chapters 1–5 of this book tell the story of the fusion of informatics, engineering, chemistry, and biology to create the research field of molecular robotics. vii

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They specifically describe how those disciplines have influenced each other and fused. In particular, molecular robotics is well characterized as the fusion of informatics in a broad sense, including system engineering, control engineering, and computer science, with chemistry and biology that deal with actual materials. Informatics provides not only tools for designing molecular systems but also methodologies for building systems, and furthermore, the reason why they should be built. On the other hand, chemistry and biology urge informatics to create new methodologies by providing enormous experimental results obtained with newly developed materials and experimental techniques. However, the story of this book does not end with the birth of molecular robotics, a fusion of many academic disciplines. The energy generated by the fusion of academic disciplines is now consumed to create various new fields from the fusion. Due to the universality of the research theme of molecular robotics, i.e., autonomous molecular systems, it is very difficult to foresee all the fields that will be created from molecular robotics. Chapter 6 of this book introduces medical applications as one of the most important fields born from molecular robotics. Chapter 7 at the end of this book touches on the social acceptance of molecular robotics, which is in fact related to the idea of singularity mentioned at the beginning. For some people, the idea of singularity may lead to the fear that the evolution of science becomes uncontrollable due to the law of accelerating returns. Chapter 7 of this book states that it is groundless. At the end of this article, I would like to express my deep gratitude to all the people involved in the writing of this book, the members of the Molecular Robotics Research Group and the Grant-in-Aid for Scientific Research on Innovative Areas “Molecular Robotics.” Thanks to all the people involved in the creation of the new research field of molecular robotics, we were able to put together the story of molecular robotics as a book. I should finally add that this story is just beginning. In other words, although this book also has an introduction, the whole book is an introduction to the long-lasting story of molecular robotics. Masami Hagiya The University of Tokyo Tokyo, Japan

Preface

If asked what the most complex system in the world is, the answer is probably living organisms, including humans. Humans consist of approximately 40 trillion cells, each of which contains approximately 20,000 different genes that are translated into various proteins to control all the different activities of life. These activities include everything from metabolism, reproduction, and muscle movement to neural activities, which can be traced back to reactions between molecules. Within a small container known as a cell, numerous molecules move around, collide with each other, and repeatedly assemble and disassemble to create new molecules and break down the existing ones, respectively. The body of living organisms is comprised of numerous cells each performing complicated reactions and aggregates as a whole behaving in a meaningful manner. This complexity is truly a stretch of the imagination. Molecular biology teaches us that all living things are made up of a common set of molecules: 8 types of nucleic acids (four types of DNA and four types of RNA), 20 types of amino acids, sugar chains, and phospholipids. With such small variety of molecules that themselves are incredibly small in size, all living organisms, from bacteria to whales, are formed. Moreover, not only are the materials common but the mechanisms of replication, transcription, and translation of DNA into proteins are also common to all species within a single framework called “Central Dogma” of molecular biology. Bacteria, plants, and animals have evolved within the same framework. This can be regarded as a history of the creation of infinite diversity from a finite number of molecular combinations. Looking at living organisms from an engineering perspective, the biological mechanism of creating various systems from a finite number of parts should be applied to artificial systems. The standpoint of molecular robotics, which is, the design, synthesis, and combination of components “from the molecular level” to create an operating “system,” deserves to be called engineering that learns from living organisms. The first artificial DNA nanostructure designed by Seaman in 1982 consisted of only 64 bases; however, with the invention of DNA origami in 2006, the number of bases has jumped up to several thousand, and as of 2017, nanostructures including more than 600,000 bases have been created. In addition, DNA computers and various ix

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functional molecular devices have been developed one after another, and development at the level of “systems” that combines these devices, such as our molecular robot prototypes, has eventually come to be oriented. The types of molecules used as materials have gradually expanded to include RNA, peptides, RNA, and artificial nucleic acids. Although these researches have not yet been put to full-scale practical use, it is expected that the field of application will expand as the technology advances. Research on molecular robots for drug delivery is already underway, and applications in drug discovery, environmental monitoring, and artificial photosynthesis are also being explored. In anticipation of these applications, discussions regarding ethical issues such as the establishment of guidelines for molecular robotics research have started. In advanced molecular design, the molecular sequence in cyberspace will be directly reflected in the material property in real world, as if the genotype of an organism corresponds to its phenotype. If computers and artificial intelligence continue to advance at this rate, we will eventually be able to perform precise molecular simulations in cyberspace and design optimal sequences based on these simulations without the need for experimentation. The synthesis of various molecules can also be automated, and it may become possible to create artificial molecular systems that are as complex and large as living organisms. The purpose of this book is to provide an overview of molecular robotics, from fundamental issues to the latest topics in molecular robotics, in an easy-to-understand manner. Because molecular robotics is an emerging field in which several completely different academic disciplines overlap, many people who have studied a particular specialty may find it difficult to grasp the terminology and other aspects of the field. In this book, after explaining the minimum necessary background knowledge, the basics of molecular robotics are described. We hope that this book will help those interested in and appreciate the potential of molecular robotics. Sendai, Japan April 2022

Satoshi Murata

Contents

1 Introduction: Welcome to Molecular Robotics! . . . . . . . . . . . . . . . . . . . . Satoshi Murata

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2 Design Theory of Molecular Robots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Takashi Nakakuki

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3 Systemization Technology for Molecular Robots . . . . . . . . . . . . . . . . . . . Shin-ichiro M. Nomura

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4 Molecular Nanotechnology for Molecular Robots . . . . . . . . . . . . . . . . . . 117 Masayuki Endo 5 Molecular Actuator for Molecular Robots . . . . . . . . . . . . . . . . . . . . . . . . 195 Akira Kakugo 6 Molecular Material for Molecular Robots . . . . . . . . . . . . . . . . . . . . . . . . 215 Akinori Kuzuya 7 Medical Application of Molecular Robots . . . . . . . . . . . . . . . . . . . . . . . . . 247 Taro Toyota 8 Social Acceptance of Molecular Robots . . . . . . . . . . . . . . . . . . . . . . . . . . . 283 Akihiko Konagaya

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Introduction: Welcome to Molecular Robotics! Its Definition, Academic Perspective, Near-Future Applications and Challenges Satoshi Murata Abstract The purpose of this chapter is to give a general idea of the field of molecular robotics and the motivation behind its research and development. We first describe the definition of molecular robots. Conventional robots can be defined as a combination of sensors, processors, actuators, and the structures in which they are embedded. Molecular robots can be defined in the same way, but the main difference is that whereas conventional robots are assembled top-down with the help of external forces, molecular robots are assembled bottom-up by the self-assembly of component molecules. Next, an amoeba-type molecular robot is taken up as a typical example of a molecular robot. In this robot, a molecular sensor, a molecular processor and a molecular actuator are embedded in an artificial cell membrane, and its movement is controlled by external optical stimuli. We also explain the historical background and inevitability of the emergence of such a discipline as molecular robotics. Molecular robotics has its origins in the cybernetics of the 1940s. It has emerged as a focal point where disciplines such as chemistry, informatics and biology intersect. The last part describes the applications of molecular robotics in various fields such as medicine, environment, energy and devices.

The purpose of this chapter is to give a general idea of the field of molecular robotics and the motivation behind its research and development. To this end, we first describe the definition of molecular robots. Conventional robots can be defined as a combination of sensors, processors, actuators and the structures in which they are embedded. Molecular robots can be defined in the same way, but the main difference is that whereas conventional robots are assembled top-down with the help of external forces, molecular robots are assembled bottom-up by the self-assembly of component molecules. Next, an amoeba-type molecular robot is taken up as a typical example of a molecular robot. In this robot, a molecular sensor, a molecular processor and a S. Murata (B) Department of Robotics, Tohoku University, Sendai, Japan e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2022 S. Murata (ed.), Molecular Robotics, https://doi.org/10.1007/978-981-19-3987-7_1

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molecular actuator are embedded in an artificial cell membrane, and its movement is controlled by external optical stimuli. We also explain the historical background and inevitability of the emergence of such a discipline as molecular robotics. Molecular robotics has its origins in the cybernetics of the 1940s. Modern robotics is based on the development of semiconductors and various elements for mechanical actuation and sensing. Molecular robotics has also been made possible by the development of elemental technologies such as artificial membranes, molecular machines, DNA nanostructures and DNA computers. In this sense, molecular robotics has emerged as a focal point where disciplines such as chemistry, informatics and biology intersect. The last part of this chapter describes the applications of molecular robotics in various fields such as medicine, environment, energy and devices that are expected in the near future and discusses the problems and challenges that need to be solved for these applications.

1.1 What is a Molecular Robot? Before explaining what a molecular robot is, let us first define what a “robot” is. What do you imagine when you think of robots? A humanoid robot? Welding robots in a car factory, drones delivering packages from the sky, or robots that clean floors; you can think of numerous other examples. All of these have something in common, the general concept of a robot. In this case, the similarity in appearance to biological creatures is not the essence. Rather, we can define it by focusing on the relationship between the elements that make up their functions, in other words, the composition of the robot as a system. The components that a robot is comprised of include motors, microcontrollers, sensors, batteries and other parts. These functional components are placed in a certain structure to form a robot system as a whole. In other words, it can be defined as a system consisting of sensors that acquire information from the external environment, computers (information processing circuits) that process the information, and actuators that act on the environment according to the processing results (Fig. 1.1). Fig. 1.1 What is a robot?

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What then is a “molecular” robot? Because a molecular robot is a type of robot, it must satisfy the above definition. When we say “molecular” robot, it means a system in which all the functions are realized by a molecular device. Ordinary robots consist of parts made of bulk metals or plastics, which are cut or poured into molds. These parts make use of the properties of the material (including mechanical strength and electrical conductivity), but these properties can only be developed when the material has a certain shape. However, in the case of molecular devices, we refer to molecules that take on specific shapes and perform specific functions, similar to proteins in living organisms. Therefore, a molecular robot consists of a molecular device that controls sensation (receptor molecules that capture physicochemical information from the outside world), molecular device that controls intelligence (molecular reaction systems that compute and process the captured information and release the resulting molecular signals) and molecular device that generates motion (molecular motors driven by molecular signals).

1.2 How to Make a Molecular Robot? Molecular devices that make up a molecular robot are synthesized by chemical reactions rather than macroscopic (bulk) processing such as machining. In living organisms, this corresponds to the reaction in which a protein enzyme synthesizes a chain of amino acids from a DNA base sequence through transcription and translation. In the case of artificial molecular devices such as DNA origami described in this book, material molecules are created by chemical synthesis, and by combining them, the synthesized molecules are folded or self-assembled into the correct shape to create a molecular device. Mechanical and electrical components cannot function as a whole unless they are properly combined. For example, in the case of gears, if gears with appropriate tooth ratios are not arranged at appropriate shaft intervals, the rotational motion cannot be transmitted. Similarly, a molecular device cannot function as a system unless it is combined appropriately. In a molecular robot, the output of one molecular device (usually a molecule) becomes the input of another molecular device, creating a network of chemical reactions, which is the substance of a molecular robot as a system. It requires a structure or container that delimits what is and is not a molecular robot; that is, the inside and outside of the robot. This is a compartment, similar to a cell membrane, as a “chassis” to hold these molecular devices.

1.3 Development of an Amoeba-Type Molecular Robot In a molecular robot, a group of molecular devices must operate organically while maintaining a certain relationship with one another. In general, it is not easy to combine different chemical reactions in the same solution space, but this can be

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achieved using biomolecules such as DNA, since DNA fragments hybridize with other DNA fragments in a sequence-specific manner, making it possible to create DNA nanostructures with complex shapes and DNA computers that resemble logic circuits. At present, nanostructures exceeding several hundred thousand bases and logic circuits containing dozens of logic gates have already been realized, and more complex systems are becoming possible to construct. In the Grant-in-Aid for Scientific Research on Innovative Areas “Development of Molecular Robots with Senses and Intelligence” (abbreviated as “Molecular Robotics”) sponsored by the Ministry of Education, Culture, Sport, Science and Technology, MEXT, Japan, which was conducted from 2012 to 2016, numerous studies on molecular robotics were conducted. In this project, the research was conducted by four planned research groups: sensor, intelligence, amoeba and slime, as well as by a recruited research group. Among them, the amoeba and slime groups are responsible for developing prototypes of molecular robots. An amoeba-type molecular robot is a system that integrates a DNA sensor molecule, DNA amplification circuit, and microtubule/kinesin molecular motor in a microcapsule (liposome) (Fig. 1.2). In this robot, transmission of the sliding motion of the molecular motor to the membrane surface is controlled using DNA computing (DNA clutch). This is the first example in which a series of functions, such as sensing, information processing and actuation, are connected by a common information molecule, DNA, and integrated into a system (see Sect. 3.2). This research caused a worldwide sensation upon its publication and was covered by various media sources. Another implementation group, the slime

Fig. 1.2 Amoeba-type molecular robot

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group, worked on the development of a new molecular computational model called a gellular automaton. Gellular automation is an extension of molecular computer calculations to the chemical reaction space.

1.4 Development of Sciences Leading to Molecular Robotics Molecular robotics focuses on the development of not only individual molecular devices and elemental technologies but also systematization technologies that link and integrate them. Unlike normal-sized machines, the assembly of molecular devices and systems is performed using bottom-up chemical reactions and self-assembly/selforganization (here, only bulk chemical manipulation is allowed) and the programming and control of the behavior must be realized by programming the chemical reaction system. This means that molecular robotics is a discipline that goes beyond the existing disciplines of chemistry, biology and nanotechnology. Highly reliable electrical and mechanical components are required to assemble an ordinary robot. In the 1940s, when cybernetics, the origin of robotics, was first proposed, there were no electronic computers and what could be done was incredibly limited. Since the transistor was invented in 1947, advances in semiconductor technology made it possible to build reliable computing circuits. Efficient electric motors and various sensors have also been developed, and by embedding them in the mechanical structures, mechatronic robots for various applications have been realized. On the other hand, the technology for building molecular robots has developed some 30 years later than this. Artificial membranes as the body of molecular robots and molecular devices as the sensors of molecular robots were both invented in 1970s in Japan. The DNA nanostructures, which form the framework for the construction of various molecular devices, began with Seaman’s work in 1982 and have made significant progress through Rothemund’s work on DNA origami in 2006. In the field of molecular computation, which is used for the information processing of molecular robots, various computational methods have been developed using DNA reactions, starting with Adleman’s DNA computer in 1986 and continuing with the recent development of large-scale molecular circuits based on seesaw gates. On the basis of these elemental technologies, it is now possible to build molecular robots. These technologies are key elements of molecular robotics, the subject of this book. Molecular robotics is the reconstruction of cybernetics at the molecular level and is truly an interdisciplinary field that transcends the existing academic fields. Accelerating the progress of this discipline will enable us to freely create largescale, complex molecular systems, which will eventually lead to a change in basic assumptions in our view of matter, information, and life (Fig. 1.3).

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Fig. 1.3 Development of sciences leading to molecular robotics

1.5 Situation Surrounding Molecular Robotics In recent years, research on molecular design and devices using DNA as a material has become active worldwide. Research and development are advancing rapidly, mainly in Europe and the United States (U.S.) and Asia is following suit. The Wyss Institute was established at Harvard University as a research base for related fields, and a research center has also been established in Denmark. Research centers have been established at the California Institute of Technology, the Technical University of Munich, and Arizona State University. In the US, large projects such as the Molecular Programming Project (MPP) have been conducted one after another. From these projects, new technologies such as spatial molecular computation, ultrahigh-resolution optical measurement using DNA origami, large-scale DNA logic gates, DNA brick technology, single-molecule measurement technology using DNA origami, in vivo use of DNA devices, and simulation of large-scale DNA nanostructures and devices have been developed. Furthermore, the International Society for RNA Nanotechnology was established in 2017, indicating that DNA nanotechnology has evolved into DNA/RNA nanotechnology. Contrarily, in Japan, although the concept of molecular robotics was proposed and has taken the lead in research, technological superiority has not necessarily been established. The Society of Instrument and Control Engineers (SICE) established the Research Group on Molecular Robotics in 2010, which implemented the aforementioned project of “Molecular Robotics.” A total of 75 researchers, mainly from the fields of chemistry, biophysics, information science, and engineering, participated in this area and vigorously promoted research and development in molecular robotics. In addition, the number of projects derived from this area, including the

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New Energy and Industrial Technology Development Organization, NEDO project of “Biomolecule-Based Artificial Muscles” and the Japan Science and Technology Agency, JST project of “Molecular Robotics ELSI.” Another characteristic of the Japanese research community is that a large number of scientists are capable of handling molecular materials other than DNA, such as RNA, peptides, artificial nucleic acids, and polymers.

1.6 Evolutionary Scenario of Molecular Robots We propose an evolutionary scenario for the development of molecular robots at the launch of the “Molecular Robotics” project (Fig. 1.4). In this scenario, the evolution of molecular robots begins with the zeroth generation of molecular spiders that incorporate functions into single molecules, followed by the first generation of amoebatype molecular robots that encapsulate molecular devices in liposomes. The second generation of molecular robot after the amoeba-type is slime-type moleclar robots that realize diversity by becoming multicellular. In addition, the fourth generation is aimed at hybridization with electronic and chemical technologies. Looking at the progress in this field, there has been much research and development on the subject of “molecular robotics,” and in fact, there is a considerable amount of research that corresponds to each stage of this scenario.

Fig. 1.4 Evolutionary scenario of molecular robots

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1.7 Future Applications of Molecular Robotics Many readers are interested in the future applications of molecular robotics. In this section, we describe the applications of molecular robotics in the near future and demonstrate how they correspond to the specific topics in this book. Molecular robotics is a highly versatile technology with the potential to replace the semiconductor industry as an industrial base for the next generation. In molecular robotics, the main themes are molecular devices made of DNA, RNA, and peptides and their systemization. Various applications are possible, especially in the field where life and artifacts are fused. Because molecular robotics is a rapidly developing field, it is not easy to foresee its future, but it is possible to make some predictions. Here, we introduce some possible applications of molecular robots in the near future. As molecular robots are mainly made of biomolecular materials such as nucleic acids and proteins, they have a high affinity for biological applications, especially in the medical field (Fig. 1.5). A typical example is the sophistication of drug delivery, which has been the subject of extensive research. Advanced diagnostic and medication logic is programmed into the molecular robot itself to perform diagnosis; treatment in the body is a typical example. In addition, there is the concept of systematic nucleic acid medicine, which is an intelligent version of nucleic acid medicine that has entered the stage of practical application in the recent situation of the COVID-19 pandemic. Unlike conventional nucleic acid drugs that target a single gene, this concept is based on an artificial reaction circuit that is injected into the body to perform diagnosis (molecular computing) using the expressed concentration of miRNAs and synthesize nucleic acid sequences that will become drugs in the

Fig. 1.5 Application of molecular robots in the medical field

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Fig. 1.6 Application of molecular robots in the environmental field

cell. Orthogonal artificial nucleic acids that do not crosstalk with biological reaction systems, design methods of diagnostic logic circuits and verification by model organism experiments are challenges in systematic nucleic acid medicine. iPSC regulation is a technology that controls the differentiation of iPSCs using artificial organelles consisting of RNA–protein complexes, which leads to the creation of artificial organs. Challenges include the development of artificial organelles with differentiation control functions, capsules for introducing artificial organelles into cells, and tissue control of cell group pattern formation. Various types of monitoring are considered for near-future applications of molecular robotics in the environmental field (Fig. 1.6). The objective was to collect information on the concentration and distribution of substances by searching for traces of substances at each location as the molecular robot moved through space. Possible environments include the outside world, such as oceans and rivers, and the inside of organisms, such as humans, livestock, and trees. Molecular Hayabusa (named after the Japanese asteroid exploration project) is the concept of an in vivo sample and the return performed by molecular robots. By bringing back molecular samples directly from human, animal and plant bodies, the robot can obtain information that is difficult to obtain using other methods, such as the distribution of miRNAs in the body and information on exosomes. Specifically, the liposome that serves as the molecular Hayabusa (see Sect. 7.4) is equipped with a peptide device for collecting molecular samples from the cell surface, and the small number of molecular samples brought back is analyzed by the nanopore system.

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In the field of chemical production and energy, rather than applying molecular robotics directly, we discuss how molecular robotics can contribute to the construction of molecular systems. Molecular robotics can be applied as a methodology for constructing systems by combining various molecular devices and nanostructures. For example, artificial photosynthesis converts solar energy into chemical energy. Figure 1.7 depicts the concept of a system that synthesizes ATP using a light-driven molecular motor. In this design, the light-driven molecular motor and ATP synthase are housed in a DNA nanostructured hexagonal cell and their rotation axes are connected to the light-driven motor. Such a complex system of molecularlevel parts is difficult to fabricate using conventional top-down technology; thus, in such a case, bottom-up construction by molecular self-assembly can be utilized. One of the near-future applications of molecular robotics in the field of devices is the construction of various information-processing systems using molecular devices (Fig. 1.8). Although it is difficult to surpass electronic technology in digital computation, the characteristics of molecular information processing have several advantages. For example, in applications where the input is the sensing of a chemical substance, and the final output is the release of the chemical substance, there are advantages to processing all the information by molecules rather than by interposing information processing by electrons. The small size and massively parallel nature of molecules, as well as their nonlinear and complex reaction dynamics, can be exploited to create a new principal computer (artificial brain).

Fig. 1.7 Application of molecular robotics in chemical production and energy

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Fig. 1.8 Near-future applications of molecular robotics in the device field

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Design Theory of Molecular Robots Takashi Nakakuki

Abstract To realize a “smart” molecular robot that works with sensors and actuators together, it is essential to design DNA circuits that transmit, integrate, and process the information obtained from these devices. This chapter outlines typical design theories and technologies in molecular robotics. We begin with the structure of a DNA strand that constitutes the DNA reaction system. Particularly, we consider how the secondary structure of the DNA strand is determined and how it can be predicted, analyzed, and designed. Then the basic framework for designing DNA circuits is outlined. Topics such as example of functioning DNA circuits, DNA amplification circuits, mathematical modeling of DNA circuits, PEN DNA toolbox, mathematical modeling of DNA circuits by the Boolean network, and simulation system for predicting the dynamic behavior of molecular robots will be explained.

To realize a “smart” molecular robot that works with sensors and actuators together, it is essential to design DNA circuits that transmit, integrate, and process the information obtained from these devices. This chapter outlines typical design theories and technologies in molecular robotics. Section 2.1 explains the structure of a DNA strand that constitutes the DNA reaction system (Dr. Kawamata). Particularly, we consider how the secondary structure of the DNA strand, which is important while designing the DNA base sequence, is determined and how it can be predicted, analyzed, and designed. In Sect. 2.2, the basic framework for designing DNA circuits is outlined, wherein three types of design methods (enzyme-free, enzyme-based, and DNAzyme) are introduced (Dr. Kawamata). Section 2.3 presents an example of a well-functioning DNA circuit—a reactive circuit for continuously responding to time-varying inputs, which is indispensable while realizing molecular robots that operate while receiving input from the environment via sensors (Dr. Kobayashi). Section 2.4 describes the DNA amplification circuit that amplifies the DNA strands from a low concentration to a high concentration, as required by the subsequent DNA circuit (Dr. Komiya). The amplification mechanism is an important module T. Nakakuki (B) Kyushu Institute of Technology, Fukuoka, Japan e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2022 S. Murata (ed.), Molecular Robotics, https://doi.org/10.1007/978-981-19-3987-7_2

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which is widely used in molecular systems. Section 2.5 presents the mathematical modeling of DNA circuits based on the reaction kinetics and discusses the difficulties of mathematical treatment as nonlinear systems (Dr. Nakakuki). In Sect. 2.6, the PEN DNA toolbox, which is a versatile framework of rational design for analog computation is discussed (Dr. Aubert-Kato). Section 2.7 presents the mathematical modeling of DNA circuits by using a Boolean network (Dr. Azuma). Various topics in the design of DNA circuits and molecular robots are introduced in Sect. 2.8 and its subsequent sections. Section 2.8 outlines the computational technology of a simulation system for predicting the dynamic behavior of molecular robots (Dr. Konagaya, and Dr. Gutmann). Section 2.9 describes the system theory that views molecular robots and their various component systems as distributed systems (Dr. Yamauchi). In the remaining two sections, certain future prospects for the theory, design, and technology of molecular robots are discussed (Dr. Suzuki, and Dr. Sugawara).

2.1 Secondary Structure Prediction and Sequence Design Technology Ibuki Kawamata Nucleic acids (DNA or RNA) are one of the promising biomaterials to build a molecular-scale system due to their predictability and designability. The capabilities are taken for granted, thanks to the development of the computational techniques of nucleic acids. In this section, such techniques are introduced together with the practical procedures to perform them. Given a DNA or RNA sequence, one may want to predict what kind of secondary structure is thermodynamically stable. Finding the most stable structure of nucleic acids from the sequence is called the “secondary structure prediction” problem. In contrast, the “sequence design problem” is the inverse problem: to design a sequence that forms a desired secondary structure. Using the case of DNA as an example, we will first explain the method of secondary structure prediction and then the method of sequence design. The primary structure of DNA is a simple string of bases (A: adenine; T: thymine; G: guanine; C: cytosine), and the secondary structure is a structure that shows which bases make hydrogen bonds with which bases (base pairing). Figure 2.1 shows the difference between the primary and secondary structures. In the secondary structure, in principle, A can form base pairs with T, and G can form base pairs with C. By changing the combination of base pairs, multiple secondary structures are possible even for DNA with the same primary structure.

I. Kawamata Tohoku University, Sendai, Japan e-mail: [email protected]

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Fig. 2.1 Difference between the primary and secondary structures of DNA. The top row shows the primary structure expressed as a string. Below that, three different secondary structures using the same base sequence are shown. In the secondary structure, the hydrogen bonds between A and T, and between G and C are represented as lines

Given a secondary structure, the Nearest-Neighbor model (abbreviated as the NN model) can be used to calculate the free energy of that structure. The specific free energy calculation is done by simply adding up the NN model parameters of the substructure. A substructure is a region bounded by base pair lines and the backbone of the primary structure, such as base pair stacking between two base pairs. As a parameter of the substructure, the values obtained experimentally by SantaLucia Jr. [1] and Sugimoto et al. [2] are widely used. When the free energies of all possible secondary structures can be determined, the most stable structure among them can be selected, and the secondary structure prediction problem is solved. In the example shown in Fig. 2.1, the bottom structure will be chosen. It is known that the secondary structure prediction problem can be solved efficiently by using an algorithm called dynamic programming [3]. The strategy of the algorithm is to recycle the computation of secondary structures with a common substructure in an exhaustive search for possible secondary structures. Please refer to the paper for details. To perform secondary structure prediction, it is common to use various software [4–6] that are available online. The usage of the Nucleic Acid Package (NUPACK) is explained below because it is widely used as a gold standard for decades in the field of nucleic acid research. Figure 2.2 shows an example screenshot of secondary structure prediction using the “Analysis” function of NUPACK [7]. In the leftmost input screen, three different DNA sequences are entered. In the middle result screen, which is displayed first after the calculation, the concentration distribution is displayed as a red bar graph. The distribution is calculated according to the free energy of each structure, so the structure with the highest amount is the most stable in terms of free energy. Clicking on the bar graph will display the secondary structure as shown on the right. The above secondary structure prediction problem is necessary when we want to predict its structure in solution for a base sequence that is known in advance. For example, if you find an RNA fragment encoded in a gene, you may want to predict how it will function based on its structure, which is important in a biological context. In the context of molecular robotics, on the other hand, it is necessary to

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Fig. 2.2 Interface for secondary structure prediction by NUPACK. The leftmost screen is the input screen. Clicking on the “Analyze” button at the bottom right of the input screen will start the calculation, and after a while, you will move to the middle screen where the results are displayed. If you click on the bar graph, the secondary structure screen on the right is displayed

solve the inverse problem, that is, to design a base sequence that will result in the desired structure. This is the case, for example, in constructing structures with a desired shape, such as DNA tiles, or in creating elements that are connected to each other, such as in DNA computation. A simple solution is to determine the sequence randomly (for complementary sequences, determining one automatically determines the other). However, in the random sequence design method, many conditions cannot be considered simultaneously. Some examples of the conditions are as follows: • Sequences that bind with a certain strength with a specified melting temperature (Tm). • Sequences with a specified Guanine and Cytosine content (GC content). • Sequences with no more than four consecutive sequences of the same base. • Sequences with no more than six consecutive purine, pyrimidine, etc., bases. • Sequences with guaranteed orthogonality. The last one, orthogonality, is especially important when designing sequences. In other words, the goal is to design a sequence in which unintended combinations of sequences do not bind together and thus crosstalk is kept to a minimum. If the orthogonality is insufficient, metastable by-products will be formed, and the reaction will not proceed as expected or the structure will not be formed as expected. It is not realistic to discover a good sequence by repeated manual trial and error until all of the above conditions are met. If the space of the sequence you want to design is small, there is a way to use a set of sequences whose orthogonality has already been guaranteed [8]. Since 37 types of DNA with 23 bases that do not interfere with each other are given in advance, the necessary sequences can be selected from them. However, the sequence set is too small to be applied to general problems and is not versatile enough. Therefore, with the aid of computers, many methods have been developed to design relatively good sequences [9–11]. Specific methods used include genetic

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algorithms that solve multi-objective optimization problems. The strategy of the algorithm is to start the search from a random sequence, repeat the secondary structure prediction many times with small modifications, and select the one that meets the desired conditions. Please refer to the paper for details. There are several sequence design algorithms available as software [12, 13]. In this description, we will use the sequence design function of NUPACK as an example to explain how to use it. Figure 2.3 shows an example of the sequence design function named “Design” in NUPACK. The left side is the input screen, where the target secondary structure and other conditions are entered. On the right is the result screen, where several specific sequence sets are output. The “Normalized ensemble defect” is a measure of how good the sequence is, the closer to 0, the better, and the closer to 100, the worse. Basically, the best sequence at the top is used. By clicking the “To Analysis” button next to the sequence, you can make another secondary structure prediction using the designed sequence. Recall that sequence design was the inverse problem of secondary structure prediction. There are some obvious parameters that can be given to the “Design” function of NUPACK, such as the type of nucleic acid (DNA or RNA) and the temperature at which the evaluation will be performed. Specifically, the secondary structure is specified in the frame of “Target Structure” using the dot-bracket-plus notation, which is a method using character strings. The notation is shown in Fig. 2.4, where the dot “.” represents an unpaired base, the corresponding left and right brackets “(“and”)” represent base pairs that are hydrogen-bonded, and the plus “+” indicates a break in

Fig. 2.3 NUPACK interface for sequence design. On the left is the input screen. Clicking the “Design” button at the bottom right of the input screen starts the calculation, and after a while, the screen on the right appears as a result

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Fig. 2.4 Example of dot-bracket-plus notation. The upper left represents a fully bound complementary double-stranded DNA. The upper right shows a structure in which a piece of blue DNA containing a hairpin is bound to a piece of red DNA. The lower part represents a secondary structure like a stick figure, formed by four DNA strands

the DNA. As shown in the example in the figure, it is a flexible notation that can be used to express complex structures. Various other constraints can be set, but please read NUPACK’s “HELP” for details. As described above, sequence design and secondary structure prediction are inverse problems of each other, and deriving the solution is an essential technique in the analysis of structures composed of DNA and RNA and in the design of systems. In this section, we explained how to use the practical algorithms, focusing on an online software called NUPACK.

2.2 Trends in DNA Logic Circuit Technology Ibuki Kawamata In the field of DNA nanotechnology, DNA logic gates and circuits have been widely used to provide information processing capability and programmability to systems. From the viewpoint of the driving principle, various strategies have been proposed, and in this article, we will introduce three types of logic circuit technology programmed around DNA: enzyme-free, DNAzyme, and enzyme-based. I. Kawamata Tohoku University, Sendai, Japan e-mail: [email protected]

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Fig. 2.5 Schematic diagram of an enzyme-free AND gate constructed from DNA. The figure shows the behavior when two inputs are given (Based on Ref. [14])

In the enzyme-free system, the DNA strand-replacement reaction (a kind of reaction that forms a double helix by exchanging hydrogen bonds) is carefully combined to implement logic gates. For example, in a study that implemented AND gates using only DNA, it was reported that the output DNA becomes single-stranded only when two input DNAs are present (Fig. 2.5) [14]. It has been reported that it is possible to design other logic gates using the same design principle, and that it is possible to make a circuit-like complexity by connecting gates to each other. In Fig. 2.6, a DNA gate called a seesaw gate is represented as a round element, and by wiring the round elements together like a digital circuit, a combinatorial circuit can be programmed at the molecular level [15, 16]. For example, the circuit constructed in the paper in [15] is capable of calculating √ square roots. For example, when a combination of DNA meaning 16 is input, 16 is calculated and DNA meaning 4 is output. In the above example, since all molecules are freely diffusing in the solution, the diffusion is the rate-limiting factor, and the reaction rate is limited. For example, it takes about 10 h to calculate the square root of the above. In addition, there is a frequent problem that the entire system does not work as expected due to crosstalk between DNAs in unexpected reactions called leakage reactions. In recent years, in the research field of enzyme-free DNA logic circuits, attempts to place DNA logic circuits on DNA origami and methods for speeding up and making the system more robust through precise sequence design have been actively studied [17, 18]. In the future, it is expected that large-scale circuits become important in the field of enzyme-free DNA computing. DNA logic gates and circuits that utilize the enzymatic activity of special DNA sequences without using the DNA strand-replacement reactions introduced so far are also being actively studied. The special DNA with enzymatic activity is called DNAzyme, and it is expected to be applied to the functionalization of systems that are difficult to achieve by strand displacement reactions alone [19]. Figure 2.7 shows an example of an AND gate using a DNAzyme that has the activity to cleave nucleic acids (DNA and RNA). It has been confirmed that the output DNA is cleaved only when both of the two input DNAs are present.

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Fig. 2.6 Schematic diagram of an enzyme-free DNA logic circuit. From the top left, the target logic circuit, the dual-rail logic circuit, and the DNA circuit with seesaw-gate elements (circle with vertical line) are represented, and the bottom right is the simulation result (Reprinted from Journal of The Royal Society Interface 2011 [16])

Fig. 2.7 An AND logic gate implemented with DNAzyme. The figure shows the DNA sequence and secondary structure required to realize the gate and the experimental results (Reprinted from Journal of the American Chemical Society 2002 [19])

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Even a system using DNAzyme can be made into a circuit by designing the output molecule to be the input molecule for the next gate. A striking example is a logic circuit that serves as a player in a tic-tac-toe game (Fig. 2.8) [20]. When a human adds the input DNA corresponding to the position of the game, the DNAzyme performs the calculation and outputs the fluorescence corresponding to another position. In the paper, the DNAzyme circuit is programmed to win or draw. Despite the complexity of the calculations described above, DNAzyme has not gone very far in increasing the complexity and scale of the system due to the small degree of freedom in the sequences that can be designed and the limited solution conditions (e.g., magnesium and zinc ions are required). In spite of these limitations, several studies tried to add programmability to the system, for example, by creating a set of DNAzyme sequences [21], which can be used as a library for efficiently designing DNAzyme gates.

Fig. 2.8 A logic circuit that serves as a tic-tac-toe player implemented with DNAzyme. The figure shows a schematic of the required gates and the experimental results of an example tic-tac-toe game (Reprinted from Nano letters 2006 [20])

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Fig. 2.9 Two-state circuit implemented by genelet. The figure shows a schematic of the synthesis and degradation of RNA by the DNA template and the experimental results of observing the states (Reprinted from ACS Synthetic Biology 2006 [23])

In contrast to the strategies described so far, it is also possible to implement DNA computational elements using enzymes that are different types of molecules from DNA. By utilizing enzymes, it is possible to synthesize and degrade nucleic acids, and further functionalization is expected. Among the reported systems, two research called PEN toolbox and genelet have attracted much attention. Since the PEN toolbox has been described in a separate article, we will introduce the genelet system here. Genelet cleverly controls the DNA sequence to initiate transcription, and switches transcription on and off. For example, a logic element called a toggle switch [22] and a two-state circuit as shown in Fig. 2.9 are implemented [23]. In a toggle switch, the system changes to only one of the two states, but switches to the other state when DNA, which means state switching, is input. Since genelets can realize gate activation and inhibition, they are good at constructing circuits inspired by Systems Biology. For example, it is possible to implement an oscillation circuit such as a ring oscillator and observe the oscillation phenomenon as shown in Fig. 2.10 [24, 25]. Despite the potential to realize a variety of dynamics, enzyme-based reaction systems have many limitations. In particular, each enzyme has its own specific working conditions, such as the concentration of ions in the solution and the solution temperature, so when using multiple enzymes, it is necessary to discover the optimal conditions through trial and error. In addition, when enzymes are purchased from reagent companies, the activity differs from lot to lot (only the minimum activity is guaranteed), and often the reaction rate constant is not the same even when the same amount of enzyme is used. Finding a way to relax the conditions and simplifying the kinetic argument may be necessary in the future.

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Fig. 2.10 Ring oscillator implemented by genelet. The figure shows the list of required DNA templates and the results of simulating oscillations (Reprinted from ACS Synthetic Biology 2014 [25])

In any of the strategies introduced in this commentary, it is already possible to fabricate two or three simple logic gates and turn them into logic circuits. In the future, larger scale, robustness, higher speed, and relaxation of solution conditions will be issues to be addressed. Please refer to the review papers [26, 27] for other research examples that could not be introduced in this commentary.

2.3 Design of Reactive DNA Circuits Satoshi Kobayashi Molecular robots repeat the process of sensing input molecules of the environment and transforming them via DNA circuits into output molecules which activate actuators. Therefore, we need to devise a reactive DNA circuit such that it changes the concentrations of output molecules responsively to the change of the concentrations S. Kobayashi The University of Electro-Communications, Tokyo, Japan e-mail: [email protected]

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of input molecules. Although there have been many studies on the design of DNA circuits, there are a few proposals for the design of reactive ones. An example of the design of reactive DNA circuits is given by Genot et al. [28], where the concentration of a DNA molecule X is changed by feeding either the molecule X (for the increase of X) or its complementary sequence X R (for the decrease of X). Since the proposed DNA circuits consist of only reversible reactions, the change of concentrations of input molecules correctly causes the change of concentrations of output molecules. The reactive behavior of the circuits was observed by biological experiments and reported in [28]. Another proposal can be found for the construction of reactive analog DNA circuits [29]. For instance, an analog adder can be constructed by simulating the following two reversible chemical reactions: k1

k1

k2

k2

A ←→ C, B ←→ C

(2.1)

Consider the steady state where the concentration of molecule C is fixed to the constant value. Then, we have k1 [A] + k1 [B] − 2k2 [C] = 0, which implies the equality [C] = 2kk21 ([A] + [B]). Therefore, if we can set the rate constants k 1 and k 2 so that k2 = 2k1 holds, then we have the relation: [C] = [A] + [B].

(2.2)

Therefore, we can implement analog DNA adders at steady states. Since equality (2.2) always holds at steady states, it holds at steady states even if we change the concentrations of input molecules A and B. This means that this analog DNA adder is a reactive one. But the question arises of how we can set the rate constants so that k2 = 2k1 holds. One of the approaches to the approximate simulation of given chemical reaction systems is proposed by Soloveichik et al. [30]. They use toehold mediated strand displacement reactions (TMSD) of DNAs for approximately simulating given chemical reactions. The proposed approximation scheme is based on the claim that the reaction rate constant of TMSDs can be controlled over 6 orders of magnitude by varying the binding strength of toeholds [31]. From the viewpoint of theoretical simulation studies, this wide range of rate constants is still not enough for designing complex analog DNA circuits. Therefore, it would be necessary to consider the use of molecules other than DNAs when implementing reactive and complex molecular circuits.

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2.4 DNA Amplification Circuit Ken Komiya The sequence-specific binding of DNA and its accompanying reactions can be utilized for the construction of nanostructures and mechanical operations via selfassembly, and multi-step logic operations. In these processes, the single-stranded DNA, which can bind to its complementary strand, plays the role of a signal that directs the operation, resulting in a versatile signal transduction system. In multi-step signal transduction, it is impossible for all DNA-binding reactions to proceed with full efficiency and specificity, and the signal gradually decays. In addition, when you build a molecular robot that detects the input of a single molecule and moves its molecular motors to respond with macroscopic motions, it is required to generate a large amount of signal DNA to direct the movement of many molecular motors in response to the input of a single molecule (Fig. 2.11). Therefore, DNA amplification circuits that can amplify signals attenuated by incomplete reactions to a level sufficient for subsequent processes, or that can rapidly amplify the amount of signal necessary for switching motions, are essential as fundamental technologies for DNA nanotechnology and molecular robotics. Polymerase Chain Reaction (PCR), which amplifies DNA under high-temperature cycling conditions using DNA polymerase, is commonly used in life science. However, since PCR amplifies double-stranded DNA and repeatedly dissociates the bound DNA strands at each temperature cycle, it cannot be used in a reaction system where unintended dissociation of DNA should be avoided. In addition, the molecular

Fig. 2.11 A DNA amplification circuit to bridge the concentration gap. For example, when a spherical molecular robot with a diameter of 20 μm detects the input of a single molecule and turns on the movement by binding signal DNA to each of the 100 nM molecular motors inside, approximately 250,000 DNA molecules need to be generated and amplified

K. Komiya Japan Agency for Marine-Earth Science and Technology, Yokosuka, Japan e-mail: [email protected]

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motors made of proteins are denatured and broken due to the high-temperature conditions. Therefore, the DNA amplification circuit used for molecular robots should be able to amplify single-stranded DNA under physiological temperature (37 °C) or room temperature (25 °C) conditions. Zhang et al. reported an entropy-driven reaction system in which the number of base pairs does not change before and after the DNA strand displacement reaction, and constructed an enzyme-free DNA amplification circuit in which DNA acts as a catalyst to amplify single-stranded DNA exponentially [32]. The amplification rate of the signal DNA was several hundred times greater than the amount of input DNA in a one-hour reaction, but even without the input DNA, a “leak” reaction amplifying the signal DNA occurred right after the reaction began. In an exponential amplification system, when even a small amount of signal is generated by accident, it is immediately amplified. The development of a method to prevent leakage is an important issue. On the other hand, DNA amplification reactions using enzymes have been intensively studied for applications in nucleic acid testing and medical diagnosis. For the purpose of detecting nucleic acids, it is not necessary to amplify the nucleic acid sequence to be detected as in PCR, but it is sufficient to amplify DNA with a distinct sequence that serves as the detection signal. A number of reactions for amplifying single-stranded DNA using DNA polymerase and nicking enzyme have been reported [33]. However, these reactions often suffer from leakage due to incomplete DNA binding (mis-hybridization) as well as non-specific DNA amplification called “ab initio DNA synthesis” that does not require any template or primer due to the inherent properties of DNA polymerases [34]. Overcoming ab initio DNA synthesis has become a major challenge. Komiya et al. designed the “L-TEAM (Low-TEmperature AMplification)” reaction, which amplifies signal DNA under temperature conditions lower than its melting temperature (T m ), and achieved suppression of non-specific amplification caused by ab initio DNA synthesis with the use of an artificial nucleic acid [35]. The L-TEAM reaction, which can amplify the signal DNA a million-fold, is a DNA amplification circuit suitable for molecular robots since the amplified DNA stably binds to the complementary strand in the same reaction vessel. In the future, it is desirable to develop a mechanism to stop amplification when the required amount of signal is generated, and a DNA amplification circuit that amplifies multiple signal DNA sequences in the programmed order.

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2.5 Design of Dynamic Reaction Circuits Based on Control Theory Takashi Nakakuki

2.5.1 DNA Circuit Based on DNA Strand Displacement Reaction DNA circuits are artificially designed DNA chemical reaction systems for processing desired information [36]. In a DNA circuit, the DNA strand is the carrier of information, and its concentration change represents the “signal” in the system. For comparison, in an electric circuit, the voltage/current change represents the signal. As with electrical circuits, there are two types of DNA circuits: an analog circuit [37, 38] in which the amount of continuous change in concentration itself has meaning, and a logic circuit [39, 40] that binarizes (discriminates) the amount of continuous change in concentration as a logical “low” or logical “high” level based on a threshold value. Figure 2.12 illustrates a conceptual diagram of two inputs and one output AND gate implemented in a DNA reaction system. In accordance with the truth table of the AND gate, the concentration of the DNA strand corresponding to output “c” transitions to a high concentration if the concentrations of the DNA strands corresponding to inputs “a” and “b” transition to high concentrations. There are two kinds of methodologies for designing DNA circuits: enzymatic design (e.g., the DNA toolbox explained in Sect. 1.6) or enzyme-free design using DNA strand displacement reactions [41]. In the enzyme-free design, which is the main subject of this section, the AND gate has a structure in which several DNA strand displacement reactions are rationally integrated [40]. For the past two decades, a wide variety of DNA circuits have been designed using DNA strand displacement reactions [42–44]. As shown in Fig. 2.1, the reaction scheme of the DNA strand displacement mechanism is relatively simple, comprising three steps. The invader strand, as input, binds to the toehold domain on the substrate strand (step 1), which triggers the branch migration process while stripping the incumbent strand from the substrate strand (step 2). Finally, the invader strand hijacks the substrate strand while releasing the incumbent strand as output (step 3). Hence, the DNA strand displacement reaction can be regarded as a molecular device with input and output ports. Then, by rationally connecting the output of a device to the input of another device, more complicated functions can be designed, which provides the basis for the bottom-up design of DNA circuits. In summary, in a DNA molecular reaction circuit, the “system” is the chemical reaction system, the “signal” is the concentration of DNA strands, and the “connection” corresponds to the binding (reaction) of DNA

T. Nakakuki Kyushu Institute of Technology, Fukuoka, Japan e-mail: [email protected]

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Fig. 2.12 Conceptual diagram of two inputs and one output AND gate using DNA strand displacement reaction

strands between two DNA circuits. The “circuit design” is achieved by designing the dynamics and combinations of DNA strand displacement reactions.

2.5.2 Mathematical Modeling of DNA Circuits Consider the DNA strand displacement reaction shown in Fig. 2.12, where the six major chemical species/structures in the reaction process are labeled X i (i = 1, . . . , 6). If the concentration of X i is denoted by xi for i = 1, . . . , 6, the reaction dynamics can be modeled by the following ordinary differential equations: x˙1 = −k f 1 x1 x2 + kr 1 x3 ,

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x˙2 = −k f 1 x1 x2 + kr 1 x3 , x˙3 = k f 1 x1 x2 − kr 1 x3 − k2 (x3 − x4 ), x˙4 = k2 (x3 − x4 ) + k f 3 x5 x6 − kr 3 x4 , x˙5 = −k f 3 x5 x6 + kr 3 x4 , x˙6 = −k f 3 x5 x6 + kr 3 x4

(2.3)

where k fi and kri are the association and dissociation rate constants, respectively, determined by various conditions such as the length of the base sequence of the toehold domain and the temperature, and k2 is the rate constant of the branch migration process in step 2 (see [45] for details). Equation (2.3) is a nonlinear differential equation, and its nonlinearity stems from the quadratic terms caused by the law of mass action. As mentioned in the previous section, a DNA circuit has a structure in which several sets of DNA strand displacement reactions are connected in a cascade and/or parallel manner. Hence, the general form of the DNA circuit is given by x˙ = f (x), x(0) = x0 ,

(2.4)

where x ∈ Dx ⊂ R n is a state vector and f : Dx → R n is a nonlinear function that includes the quadratic terms x∗ x∗ . Generally, for DNA circuits based on DNA strand displacement reactions, it is necessary to prepare a sufficient initial concentration for some specific DNA strands in order for the circuit to operate properly. For example, for the amplifier circuit designed in [37], it was shown that by setting the initial concentration of DNA strands X 2 and X 5 in Eq. (2.3) to xi (0)  x j (0) (∀i = 2, 5, ∀ j = 1, 3, 4, 6), the circuit functions as an amplifier with a relationship of x6 (∞) = (x5 (0)/x2 (0)) · x1 (∞), where the input and output strands are X 1 and X 6 , respectively. In this sense, DNA strands X 2 and X 5 can be considered “fuel” and DNA strands X 1 , X 3 , X 4 , and X 6 can be considered “signals” in the DNA circuit. The following definition characterizes the role of DNA strands. Definition 1 Fuel and signal DNA strands [46] Let I f and Is be index sets such that xi (0)  x j (0) for all i ∈ I f , j ∈ Is and  If Is = {1, . . . , n}. The symbol X i is called a fuel strand for i ∈ I f and a signal ◼ strand for i ∈ Is . Next, we discuss the DNA circuit in which the fuel and signal DNA strands are defined.

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2.5.3 Three Major Problems in DNA Circuit Design The bottom-up design of a DNA circuit with the desired function, established by combining DNA strand displacement reactions as elemental components, is the backbone of rational design. However, because the elemental component, the DNA strand displacement reaction (2.3), is already nonlinear, the interconnected circuit generally possesses complex characteristics, which complicates the circuit design because of the following three problems. Positivity: The DNA circuit (2.4) belongs to the class of positive systems because the concentration as a state in the system cannot have a negative value. Designers are required to be creative in handling negative values when designing DNA circuits that perform various types of information processing. More seriously, the theoretical characterization of nonlinear positive systems is an open problem, whereas the theoretical framework of linear positive systems has been thoroughly studied. Modularity: When designing a combinational circuit by connecting some logical gates such as AND and OR, the behavior of the whole combinational circuit should be predictable and should follow a truth table determined by Boolean algebra. This requires the circuit to be independent, and it is not acceptable for individual circuit functions to be altered by circuit connections. The invariability of the circuit functions against circuit connections is called “modularity”. In the electric circuit shown in Fig. 2.13 (upper), the modularity of the circuit is ensured by appropriately adjusting the input and output impedances (Ru , Rx(in) , Rx(out) , Rv ) of each circuit. In contrast, in the DNA circuit shown in Fig. 2.13 (lower), the connection of the circuit is realized via the reaction between the DNA strands responsible for the input and output ports. Because the modularity of the DNA strand displacement reaction as an elemental

Fig. 2.13 Circuit modularity: electric (upper) and DNA (lower) circuits

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component is known to be low [47], DNA circuits that ensure modularity should be designed. Finiteness: Normal operation of the DNA circuit is guaranteed under the condition that the reaction field contains a sufficient amount of fuel DNA strands. Because the initial concentration of the fuel strands is consumed gradually in the reaction with the signal strands, the DNA circuit operates normally only within a limited period of time. In addition to establishing a means of refueling the fuel strands externally, it is necessary to consider an “energy-saving” circuit design to efficiently use the limited fuel strands.

2.5.4 Example: Role of Control Theory in the Design of a Concentration Regulator Circuit One of the attractive applications of DNA circuit design is the development of a feedback controller for a molecular robot [48]. A molecular robot is a microscale autonomous mobile system consisting of biomolecules. Figure 2.14 schematically illustrates the system configuration of a molecular robot based on the blueprint of the Molecular Robotics Project in Japan [49]. The body, which is a capsule consisting of a phospholipid bilayer such as that of an amoeba, has a receptor-type sensor mounted on the surface and contains an internal molecular actuator. The inside of the body is filled with buffer to provide a reaction field for DNA reactions. This system is architecturally similar to a mechatronic robot, in the sense that the actuator operation is performed by the feedback controller based on the sensor information.

Fig. 2.14 Conceptual diagram of concentration regulator in a molecular robot

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The receptor-type sensor receives an external stimulus that encodes the command and releases a reference strand R in the reaction field. The molecular actuator is driven by a control strand U released from the feedback controller, and it releases an output strand Y depending on its operational status, where the concentrations of R, U , and Y , denoted by r (t), u(t), and y(t), respectively, are the signals in the feedback system. From the viewpoint of practical applications, a major control problem with a molecular robot is maintaining the activity of the actuator operation at the desired level. This requires the feedback controller, which maintains the output y(t) at the desired concentration r ∗ , to be appropriately designed on a DSD circuit. Here, we refer to such a controller as a “concentration regulator”. Although this control problem seems to be typical, the fact that a DNA circuit is a chemical reaction system means that there are some critical differences between molecular and mechatronic robots [46], and these differences complicate the controller design. Solution for the positivity problem: The general strategy for realizing a regulator is to continuously adjust the control signal u(t) based on the error signal e(t) = r (t) − y(t). Although the error signal can be positive or negative, there is still no ideal subtractor that responds continuously to DNA circuits. In this case, a design policy using the DNA comparator [50] shown in Fig. 2.15 can be a candidate concentration regulator [47]. In the comparator circuit, the two inputs r (t) and y(t) are continuously compared; in the case of r (t) > y(t), the control signal u(t) is generated as output to increase the actuator activity. In contrast, in the case of y(t) > r (t), the complementary strand U of the DNA strand U is released from the comparator circuit. The complementary strand U hybridizes with the DNA strand U present in the reaction field, thereby decreasing the control signal u(t) and lowering the actuator activity. In other words, the concentration regulator using the comparator circuit switches the control signal on and off near the operating point. This type of control law is widely used in mechanical and electrical systems. Solution for the modularity problem: The DNA comparator operates normally when it is tested independently as a single module. However, as shown in Fig. 2.16, the simulation result of the whole circuit connected to the comparator and the actuator shows that (i) the output signal y(t) is not maintained at the target concentration r ∗ , and (ii) the reference signal r (t) also decreases dramatically from the initial value r (0) = r ∗ . As a result, the concentration regulator fails to operate normally. In general, the modularity of DNA strand displacement reactions is not high enough;

Fig. 2.15 Design of concentration regulator using the DNA comparator

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Fig. 2.16 Simulation result without consideration of modularity (failed case)

therefore, the modularity of DNA comparator circuits, which are made by connecting DNA strand displacement reactions in cascade and in parallel, is also insufficient for the comparator to be integrated into the whole circuit. By applying the retroactivity theory, we can analyze how the modularity of a circuit is altered depending on the initial concentration of DNA strands and the reaction rate parameters (refer to [47] for details of the quantitative evaluation method for determining the modularity of DNA circuits). Figure 2.17 shows an example of an improved concentration regulator based on retroactivity theory. The appropriate insertion of an autocatalytic amplifier circuit (e.g., seesaw gate [40]) as an insulation module can enhance the modularity of each circuit. As a result, tracking of the output signal y(t) to the target concentration r ∗ is achieved, as shown in Fig. 2.18. Solution for the finiteness problem: Fig. 2.19 shows the simulation results when the concentration regulator operates for a long time. The fuel strand concentration in the reaction field must be sufficient to drive the concentration regulator normally, but this concentration decreases rapidly during circuit operation (single-dotted line). As the fuel strand concentration begins to be depleted, although the reference signal r (t) is maintained at around the target concentration r (0) = r ∗ , the output signal y(t) can no longer continue to follow the reference signal. The improvement of control performance in terms of energy saving is one of the most important requirements

Fig. 2.17 Design of concentration regulator using the DNA comparator along with insulators

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Fig. 2.18 Simulation result (improved case)

Fig. 2.19 Long-term simulation

for mechanical and electrical systems. In DNA circuits as well, optimization of the circuit design (reaction scheme, reaction rate constants, initial concentrations, etc.) based on control theory would contribute to improving the circuit performance more effectively, enabling the concentration regulator to work for prolonged periods with a limited fuel strand concentration.

2.5.5 Concluding Remarks In this section, the characteristics and mathematical models of DNA circuits are outlined, and three major problems that exist in circuit design are discussed. These problems and their solutions have been considered in the context of control theory, using the design example of the concentration regulator. In the field of control engineering, since the Industrial Revolution in the eighteenth century, many practical analysis and design methods have been developed to improve the stability, transient, and steady-state characteristics, optimality, and robustness of systems. Although DNA circuits differ from mechanical/electrical systems because of their nonlinearity, the challenge of applying control theory to DNA circuit design is being actively addressed.

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2.6 PEN DNA Toolbox Nathanaël Aubert-Kato The Polymerase-Exonuclease-Nickase Dynamic Network Assembly (PEN DNA) toolbox, created by Montagne et al., is based on interactions between DNA molecules and enzymes. This approach provides a framework for out-of-equilibrium designs with unique capabilities. Applications range from the creation of Turing patterns to the design of molecular robotics controllers. Mimicking Gene Regulatory Networks In every living cell, there is a complex computing system that uses a multistage process to transcript DNA (Genes) into messenger RNA (mRNA) that, in turn, will be used to create proteins. Those proteins may activate or inhibit the activity of other genes, forming complex networks named Gene Regulatory Networks (GRN). Complex behaviors, such as oscillations [51] or memories [52], have been observed and engineered in GRNs. GRNs also act as controllers (i.e., brains) for bacteria [53] and can thus be expected to have the computational power to control micro-robots. However, the whole process (DNA → RNA → protein) makes such systems slow and hard to customize. Montagne et al. [54] came up with a strategy to use a combination of DNA and enzyme generating the same complexity: • “Long” (approx. 25 nucleotides) DNA strands, called templates, act as genes, producing shorter DNA strands with the help of enzymes. • The shorter (11–13 nucleotides) DNA strands, called signal strands, hybridize with target templates and activate or inhibit them. • Signal strands are degraded over time by an exonuclease enzyme, keeping the system dynamic. Since the original design of the PEN DNA toolbox, multiple additional modules have been proposed: a predator–prey system allowing signal strands to reproduce from other strands [55], a pseudo-template approach, which increases the degradation rate of target signal species [56], and downstream transduction, where the output of a template is a DNA strand not related to the PEN DNA toolbox, giving us the possibility to interface with other molecular programming paradigms [57]. Working of DNA templates The DNA templates, central to the DNA toolbox, are made of two domains: an input and an output. When the appropriate signal attaches to the input domain, it gets elongated by an enzyme called polymerase. The result is a fully double-stranded

N. Aubert-Kato Ochanomizu University, Tokyo, Japan e-mail: [email protected]

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Fig. 2.20 Ideal working of a template: 1. Hybridization, 2. Elongation, 3. Nicking, 4. Denaturation

template. Templates contain the recognition sequence of a nicking enzyme, positioned so that the enzyme will then separate the input and output, eventually freeing them (Fig. 2.20). A core aspect of the DNA toolbox is that the output of a template can be a signal itself. It is then possible to connect such templates, generating GRN-like networks. As mentioned in the previous paragraph, exonuclease degrades single-stranded signal strands. However, templates are protected against this effect thanks to backbone modifications near its 5’ end. • Modules in the DNA toolbox There are three main modules in the DNA toolbox: activation, autocatalysis (or self-activation), and inhibition. Figure 2.21 presents their working, behavior, and graphical representation. When representing such modules, nodes represent signals, arrows represent templates, and bar-headed arrows represent inhibition. The third module, inhibition, is done by a specific type of DNA signal strand called an inhibitor. It is designed to attach to most of its target template but does not trigger the polymerase (due to mismatch) or nickase (due to incomplete recognition site). This configuration inactivates the template until the inhibitor denatures. Note that inhibitors cannot be used as inputs.

Fig. 2.21 Modules of the DNA toolbox

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Fig. 2.22 Montagne et al.’s Oligator and experimental results (from [54])

Montagne et al. used this strategy to create an oscillator [54] (Fig. 2.22), while Padirac et al. made a bistable circuit as well as a bistable switch [58]. All those systems have been implemented experimentally and formed the basis for more complex theoretical constructs [59]. A function in the PEN DNA toolbox is implemented through the interactions between those different modules (Fig. 2.23). The inputs of that function are designated species either directly injected into the system or generated by another molecular program. Eventually, an output signal is released. The standard way to measure the output is to use fluorescence monitoring. Simple model Due to the nonlinearity of DNA toolbox systems, it is hard to predict the exact behavior of a given system. At the lowest level of detail, we consider template species as black boxes (Fig. 2.24). Those boxes have a simple transfer function inspired by Michaelis–Menten reaction rates. The rationale behind this model is to assume that hybridization and denaturation (two complementary DNA strands attaching and detaching, respectively) reach equilibrium much faster than enzyme-based reactions. This approximation yields the transfer function in Fig. 2.24b for each module. The complete set of equations can thus be directly built from the graph representation of a system by summing over templates generating each species, with the addition of a first-order approximation of the exonuclease activity shown in Fig. 2.24d.

Fig. 2.23 Function in the PEN DNA toolbox

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Fig. 2.24 Black box representation and equations for the DNA toolbox. The vertical bar represents the restriction, [.] represents concentration, and α and β are parameters. Exo is the exonuclease activity

This model of the DNA PEN toolbox was first introduced in Padirac et al. [58]. One of the main advantages of this model is that the simulation of large systems is extremely fast, thanks to the simplicity of the equations. As such, the simple model is suitable for the evolution of complex DNA toolbox systems requiring thousands of separate evaluations [60]. While the simple model presents a reality gap compared to the full model [61], evolutionary optimization will still find reusable design patterns [62]. Full model: DACCAD The major limitation of the simple model is that template states are not explicit. It is thus impossible to account for the protection against the exonuclease that comes from being hybridized into a template. It is also impossible to compute the saturation of enzymes since the actual concentrations of their substrates are unknown. Finally, the delay induced by the hybridization-extension-nicking cycle is lost. To solve those problems, Padirac et al. proposed a full domain-level model [58] that keeps track of the strands hybridized to each domain of the templates. This model was further refined and extended by Aubert et al. [59, 61]. Each template is modeled by five variables: with input (tempin), with output (tempout), with both (tempboth), fully double-stranded (after step 2 in Fig. 2.1; tempfull), and an optional variable for inhibited template (tempinhib). The concentration of free templates can be computed from the total concentration added to the system minus the sum of the other five variables, and thus does not need to be expressed as a differential equation. Those equations are given in full in [61]. Once we have expressed the equations for all templates in the system, we can write the flux for the input, output, and inhibitor species with respect to that template:   in ϕs,temp = kduplex K s [tempin] + stacktemp · [tempboth]   + kduplex · i temp · [tempin] − kduplex [s] · ([tempalone] + [tempout] + λin · [tempinhib])

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  out ϕs,temp = kduplex K s [tempout] + stacktemp · [tempboth]   + kduplex · i temp · [tempout] − kduplex [s] · ([tempalone] + [tempin] + λout · [tempinhib]) + poldispl · [tempboth]   inhib ϕtemp = αkduplex K i [tempinhib] − kduplex i temp ([tempalone] + [tempin] + [tempout]) + kduplex [tempinhib] · (λin [sin ] + λout [sout ]) k duplex is the association constant, K s the stability constant for species s, stack is the stacking slowdown of the template, λin and λout are the displacement slowdowns of the inhibitor (from the input and output side, respectively), poldispl is the activity of the polymerase while displacing an output, and α is the stability malus for the inhibitor, as it isn’t fully double-stranded to its target template. We then get the full equation for a given signal species:  d[s]  in = ϕtemp + ϕ out − exos · [s] for an activating signal temp∈I temp∈O temp dt  d[i] inhib = ϕtemp + ϕ out − exoi · [i] for an inhibiting signal temp∈O temp dt Due to the massive amount of combinations and possible reactions considered in the model, it is difficult to manage the simulation by hand. Aubert et al. introduced a graphical user interface named DACCAD to streamline this process [59]. The program is freely available online. Evolving complex systems: BioNEAT While DACCAD can help quickly iterate over versions of a given system, it still requires a human designer to have an idea of the correct design. That might not be the case when the target behavior is complex, or not easily understandable as a combination of basic modules. In that case, we are faced with an inverse problem: rather than trying to find the behavior of a given system (simulation), we are trying to find systems matching a given behavior. Using an evolutionary algorithm is a standard approach to solving such inverse problems. Huy et al. introduced an algorithm called BioNEAT [60, 62], which is specially adapted to find and optimize designs for the PEN DNA toolbox. Figure 2.6 shows the working of the algorithm: starting from a set of basic systems, the algorithm progressively increases the complexity of their structure, selecting those that are closer to the target behavior. The optimization continues either for a fixed number of iterations or until the distance to the expected behavior is below a given threshold (Fig. 2.25).

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Fig. 2.25 Evolving a robust oscillator

Wet lab implementation Once a system has been correctly put together and simulated, the next step is to design its actual DNA sequences. A thorough explanation of this particular workflow has been described by Baccouche et al. [63]. That article also describes the basic experimental setting required, as well as solutions to commonly occurring problems. Additionally, Baccouche et al. also proposed a parameter scanning strategy using a microdroplets approach [64]. Their approach generates tens of thousands of droplets, each with a different value for up to three parameters. Each droplet also contains a fluorescent bare code that allows recovering the parameters it was primed with. Their approach allowed them to identify the working range of 2 standard systems: Padirac et al.’s bistable circuit [58] and the Predator–Prey system from Fujii and Rondelez [55]. Reaction–diffusion systems While well-mixed environments have a certain computational power, they also have restrictions due to competition for enzymes, equivalent to the processing units in standard computers. Moving to a heterogeneous environment could remove this limitation, as different areas perform separate computations in parallel. Moreover, in the case of spatially heterogeneous systems, the PEN DNA toolbox can be used to create patterns. A specific type of spatio-chemical patterns, called Turing patterns, is believed to be a driving mechanism for morphogenesis, such as the formation of stripes on the fur of Zebra and digits in mammals. Synthetically engineered reaction–diffusion systems could have applications ranging from the creation of novel materials to the development of artificial organs.

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• Model for reaction–diffusion Spatially heterogeneous chemical systems are called reaction–diffusion systems, as the diffusion of molecules now has to be taken into account. The set of ODE describing a given system has to be extended to include the diffusion of species in the environment. For each species s, the derivative at point (x, y) becomes d[s] = R(s) + Ds ∗ ∇ 2 (s) dt where R(s) is the reaction flow for s (from the well-mixed model, using either simple or complex equations), Ds is the diffusion rate of s, and ∇ 2 (s) is the concentration gradient (Laplacian) of s in (x,y). That set of equations thus describes a Partial Differential Equation (PDE) system. Like ODE systems, PDE systems cannot be solved analytically, except in very simple cases. Instead, we must rely on numerical simulation. Note that programs solving that type of problem are freely available (see, for instance, ReaDy [65]). • 1D The simplest type of RD system is one where one dimension of the reactor is much larger than the other two. In practice, such a system can be effectively considered unidimensional. Moreover, reactors with those characteristics can be easily made using microfluidics fabrication techniques. Zadorin et al. have checked the basic behavior of the PEN DNA toolbox in that environment. They started from a long reactor filled with a free autocatalytic template and added trigger signal strands on one side. Due to the catalysis, the propagation speed is faster than simple diffusion. Zadorin et al. showed that the speed of the wave is similar to what is expected from theory [66] (Fig. 2.26). Moreover, two diffusion fronts from independent templates going in opposite directions did not affect each other. However, upon collision, the fronts had a slight decrease in propagation speed, most likely due to enzymatic saturation.

Fig. 2.26 Fluorescence profile at different times (top) and propagation of the front (bottom) from [66] (left) and simulation (right)

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Fig. 2.27 French flag pattern. Top: reaction network. Second line: morphogene concentration over x. Third line: kimograph of the stable zone. Bottom: fluorescence profile of autocatalytic signals. Adapted from [67]

They later extended that approach to create a standard reaction–diffusion pattern made of three consecutive bands [67]. They used a morphogene: a stable chemical gradient in the environment impacting the behavior of the autocatalytic fronts. When the concentration of the morphogene reaches a threshold, the activity of the left autocatalyst stops. Conversely, when the morphogene is below another threshold, the right autocatalyst stops. By tuning those thresholds, they can create a variety of conditions, such as left active—none active—right active or left active (bistability)— both active—right active (Fig. 2.27). • 2D The most standard reactors are 2D: a thin layer of reaction mix is spread across a surface, and kept between sealed glass slides (or other transparent material) to prevent evaporation. Padirac et al. used a very simple approach to prepare such reactors, using parafilm and glass slides [68] (Fig. 2.28): – – – – – –

Cut parafilm into the desired shape (here a circular area). Put on a glass slide. Bake to attach the parafilm to the glass. Fill in the reactor. Put on the top glass layer. Bake again to seal by melting the parafilm.

Putting an oscillating system in that environment yielded traveling waves, similar to theoretical results [68]. While those results could be achieved with other chemical oscillators, such as the BZ reaction, the PEN DNA toolbox gives us much more flexibility in terms of programmability. • Molecular robotics

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Fig. 2.28 Padirac et al.’s preparation of 2D reactors. Reproduced from [68]

Gines et al. [70] provided a strategy to control the localization of PEN DNA toolbox templates in a reaction–diffusion context. Their approach was to use sepharose beads (agarose beads with cross-linked streptavidin) to capture templates chemically bonded to a biotin molecule. They used large beads (~50 um) that were static but could capture and release signal strands in the environment. Such a system thus behaves in a way similar to ad-hoc communication networks. To create molecular robots, two elements were missing: mobility and actuation. Mobility was achieved by scaling down the beads to ~5 um, making them mobile through Brownian motion. Actuation was provided by an extension to the PEN DNA toolbox: the production of a special strand (anchor) able to connect two beads together [67, 69] (Fig. 2.29). • Conclusion The PEN DNA toolbox is a versatile framework for analog molecular computing. Its modularity allows the user to create large, complex reaction networks in vitro while existing software can help with ensuring the correctness of the design. In the past few years, the PEN DNA toolbox has been used to program reaction– diffusion systems, as well as simple molecular robots.

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Fig. 2.29 Localization of a PEN DNA toolbox system on beads and bead-binding mechanism. Reproduced from [69]

2.7 Design of Chemical Reaction Networks with Unknown Reaction Dynamics Shun-ichi Azuma For molecular robots, it is typical to implement a controller, which governs the dynamics of the robot, on a chemical reaction network. Thus, the design of a chemical reaction network is an important issue. Meanwhile, one often encounters the situation where the network structure can be explicitly designed but the node dynamics, which is the dynamics of individual reaction, cannot. In such a case, it is preferable to specify the dynamics of the chemical reaction network by designing only the network structure [71]. Here, we introduce a solution to such an issue for a mathematical model of chemical reaction networks, called the Boolean networks [72]. A Boolean network [73] is given by xi (t + 1) = f i



   x j (t) j∈Ni , x j (t) j∈N i (i = 1, 2, . . . , n),

where xi (t) is the state of node i, which takes a binary value (i.e., 0 or 1), f i is a monotone Boolean function, and Ni and N i are the index sets of the neighbors of node i satisfying Ni ∩N i = ∅. The set Ni specifies  the neighbors whose state directly affects the update of the state of node i and x j (t) j∈Ni denotes the vector composed of the states of the neighbors listed in Ni . Meanwhile, N i specifies the

S. Azuma Nagoya University, Nagoya, Japan e-mail: [email protected]

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  neighbors whose negated state affects the node i and x j (t) j∈N i is the vector defined   in a similar manner to x j (t) j∈Ni . The network structure of the system is expressed by the edge-labeled directed graph G with the node set {1, 2, . . . , n}, the edge set {( j, i)| j ∈ Ni ∪ N i }, and the labeling function L such that L(( j, i)) = 1 for j ∈ Ni and L(( j, i)) = −1 for j ∈ N i . By letting F denote the node dynamics, i.e., a tuple of f 1 , f 2 , ..., f n , the system can be considered as the pair (G, F), and thus it is referred to as the system (G, F). The system (G, F) is said to be monostable if it has a unique attractor and the attractor is an equilibrium. Furthermore, the system is said to be structurally monostable if it is monostable and the system (G, F ∗ ), which is a modified version of the system (G, F) in terms of the node dynamics, is monostable for any possible F ∗ . Note that the latter property depends only on G. Even if we have no knowledge of F for the system (G, F), we can guarantee that the system is monostable, provided that its network structure G is constructed so that the resulting system is structurally stable. This system property is useful for the aforementioned situation where the network structure of the chemical reaction network can be only explicitly designed. Now, when is the system (G, F) structurally monostable? An answer is given as follows [72]: G is 8-shaped, the length of one simple cycle is the integral multiple of that of the other simple cycle, and the longer simple cycle (if the simple cycles have different lengths) or either simple cycle (if the simple cycles have the same length) has an odd number of the edges with label −1 and the other cycle has an even number of the edges with label −1.

2.8 Real-Time Visualization of Swarm Molecular Robot Dynamics Gutmann Gregory Spence andAkihiko Konagaya

2.8.1 Introduction Rational molecular robot design requires not only molecular design facilities for supramolecular assemblies such as DNA-origami and protein complexes but also molecular simulation facilities to reproduce experimental results of molecular robot dynamics. As for the dynamics of individual molecular robots, three pioneering G. G. Spence · A. Konagaya Molecular Robotics Research Institute, Co., Ltd., Tokyo, Japan e-mail: [email protected] A. Konagaya e-mail: [email protected]

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works have been reported so far: (1) linear molecular dynamics with microtubules and motor proteins in a giant liposome [74], (2) nematic alignment dynamics of confined actin in a giant liposome [75], and (3) peptide nanofiber dynamics to propel a giant liposome [76]. On the other hand, swarm dynamics of molecular robots have been studied in the category of artificial molecular muscle [77, 78]. The emergence of molecular robot swarm dynamics strongly depends on the complex interaction of thousands of molecules. In addition, these interactions, which result in swarms, often continue to evolve and develop over the course of minutes to hours. However, both of these points, the number of objects and the time scale, pose significant challenges for simulation. With the rapid increase in CPU and GPU performance, memory capacity, and inter-processor network performance, we are able to deal with more than 10 million atoms in molecular dynamics (MD), simulating a femtosecond time step [79]. However, the simulation time scale that MD simulations deal with is at most nanoseconds to microseconds on conventional computers. This implies that coarse-grained molecular dynamics, which deals with a chunk of atoms as a particle, is needed to reproduce molecular robot swarm dynamics and the emergence of larger scale patterns. Recently, a particle simulation system with real-time visualization capabilities has been developed as one of such platforms to observe microtubule swarm dynamics [80]. This system simulates all particle–particle interactions and visualizes the resulting behavior of the particles at a rate of 30 frames per second, the standard frame rate for movies, providing smooth simulation and visuals of molecular swarms. The following section describes the concept and experiences of the real-time particle simulation system from the viewpoint of molecular modeling, swarm pattern formation, real-time visualization, and real-time high-performance computing using microtubule motility dynamics as an example.

2.8.2 Microtubule Particle Modeling Microtubule motility assay is a well-known experiment to observe swarm dynamics of microtubules propelled by motor proteins fixed on a glass surface with a fluorescence microscope. Since the movement of motor proteins is random, microtubules move independently under sparse conditions. However, under dense conditions, when microtubules are dosed with linker molecules, swarms start forming linear and/or spiral motion patterns. In order to simulate microtubule interactions, coarse-grain modeling is necessary due to the number of atoms in a microtubule. A microtubule is a hollow tube-like protein complex whose diameter is about 25 nm and whose length ranges from 5 to 50 μm in general. The hollow tube consists of 13 long sequences of tubulin dimers. Each tubulin dimer forms an 8 by 5 nm spheroid whose molecular weight is about 11 thousand. Therefore, a 5-μm length microtubule consists of about 8 thousand tubulin dimers bringing the total molecular weight to about 900 million.

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Fig. 2.30 Conceptual image of particle–particle interactions of microtubule overriding (CC BY 4.0 Reprinted from “Introduction to Molecular Robotics Creating a system with molecular design” (in Japanese), 1.1.8 Fig. 30, p. 52, CBI Society eBook Vol. 3, 2019)

Interestingly, more than 2000 microtubules were necessary for the recreation of microtubule swarm behavior in our particle simulation. In order to deal with such a massive number of microtubules in simulation, each microtubule was represented by a sequence of 100–200 particles, where each particle represented a 25-nm segment of the microtubule, which is also equal to the diameter of microtubules. In our microtubule motility assay simulation, we found that one of the primary factors that determine whether or not large-scale collective motion would emerge was the rate of overriding versus snuggling. Two microtubules will undergo overriding or snuggling depending on the crossing angle of microtubule interaction [80]. In order to deal with such microtubule interactions in a particle simulation, a Lennard–Jones potential among particles was introduced (Fig. 2.30). As a result, various motion patterns could be reproduced by tuning the Lennard–Jones potential.

2.8.3 Motion Pattern Formation It is widely known that swarms of fish and birds form complex motion patterns caused by interactions between neighboring individuals and not by some form of global control. In other words, complex swarm motion patterns could be reproduced by a simple local rule, such as keeping a distance equally from neighbors for each individual in the swarm. The Vicsek model [81] is one of such mathematical models which can reproduce swarm motion patterns and is defined as follows. xi (t + 1) = xi (t) + vi (t)t. In this equation, xi (t) and vi (t) represent the location and velocity of individual xi at a time t. vi (t) has two parameters of swarm speed v and angle θ(t + 1) for turning. For each individual, the next turning angle θ(t + 1) is calculated by the average

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Fig. 2.31 Vortex motion patterns in microtubule swarm dynamics (CC BY 4.0 Reprinted from “Introduction to Molecular Robotics Creating a system with molecular design” (in Japanese), 1.1.8 Fig. 31, p. 53, CBI Society eBook Vol. 3, 2019)

speed of other individuals in a radius r, and noise term θ extracted from a uniform distribution [−η/2, η/n]. The Vicsek model is very simple but powerful enough to reproduce complex microtubule motion dynamics. Within the real-time visualization particle simulation, various sizes of vortex motion patterns can be reproduced by introducing the neighbor radius-based interactions and turning the angle parameters for microtubules similar to the ones in the Vicsek model (Fig. 2.31).

2.8.4 Real-Time Visualization The rapid increase in GPU performance has made it possible to perform real-time visualization and simulation in parallel at conventional video frame rates. This enabled us to observe simulation processes on the fly, as well as end the simulation early if the results were not favorable or needed. These are significant merits of real-time visualization from the viewpoint of time-and-cost saving, in contrast to conventional off-line visualization, which needs to wait for the entire simulation to be finished before starting visualization and analysis. Another merit of real-time visualization is the discovery of strange but interesting behaviors by the human eyes. Strange behaviors such as towering microtubules and flying microtubules occurred at times in our microtubule motility simulation. Real-time visualization has enabled us to observe such strange behaviors deeply by zooming in on specific areas. In some cases, the strange behaviors turned out to be artifacts caused by incorrect simulation models or parameter settings. However, the discovery of such artifacts was very useful to accelerate a cycle of modeling, simulation, and testing.

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Fig. 2.32 Complex vortex motion patterns emerged in microtubule swarm simulation (CC BY 4.0 Reprinted from “Introduction to Molecular Robotics Creating a system with molecular design” (in Japanese), 1.1.8 Fig. 32, p. 54, CBI Society eBook Vol. 3, 2019)

The largest merit of real-time visualization is on-the-fly simulation parameter tuning. It played a very important role in finding complex motion patterns which were able to reproduce an experimental result, as seen in Fig. 2.32. The behavior of the simulation dynamics strongly depended on simulation parameters and initial microtubule distribution. Most parameters were not so sensitive, but some were very sensitive for motion pattern emergence. Parameter-parameter dependency also made it difficult to find an optimal simulation parameter set that reproduces experimental results. Metasearch might be applicable when an evaluation function is available; however, no good evaluation function is currently known for microtubule motion pattern discovery. So, we strongly believe that real-time visualization could be one of the most powerful ways to study microtubule motility dynamics.

2.8.5 Real-Time High-Performance Computing Real-time high-performance computing is different from conventional highperformance computing due to the extreme latency requirements of real-time viewing and interactions. Conventional high-performance computing aims to achieve maximum computational throughput, often by dividing work into large tasks to minimize communication overhead at the cost of increasing latency, the time required to complete each distributed task. Real-time high-performance computing also seeks to reach maximum computational throughput; however, it also requires each task to be completed at interactive rates. Thus, alternative methods for task scheduling and communication are required for minimizing both the scheduling time and the amount of data that is needed to be communicated during each simulation step. Intensive use of distributed GPGPU processing has enabled us to achieve real-time visualization

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Fig. 2.33 GPU scalability for Real-time visualization particle simulation (CC BY 4.0 Reprinted from “Introduction to Molecular Robotics Creating a system with molecular design” (in Japanese), 1.1.8 Fig. 3, p. 55, CBI Society eBook Vol. 3, 2019)

of massive microtubule particle simulations at a rate of 30 frames per second for 2D flat displays and 90 frames per second for virtual reality head-mounted displays. As for the computational performance, the latest GPUs such as TITAN X and P100 contain more than 3000 cores and can achieve 10 Tera FLOPS in singleprecision floating-point decimal calculation performance. By dividing up the simulation process into several massively parallel tasks of local particle interactions, we are able to make full use of many GPUs to achieve the required performance for largescale real-time simulation. Linear scalability up to 10 GPUs was achieved by means of space decomposition and GPU task scheduling on a shared memory architecture, as seen in Fig. 2.33. As for real-time visualization, GPU also played an important role in 3D object rendering. The use of instanced rendering, batched parallel drawing, has enabled us to render more than a million particles in real time.

2.8.6 Summary Real-time visualization of microtubule particle simulation has enabled us to reproduce microtubule motion patterns very similar to the ones observed in microtubule motility experiments. In the simulation, complex swarm patterns emerged from the

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local interaction of particle sequences representing microtubules. We strongly believe that real-time high-performance computing would also be applicable to study other dynamics of molecular robots. In order to achieve this, a VR particle simulation system with interactive operation capabilities is currently under development.

2.9 Molecular Robot System as a Distributed System Yukiko Yamauchi A distributed system consists of autonomous computing entities that cooperate with each other. Its application covers a variety of systems such as a network of computers, a robotic swarm, a human society, and a natural system, where the global behavior of a system is generated by local computation and interaction of small computing entities. Theoretical studies on distributed systems generally start with assumptions on computing entities (i.e., a system model) and a description of global behavior (i.e., a problem definition). Existing studies demonstrate impossibilities due to locality, asynchrony, and parallelism, and how to realize distributed coordination (i.e., distributed algorithms) for solvable cases. A molecular robot system can be considered as a distributed system but there are many choices for a system model. For example, molecules, DNA strands, and cells can be considered as autonomous computing entities. Common properties of such distributed systems are the large size of the system and anonymity, uniformity, and asynchrony of computing entities. That is, a large number of indistinguishable computing entities follow a common computation rule. They asynchronously perform computation and interaction that yield global behavior of the distributed system. Self-organization is one of the most important issues because the behavior of such an anonymous distributed system is represented by the shape of the system. In distributed system theory, one of the most important issues is the capability of each computing entity that enables a specific global behavior of the system. As an example, we briefly survey the self-organization of a swarm of autonomous mobile robots. Each robot is an anonymous point in the 2D space and autonomously moves while executing a common algorithm. It observes the positions of other robots, computes its next position based on the current observation, and moves to the next position. Each robot asynchronously repeats this unit action called a Look-Compute-Move cycle. We consider a distributed system of oblivious robots, whose local memory is reset at the end of each cycle. The robots have no explicit communication medium, and they interact with each other solely by observation and movement. The pattern formation problem requires the robots to form a specified shape, for example, a

Y. Yamauchi Kyushu University, Fukuoka, Japan e-mail: [email protected]

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point, line, and circle. It has been shown that when a common algorithm is deterministic, the symmetry of an initial configuration of the robots determines formable shapes irrespective of asynchrony and obliviousness [82–84]. In addition, when the robots can access randomness, asynchronous oblivious robots can form any arbitrary pattern [85]. Most existing results consider robots in 2D space [82–85], and it has been shown that they can be naturally extended to robots in 3D space [86]. Additional capabilities are considered for autonomous mobile robots. A luminous robot is equipped with a light that can take a constant number of colors [87]. The light is an abstraction of local memory and communication. The colored robot model considers a heterogeneous mobile robot system [88]. Many related problems are investigated such as the formation of a sequence of patterns [89], team assembling [88], leader election [90], and exploration [91]. See [92] as a survey of existing studies on the autonomous mobile robot model and related models. Recently, a variety of distributed system models inspired by natural systems have been proposed. The population protocol model focuses on global behavior generated by pairwise interactions of agents [93]. The Amoebot model is inspired by the motion of amoeba and considers programmable particles in the triangular grid [94]. The tilt assembly model considers particles that move under external forces and bond together [95]. The metamorphic robotic system model consists of autonomous modules that can perform sliding and rotation by keeping global connectivity [6, 96]. These new models consider different types of memory, mobility, and interactions. However, existing studies demonstrate that self-organization is possible in these models when anonymous, uniform, asynchronous computing entities have access to randomness.

2.10 Toward the Molecular Artificial Intelligence Yasuhiro Suzuki We realized the intelligence of molecular robots, based on the philosophy of intelligence of living systems by Millikan [97]. The philosophy claims that an intelligent system must have a purpose, which arises spontaneously from the inside of the system, not to be given from the outside. We set the purpose of the system to keep reproducing the same molecules. We used a seesaw-gate reaction [98], and investigated whether the reaction could be maintained when mutations were introduced into the input molecule. We found that the reaction system works even with the mutations in the input molecule. We confirmed that if the concentration of the input molecule without mutation is equal to or greater than the input molecule with mutation, the seesaw-gate reaction selects the input molecule without mutation. However, if the concentration of Y. Suzuki Nagoya University, Nagoya, Japan e-mail: [email protected]

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Fig. 2.34 1-bit memory using seesaw-gate reaction: i’ is the concentration of input sequence with mutation, and i is the input sequence without mutation. By varying the concentration of each sequence, 2-bit memory is realized

input molecules with mutations is more than twice that of those without mutations, the seesaw-gate reaction selects input molecules with mutations. The seesaw-gate reaction selects the available input molecules with mutations if the concentration of input molecules without mutations is low. In other words, the reaction can adapt to the environment. We regard this environmental adaptation as “intelligence”. The seesaw-gate reaction is based on the chain-replacement reaction, which has a scaffold sequence, called a toehold. We confirmed that this adaptive behavior does not change even if the toehold can be wholly removed. In comparison with a related study [99], which investigated the strand-replacement reaction of mismatch sequence, we found that the reaction time was faster when the mismatch sequence (input molecule with mutation) coexisted with the full-match sequence. Based on this finding, we can compose memory, by varying the concentration of mutated and unmutated input molecules (Fig. 2.34); in the following, input molecules with mutation are denoted as i’ and their concentration as [i’], input molecules without mutation and their concentration are denoted as i, [i], respectively. The system has three equilibrium states, [i] = [i’], [i] = a[i’] (a > 1), [i’] = a[i], and a[i] = a[i’] [4]. Let the equilibrium state [i] = [i’] be (0, 0), [i] = a[i’] (a > 1), [i’] = a[i] (0, 1), and a[i] = a[i’] (1, 1), resulting in a 2-bit memory [100]. Bit on/off is implemented by increasing or decreasing [i] or [i’].

2.11 Molecular Robots as Emergent Systems Ken Sugawara Emergence is a phenomenon in which local interactions between elements or between elements and the environment in a system composed of many elements generate global properties and functions that are greater than the simple sum of their parts. Even if the properties and behaviors of individual elements are simple, there is a K. Sugawara Tohoku Gakuin University, Sendai, Japan e-mail: [email protected]

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possibility that advanced functions emerge as a group. On the other hand, there is a problem of design difficulty in emergence, i.e., what characteristics should be given to each element to achieve the desired function, and it is still an open question. Molecular robotics is very interesting from the point of view of emergent systems. It would be possible to make intelligent molecular robots utilizing chemical reactions or physical properties of the molecules. However, its intelligence must be limited because each robot consists of limited sensors, limited actuators, and a simple processor. Therefore, it is necessary to make the best use of the interaction between robots or between robots and the environment and to generate useful functions for the system as a whole. This is the interesting point of molecular robots as emergent systems. Molecular robots may also shed new light on hierarchy, which is often mentioned in the discussion of emergent systems. Hierarchy is a phenomenon that can often be found in natural phenomena, but there are very few examples that produce hierarchy in artificial systems. One of the reasons is that in conventional artificial systems, it has been difficult to create enough elements to discuss hierarchy. In the world of molecular robots, however, it is possible to construct a swarm of Avogadro’s number of robots. In this sense, molecular robots can provide the basic framework for emergent systems. Molecular robotics could have a remarkable property that is essentially different from conventional robotics. In conventional robotics, it is possible to make a clear distinction between robot, environment, and information. However, in molecular robotics, the robot itself is a molecular-scale object, and the environment surrounding it is also a molecular-scale object, and the medium of information transmission is a molecular-scale object, and they could have indivisible relationships (Fig. 2.35). This property must be definitely important and essential for molecular robotics. This nature of molecular robots is also expected to give concrete examples of emergent systems.

Fig. 2.35 Molecular robotics requires the inseparability of robot, environment, and information

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Chapter 3

Systemization Technology for Molecular Robots Shin-ichiro M. Nomura

Abstract This chapter describes the practical examples of efforts in designing molecules and their combinations to work as a system and obtain higher-level spatiotemporal structures. While life on earth has evolved through and even defined by a series of practical and environmental challenges over 4 billion years, our synthetic systems can be designed and implemented without the constraints of the earth’s ambient conditions. Here, we introduce the overview of research on artificial cells and molecular robots including the systemization of amoeboid (artificial cell type) molecular robots. Then we describe gellular automaton and molecular computing where a variety of information is propagated in the gel, and changes in their physical properties and material production are constantly repeated. Next, we explain the use of microfluidic devices in manipulating water droplets containing the desired molecules for the controlled assembly of molecular robots at the micron scale. We also describe the practical applications of using nanopores as molecular gates in lipid membrane. Finally, we give an overview of synthetic biology, an important research field connecting molecular robots and artificial cells.

This chapter describes the practical examples of efforts in designing molecules and their combinations to work as a system and obtain higher-level spatiotemporal structures. As molecules are not isolated in space, they have the capacity to both influence and are influenced by neighboring molecules, such interactions are governed by both their chemical properties and physical structure, the complex interplay of which leads to interesting higher ordered assemblies. For example, lipid molecules with spatially separated hydrophobic and hydrophilic sections are capable of forming closed structures in water, delineating an internal and external environment. Charged molecules interact strongly with one another, with opposite charges attracting and coordinating with each other, and like charges repelling and orientating themselves away. Macromolecules such as polymers accordingly have many more potential molecular interactions both externally (intermolecular) and internally self-interacting (intramolecular). S. M. Nomura (B) Department of Robotics, Tohoku University, Sendai, Japan e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2022 S. Murata (ed.), Molecular Robotics, https://doi.org/10.1007/978-981-19-3987-7_3

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Such interactions can lead to conformation changes, the degree and resulting shapes of which are determined not only by attractive and repulsive forces but their spatial order in the macromolecule sequence. Proteins, for example, encode their spatial structure by their unique amino acid chemical sequence which in turn produces their unique folding pattern and their resulting functional shape. Similarly, DNA and RNA also encode information and function in their base pair sequences. Interestingly, some combinations of interactions between single molecules are known to give rise to self-assembly, which in turn leads to higher-order self-organized structures, resulting in a hierarchy that moves from the molecular building blocks all the way up to the macroscopic scale. Here too, the specific molecular shapes and interactions lead to a wide variety of molecular assemblies, including crystals, folders, micelles, vesicles, liquid crystals, clusters, phase-separated structures, droplets, and gels. In addition to the function of individual molecules, their spatially assembled and spatio-temporal organized structures have the potential to be controlled in both space and time, depending on their molecular design. While life on earth has evolved through and even defined by a series of practical and environmental challenges over 4 billion years, our synthetic systems can be designed and implemented without the constraints of the earth’s ambient conditions. In this chapter, we show the following examples of this molecular systematization approach. In Sect. 3.1, Nomura introduces the overview of research on artificial cells and molecular robots; in Sect. 3.2, Sato describes the systemization of amoeboid (artificial cell type) molecular robots. In Sect. 3.3, Hagiya describes gellular automaton and molecular computing where a variety of information is propagated in the gel and changes in their physical properties and material production are constantly repeated. In Sects. 3.4 and 3.5, Arimura et al. describe an example of its realization in real space and its movement mechanism, and in Sect. 3.6, Kawamata describes an attempt to move the gel automata. In Sect. 3.7, Takinoue explains the use of microfluidic devices in manipulating water droplets containing the desired molecules for the controlled assembly of molecular robots at the micron scale. In Sect. 3.8, Takeuchi et al. describe the practical applications of using nanopores as molecular gates in lipid membranes, allowing passage and even measuring of molecules as they move across the through the nanopore. Finally, in Sect. 3.9, Kuruma gives an overview of synthetic biology, an important research field connecting molecular robots and artificial cells. Through this chapter, we hope that you will see how the design of molecules and their systems can achieve structural organization and functionalities, expanding the possibilities of bridging from basic science to application.

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3.1 Artificial Cell Research and Molecular Robotics Shin-ichiro M. Nomura S. M. Nomura Department of Robotics, Tohoku University, Sendai, Japan e-mail: [email protected] Artificial cell research has a long history, and a wide variety of spatio-temporal structures have been proposed [1]. Nano- and micro-sized structures, called liposomes or vesicles, are established models for cells [2]. These are microcapsule-like structures that spontaneously assemble from amphiphilic lipid molecules dispersed in water, and have recently attracted attention in combination with molecular design in DNA/RNA/protein nanotechnology. The molecular design field is expected to scale up from single molecules to supramolecules and supramolecular complexes with unique functions. This chapter reviews the area of artificial cells of lipid vesicles research and related molecular robotics.

3.1.1 Introduction Since long before alchemy, the origin of chemistry, humans have wanted to create life with their own hands. To create a phenomenon that can be called life, by a method other than the self-replication performed by living organisms, would be a liminal technological breakthrough in engineering, and fundamentally groundbreaking in science, opening windows into a closer examination of what the essence of life is. Experimental research on artificial cells aims to reach this goal. On the other hand, as described in this book, molecular robotics research aims to systematize molecules and make them work and can be characterized as ‘making machines that look like living things’. Both artificial cells and molecular robotics belong to engineering. Engineering is a part of human culture; it is the study of creating better things under unbreakable rules. Since the rules are based on those revealed by the natural sciences1 , a Venn diagram relating the natural sciences to artificial cell and molecular robotics research could be drawn, as shown in Fig. 3.1. Engineers are in the luxurious position of being able to decide which knowledge to pick up from the entire Venn diagram, based on their own personal interest. As of the year 2021, engineering stands between living organisms and materials at the star in Fig. 3.1, where artificial cells are closer to living organisms, and molecular robotics is nearby but concerned with non-living matter.

1

Although the natural sciences are also part of human culture, their motivation slightly differs from that of engineering in aiming to solve unexplored mysteries for humanity.

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Fig. 3.1 Epistemic relationship between artificial cell engineering and molecular robotics

3.1.2 Artificial Cell Research There are three main purposes of studying artificial cells: (1) (2) (3)

To realize a possible model of life. To understand the origin of life on earth. To reconstruct cells that are representative of existing life forms.

(1) and (2) are fascinating themes in science. The desire to mimic cells from matter and, if possible, to construct real cells, has led to the construction of various models, such as by Traube or by Oparin, in considering the origin of life and astrobiology. The materials and conditions that can be used for biosynthesis are the elements and compounds that exist in the known universe, and there are an infinite number of combinations that can be used to systematize the reactions. Ordinary engineers would not choose such a job, because they have been trained to think of the shortest route and devote finite resources to reach the set goal. New approaches, however, have begun, such as the Evolution2.0 Prize (https:// www.herox.com/evolution2.0), a contest that uses machine learning to help identify prospective materials and paths. Let us look forward to future developments. Regarding point (3), it is obvious that the cell, which is considered the carrier of life, is actually an aggregate of matter. However, the pathway connecting cells and matter is only one-way, that is, when cells are decomposed, they become matter, while the reverse has not been successful. In order to resolve this lack of symmetry, artificial cells, which will be called simply ‘cells’ here, have been created from matter, and their behavior has been checked. In contrast to artificial life models in computers, where everything can be programmed, the shortest path to realizing a working model in physical reality is to emulate something that is already working. Biopolymers and other cell-compatible materials are used to construct a molecular system that can reproduce known cell functions. The two questions—how matter and its systems behave in life, and how matter and its systems behave to become something that can be called life—can both be clarified not only by observing and breaking cells but also by creating and driving a ‘cell-like’ model.

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3.1.3 Synthetic Cell Research Here, let us briefly mention synthetic cells, a topic that has been developing rapidly in recent years. This field aims to design artificial molecular systems and molecular circuits as genetic circuits, and, through the actual operation of these systems and circuits, remodel living cells to make possible what is impossible in natural organisms, and to approach the design principles of living organisms. The latter type of research has been known as constructive biology in Japan. In terms of applications, bioengineering and agricultural chemistry have benefited from these technologies, and there are high expectations for them in various domains such as energy, food, medicine, and housing. One of the best known examples is iPS cells, in which artificially introduced genes are used to reset cells to an undifferentiated state. In addition, the Craig Venter Institute (CVI) in the US has created artificial mycoplasmas by introducing artificially designed and synthesized full-length genomes [3]. These are sometimes referred to as ‘engineered cells’. Recently, highly efficient genome editing technologies such as CRISPR/Cas have emerged, and the technology for creating synthetic organisms is developing further. On the other hand, the Boston group, where synthetic biology originated, set as one of its early goals the construction of artificial cells with a minimum set of genetic information (minimal cells). In contrast to the bottom-up approach of artificial cells and molecular robotics, which aims to start biological phenomena from scratch (fully scratch-built), this is a top-down approach that asks how many unnecessary elements of living cells can be removed. The disadvantage of this approach is that molecular systems with unknown functions remain as black boxes (for example, 149 genes with unknown functions were needed to run a mycoplasma with a minimum genome of 473 genes synthesized from the entire genome [4]). The advantage is the use of a superior self-renewal mechanism. For details, please refer to book [5, 6] and Chap. 2. In constructing an artificial cell with a minimum set of genetic information, it is essential to achieve a working link between genotype and phenotype. The link is the so-called ‘Central Dogma of Life Science,’ which is a series of steps: RNA synthesis from genetic DNA (transcription), protein synthesis from RNA (translation), metabolism through functional expression of folded proteins, establishment of individuals, and self-replication. As for transcription and translation outside the cell, starting with the protein synthesis system using cell extracts in the 1960s, Shimizu et al. reported PURE SYSTEM, a molecular system containing only the factors necessary for protein synthesis of E. coli without a black box [7], and it is commercially available. Recently, Suetsugu et al. reported an exponential amplification of cyclic DNA using a set of only essential factors that reconstitute the genome replication cycle [8]. Advances in biochemistry and molecular biology are thus bringing us closer to the level of cellular systems, as we attempt to reconstruct and realize the molecular systems that realize the essential functions of the cell outside the cell, that is, in solution in a test tube. A cell is a small, compartmentalized unit of the whole, given its origin. A microcapsule, which is a model of a cell, is used as a vessel to encapsulate

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functional molecular systems that are highly compatible with cells, as described above, and to realize the essential functions that constitute a cell. To date, liposomes, lipid bilayer vesicles, have been the most-used vessel or body for this purpose.

3.1.4 Lipid Bilayer Vesicles: As Body of Artificial Cells and Molecular Robots Lipid molecules are mainly amphiphilic, i.e., they have both hydrophilic and hydrophobic functional groups. When dispersed in water, they form an aggregate with hydrophobic molecules folded inside, thus acting as interfaces between water and oil. Depending on the aggregation state and shape of the lipid molecules, they exhibit various forms such as spheres (micelles), rods (micelles), and plate-like (membranes). The molecules, assembled with forces weaker than covalent bonds, are dealt with by supramolecular chemistry [9]. Along with polymer chemistry and chemical thermodynamics, supramolecular chemistry is an essential field for molecular design. Among the lipid assemblies, the most-used artificial lipid membranes are monolayer Langmuir–Blodgett membranes, bilayer planar black membranes, water-in-oil (w/o) emulsions with a monolayer formed on a spherical oil–water interface, and liposomes with lipid bilayer vesicles. Some typical structures are illustrated in Fig. 3.2. Liposomes, Greek roots lipos (fat) and soma (body or object), are vesicles in which the lipid bilayers are closed, containing an interior. Liposomes, also called vesicles, were first reported by Bangham in 1964. They have a lipid bilayer structure

Fig. 3.2 Liposomes and droplets (w/o emulsions) as artificial cell models

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like that of cell membranes, and are being actively studied for use in biomembrane models, cosmetics, drug delivery systems (DDS) [10], and engineered cells used to reconstitute cells [11]. Liposomes are classified as small, large and giant unilamellar vesicles (SUV, LUV, GUV), and multilalamellar vesicles (MLV) according to their size and topology. Liposomes made from polymers as their amphiphilic raw materials are called polymersomes; these are attracting attention because of their robustness and the ease of adding functions. GUVs are characterized by a diameter of several micrometers or more and have been reported as a cell model in recent years. The thickness of the lipid membrane is usually 5–7 nm, which is so much smaller than the wavelength of visible light that even the presence of the membrane cannot be observed with an ordinary microscope. Since the 1980s, however, Hotani et al. have pioneered the direct observation of lipid membranes using dark-field microscopy with scattered light [12], and the development of high-precision microscopy methods using fluorescently labeled hydrophobic molecules has made it possible to observe and manipulate lipid membranes in the same way as cells. Recently, advances in microfabrication technology and advanced devices have made it possible to produce cell-sized liposomes of uniform size. Thus, many attempts have been made to prepare, track, and evaluate biochemical reaction systems inside liposomes as artificial cells [13].

3.1.5 Functional Artificial Cells Here, we note some examples of artificial cell research using lipid containers. Evolution: in artificial cells, Ichihashi et al. reported a system that reproduces Darwinian evolution by placing RNA replication reactions by protein synthesis in a w/o droplet, and introducing mutations and selection laws externally [14]. Movement: Takiguchi et al. found that actin motor proteins are encapsulated in liposomes at high concentrations, and external light stimulation causes large-scale deformation that can only be called movement [15]. Multi-cellular self-organization: Toyoda et al. have shown that artificial multicellular states can be generated by the coexistence of amphiphilic polymers [16]. Self-reproduction: Sugawara et al. reported an artificial system that couples membrane replication and internal DNA replication by creating a vessel that can synthesize membrane molecules in situ by catalytic reactions using organically synthesized lipid molecules [17]. Let us look at an example of research that aims to reconstitute the entire cellular function using the same molecules that biological cells actually use. A turning point was achieved by encapsulating E. coli extracts or the PURE system (described above) in liposomes to realize a protein synthesis system [18, 19]. Similarly, membrane protein structures such as channels, which are responsible for the exchange of materials across membranes, were synthesized and embedded in membranes to realize the exchange of molecular information between liposomes and their surrounding environments [20, 21], and an artificial cell evolution system in the form of liposome display using membrane proteins was reported by Matsuura et al. [22]. A system

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that realizes the reverse transcriptase reaction to synthesize DNA from RNA in liposomes has also been reported [23]. Recently, Danelon et al. reported the realization of genome replication in liposomes [24]. Self-replication is completed when the replication reaction of the gene couples with the replication reaction of the vessel. On the vessel side, the lipid membrane of GUVs has been found to be deformed on a large scale by internally synthesizing a set of membrane proteins that divide E. coli [25]. When the cell-free synthesis in liposomes succeeds in reconstituting the set of molecules that define the starting point of division where these proteins assemble, or in reconstituting the system that performs the synthesis of the lipid molecules themselves, the word ‘reconstitution’ will be removed, and the replication reaction of the vessel will be realized, and gene replication and coupling will be achieved. A fierce race is quietly underway to perfect the bottom-up method of dividing artificial cells. In addition to independent vesicles working in a static in vitro environment, ‘open’ models are being developed in combination with MEMS. A system in which a chamber with an artificial cell membrane is fused to an E. coli bacterium with its outer membrane removed, so that it can be treated as a hybrid artifact, [26] has also been reported. Thus, research on artificial cells, which bridge the gap between life and matter, has been active both in Japan and abroad. In Japan, the ‘Creating Cells’ research group was established in 2005 [http://www.jscsr.org]. Recently, a project to create and use genome-scale DNA has been underway, which is beginning to be linked to artificial cell research. In the United States, there is CVI [https://www.jcvi. org/], and a project called Build-a-cell [https://www.buildacell.org/], and in Europe, there is a large project called BaSyC (Building a Synthetic Cell), mainly in the Netherlands [https://www.basyc.nl/].

3.1.6 Artificial Cells and DNA Nanotechnology Finally, I would like to introduce a recent study that can be called a fusion of DNA nanotechnology, one of the basic technologies of molecular robotics, and artificial cell systems. As a static model, for challenging the artificial cell’s fragility, Yanagisawa et al. reinforced liposome membranes by using tiling patterns formed on the surface of the membranes by the aggregation of Y-motif DNA as a backing structural material [27]. Examples of the dynamic model systems are shown in Fig. 3.3. Tadakuma et al. constructed a gene expression chip (Fig. 3.3a), in which RNA polymerase and protein-coding DNA sequences are placed at appropriate positions on DNA origami [28]. By adding this chip to a cell-free protein expression solution, protein synthesis occurs. The expression efficiency depends on the distance D between the transcription unit (left) and the gene (right). The chipset can be introduced into a w/o droplet and made to work, and sensing of specific miRNAs using DNA logic gates

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Fig. 3.3 Examples of artificial cell models using DNA nanotechnology. a Gene expression chip placed on a DNA origami (adapted with permission from [27], © 2018, Springer Nature). b Artificial cell system with built-in DNA computer (adapted with permission from [29], © 2017, American Chemical Society). c Insulin-secreting artificial cell model (adapted with permission from [30], © 2017, Springer Nature). d Amoeba-type molecular robot (adapted with permission from [31], © 2017, American Association for the Advancement of Science). The scale bar is 10 μm

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Fig. 3.3 (continued)

has been achieved. The conceptual diagram of the artificial cell ‘system’ with a builtin DNA computer by Kawano and Takinoue et al. is shown in Fig. 3.3b [29]. The AND operation was performed in the DNA circuit, and the output RNA molecules were detected by electrochemical measurement as they penetrated the nanopores on the lipid membrane. Figure 3.3c shows the ‘anti-diabetic’ artificial cell model reported by Chen et al. that secretes insulin in response to an increase in external glucose concentration [30]. PEG is bound to the surface of insulin-encapsulated small liposomes via DNA I-motifs. When the I-motif is removed in response to an increase in pH in the large liposome, the membrane of the small liposome fuses with that of the large liposome, releasing insulin to the outside. Figure 3.3d shows an amoeboid molecular robot reported by Sato et al. [31], which switches between stop mode (left) and locomotion mode (right) using a ‘molecular clutch’ that binds/separates the lipid membrane of the GUV and kinesin (motor protein) by hybridization of DNA molecules. For details, please refer to Chap. 2.

3.1.7 Conclusion It is fun to think about how to create artificial cells that go beyond real live cells. DNA, lipid membranes, liposomes, cells, and organisms are material entities that can all be touched and be manipulated by our human selves. Life, however, is a word, an intangible concept. Word and concept depend on the situation or definition. Physicist Richard Feynman once asked, ‘Can you explain in words how the gears mesh and turn?’ His question means that humans can create things that are difficult to describe in words.

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Artificial cells and molecular robotics go hand in hand with each other because of the high compatibility of their materials, and the fundamental purpose of making molecules work as a system. Based on them, it should be also fun to create other life-like systems that are difficult to describe in words. Both research fields will be developed while stimulating each other, and will open new frontiers of the human environment.

3.2 Amoeba-Type Molecular Robot Prototype Yusuke Sato Y. Sato Faculty of Computer Science and Systems Engineering Department of Intelligent and Control Systems, Kyushu Institute of Technology, Fukuoka, Japan e-mail: [email protected] Establishing the methodology to assemble multiple molecular devices into a system is one of the most fundamental challenges in molecular robotics. For the construction of meter-sized robots, we can assemble parts and devices with our hands or tools. On the other hand, such manual assembly is almost impossible in molecular robotics because a few nanometer-sized molecular devices are too small to manipulate. The use of self-assembly is a possible means to construct a molecular robot. If interactions of each molecular device are programmed, the devices can be spontaneously assembled and integrated into a designated system. However, even though an assembled molecular system (molecular robot) works in test tubes, when the molecular robot is transferred from the tubes to a different environment such as inside of body or sea, the integrated multiple devices will disperse, leading to the disappearance of robot’s functions. Mimicking living cells is one of the feasible approaches to solve the problem. In cells, various types of molecules are compartmentalized by cellular membranes. Compartmentalization prevents intracellular molecules from dispersing in solution, which allows cells to perform and maintain their functions. Thus, compartmentalization of molecular devices into a chassis to keep the integrated state of the devices is a feasible approach to constructing molecular robots. The ‘amoeba-type’ molecular robot is the generic name of molecular robots in which multiple molecular devices are compartmentalized and integrated into an artificial lipid bilayer vesicle (liposome). The amoeba-type molecular robot was proposed as the 1st generation of molecular robots [32]. It was expected that amoeba-type molecular robots would have higher functionality than those of single molecule-type molecular robots (0th generation), such as DNA walkers [33] or molecular spiders [34], because multiple molecular devices will be integrated into one compartment. Not only as a concept but a prototype of the amoeba-type molecular robot was developed in 2017 [35]. In this section, the design, function, and outlook of the amoeba-type molecular robot are described.

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3.2.1 Design of the Amoeba-Type Molecular Robot The amoeba-type molecular robot (amoeba robot) recognizes DNA with a specific base sequence as a signal. It switches its states between active (shape-changing) and inactive (spherical) states in response to the input signals (Fig. 3.4a, b). The amoeba robot was composed of a cell-sized liposome, a rod-shaped protein (microtubule), a motor protein (kinesin), and a control device (molecular clutch) that regulates the initiation and termination of the shape-changing behavior. Microtubule is one of cytoskeletal proteins and has the highest stiffest in all of them [36, 37]. Kinesin is a motor protein that converts chemical energy from ATP hydrolysis into mechanical energy for actuation. Kinesin has the motor domains, called “head,” and walks on microtubule toward one direction for cargo transport or shaping in cells [38]. By immobilizing the kinesin on a substrate so that the heads face upward, microtubules are moved. This technique is called a motility assay (or gliding assay) [39]. In the amoeba robot, kinesins were placed on the inner leaflet of liposomal membranes, similar to the motility assay. It allowed for microtubule movement on the inner leaflet, which pushed the membrane and caused the deformation of liposomes (Fig. 3.4b, d). The localization of the kinesins on the inner leaflet was controlled by the molecular clutch devices (Fig. 3.4e, f). The kinesins used in the amoeba robot were biotinylated. Kinesin-DNA conjugates (‘motor units’) were prepared using biotin-avidin binding by mixing biotinylated-DNA, biotinylated-kinesins, and neutravidin. The motor units were introduced onto the membrane by ‘anchor units,’ composed of cholesterolmodified DNAs and a linker DNA that connects three cholesterol-modified DNA. The motor and anchor unit can be joined via a “connector” DNA signal. When both units are connected, the force of kinesins that drives microtubules transmits to the membrane; namely, clutch becomes ON state (Fig. 3.4f). The connector DNA signal has a toehold region. Therefore, the connection of the motor and anchor units can be released through toehold-mediated strand displacement reaction by adding a “releaser” DNA signal with a complementary sequence to the connector DNA. It makes the molecular clutch OFF state (Fig. 3.4e) in which the force of kinesin to move the microtubules does not induce the shape change of the liposomes (Fig. 3.4c).

3.2.2 Robot Production The amoeba robots were produced by an inverted emulsion transfer method [40], a typical method of generating cell-sized liposomes with designated inner composition. Adding robot components, including DNA, microtubules, kinesins, ATP (fuel), and other necessary components (over 25 species in total), in the correct amount and order into a test tube was essential in the robot production. Besides, the membrane composition of liposomes was also an essential factor because the fluidity of the membrane affected the force transmission of kinesins on the membrane. Please see

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Fig. 3.4 Design of the amoeba-type molecular robot. a, b Schematics of the robot in the inactive and active state. c, d Magnified schematics of the liposomal membrane. e, f Schematics of the molecular clutch mechanism (From [35], reprinted with permission from AAAS)

the original article for more detailed protocols or discussion about the membrane fluidity effects [35].

3.2.3 Robot Performance: Active and Inactive State The robot’s state (active or inactive) is determined by the ON or OFF of the molecular clutch. The clutch function was examined by premixing the signal DNAs (only connector or both connector and releaser) in the inner solution of the liposome. Figure 3.5 shows sequential images of the amoeba robot, visualized using a confocal laser scanning microscope. When the connector DNA signal was premixed, namely

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Fig. 3.5 Microscopy image sequences of robots. a The robot in the active state (clutch ON): green and magenta show kinesin and microtubules, respectively. White triangles in the images indicate the microtubules on the membrane. b The robot in the inactive state (clutch OFF). Scale bars: 20 μm (From [35], reprinted with permission from AAAS)

clutch was ON, kinesins and microtubules localized on the membrane, and continuous shape-changing behavior of the amoeba robot (active state) was observed (Fig. 3.5a). When both the connector and the releaser DNAs were premixed at equimolar ratio (clutch OFF), the kinesins and microtubule were not on the membrane, and the robot remained the spherical shape (inactive state) which is generally the most stable shape of liposomes. These results demonstrated that the state of the amoeba robots (active or inactive) could be controlled by switching the molecular clutches between ON and OFF in response to the signal DNAs.

3.2.4 Switching the Robot State If the signal DNA was input into the active or inactive robots, switching from active to inactive or vice versa will be realized. However, even if the signal DNA is added to the solution containing the amoeba robots, the signal is not input to the robots’ inside because DNA molecules cannot path through the lipid bilayer. Therefore, photo-responsive signal DNAs were equipped inside the robot. In this section, photoresponsive DNA means DNA with photo-cleavable (PC) spacers [41], which can be cleaved by ultraviolet (UV: 300–350 nm) irradiation. The photo-responsive signal DNA was hairpin-shaped DNA in which the signal sequence was covered with a complementary sequence with PC spacers before the UV irradiation. The covering parts are fragmented and detached after UV irradiation, leading to the production of the signal DNAs (connector or releaser DNA). For the ON–OFF switching of the molecular clutch, photo-responsive connector (from OFF to ON) or releaser signal DNA (From ON to OFF) was prepared and equipped inside the robot. Experimental results showed the successful robot’s state switching (Fig. 3.6). Before UV irradiation, the robot containing the photo-responsive releaser DNA exhibited shape-changing behavior (active state). Localization of microtubules and kinesins was observed. After the signal input, i.e., UV irradiation, the robot eventually

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Fig. 3.6 Switching the robot state using photo-responsive signal DNA. a, b Schematics of photoresponsive releaser (a) and connector (b) DNA. c, d Robot image sequences show the state switch from active to inactive (c) and from inactive to active (d). The signal was input by UV irradiation at t = 300 s. White triangles in the images show microtubules on the membrane. Scale bars: 10 μm (From [35], reprinted with permission from AAAS)

became spherical shape (inactive state). Similarly, the spherical robot containing the photo-responsive connector DNA initiated the shape-changing behavior after the UV irradiation (from inactive to active state). These results demonstrated that a prototype of the amoeba molecular robots capable of fundamental robotic function (sensing inputs and controlling actuators) was successfully achieved by integrating molecular devices into the compartment.

3.2.5 Outlook: From Prototype to Advanced Molecular Robots The constructed amoeba robot would become more functional by combining with various molecular devices. For instance, the repetitive switching of the robot’s state was still not demonstrated; however, incorporating azobenzene- [42] or cnvKmodified DNA [43] into the molecular clutch may allow for the repetitive state

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Fig. 3.7 Shape-changing behavior of the robot after freezing and thawing. The robots were mailed to Nagoya University from Tohoku University, Japan. The images were kindly provided from Dr. Kingo Takiguchi and Dr. Masahito Hayashi

switching in response to irradiation of different wavelength light. Equipping artificial nanopores in the liposomal membranes [44] would enable the amoeba robots to recognize molecular signals or acquire fuel molecules (ATP) from surrounding solution. DNA computing devices may increase the information processivity of the robot [45]. Bundle formation of microtubules by DNA may increase the actuator’s power [46]. In addition to these possibilities, it should be noted that the amoeba robot could be frozen and continued the function even after thawing, suggesting that the amoeba robot could be distributed to other laboratories by mail. Indeed, mailed robots showed similar shape-changing behavior in another laboratory (Fig. 3.7). This fact enables us to envision that researchers can design, install, and test their original molecular devices on the amoeba robot. Although several possible advancements of the amoeba robot were listed above, the current robot’s function is only to initiate or terminate the shape-changing behavior in response to DNA signals. In other words, it is currently basic and far from practical application. However, achievement of basic functions will open the door for further developments. For example, in the early stage of biped robot’s studies, achieving even just ‘walking,’ a basic motion in human beings, was technologically challenging. A biped robot in the 1960s required 90s for a step [47]. Nowadays, the achievement of the basic function (slow walking) led to construction of various biped humanoid robots that can be seen close to our daily life; as a toy [48] or practically helping the cargo move [49]. Given the development in biped robots, it would be acceptable to expect that the amoeba robot with the basic function leads to more functional and practical molecular robots. The few micrometer-shape changes of the amoeba robot will be a basis in future advancements of molecular robots.

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3.3 Gellular Automata and Molecular Computing Expanding to Space Masami Hagiya M. Hagiya The University of Tokyo, Tokyo, Japan e-mail: [email protected] After artificial cells or molecular robots have been constructed, it is natural to think about a collection of cells or robots that communicate with one another. Of course, such a collection of cells is expected to exhibit more advanced behaviors than a single cell. Cellular automata are a theoretical framework for studying such collections of cells. This section introduces gellular automata, which are cellular automata that satisfy some conditions for implementing collections of molecular robots made of gel materials.

3.3.1 Cellular Automata Cellular automata are a computational model inspired by multicellular organisms [50]. A state machine called a cell is placed at each lattice point. Each state machine determines its next state according to its own current state and the current states of the state machines at its adjacent lattice points. Especially in a synchronous cellular automaton, each state machine transitions to its state at the same time. Conway’s Game of Life is a well-known example of synchronous cellular automata [51]. In the Game of Life, cells with states 0 or 1 are arranged at the lattice points of the square lattice. As adjacent lattice points, four points in the diagonal direction are considered in addition to the points on the top, bottom, left, and right. Generally, a set of adjacent lattice points is called a neighborhood. The neighborhood consisting of four lattice points on the top, bottom, left and right is called the Neumann neighborhood, and the neighborhood consisting of eight grid points including the diagonal directions is called the Moore neighborhood. In the case of a square lattice, the cells at each lattice point are displayed as squares, and the states are indicated by their colors. In Fig. 3.1, the cells in the neighborhood of the black cell are shown in gray (Fig. 3.8). In Game of Life, state 0 means that the cell is dead, and state 1 means that the cell is alive. Then, under the Moore neighborhood, each cell determines its next state as follows [51] – A cell whose current state is 0 transitions to state 1 if there are two or three adjacent cells in state 1. – A cell whose current state is 1 transitions to 0 if there is one or no adjacent cell in state 1, or if there are four or more adjacent cells in state 1.

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Fig. 3.8 The Moore neighborhood and the Neumann neighborhood

Moore neighborhood

Neumann neighborhood

– In the other cases, the state remains in the current state. Figure 3.9 shows a change in a pattern called a glider. The cells in state 1 are displayed in black, and the cells in state 0 are displayed in white. The state transition of each cell in the configuration shown in the upper left figure is shown in the upper middle figure. The red cells are the cells that transition from state 1 to 0, and the blue cells are the cells that transition from state 0 to 1. The states of other cells do not change. The result of the synchronous transition is shown in the upper right figure. The same pattern as the upper right figure is shown in the lower left figure, the transition is shown in the lower middle figure, and the transition result is shown in the lower right figure. This pattern moves in the lower right direction by repeating the transitions. That is, in every 4 steps, it goes to the right by one cell and goes down by one cell. In the transition rules of the Game of Life, the next state of a cell is determined by its current state, the number of cells in the state 0 among its neighboring cells,

Fig. 3.9 Conway’s game of life

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and the number of cells in the state 1 among its neighboring cells. That is, they do depend on the direction of neighboring cells. Such a transition rule is called an outer totalistic rule. In the Game of Life, many patterns have been discovered that exhibit extremely complex behavior. By combining such patterns, various computation and information processing can be performed by the Game of Life. Specifically, the Game of Life can be used to simulate logic circuits and Turing machines [51]. In this sense, Conway’s Game of Life is known to be computationally universal.

3.3.2 Gellular Automata Gellular automata are a kind of cellular automata that impose various constraints based on the assumption that they will be implemented by gel walls and reaction solutions. The Game of Life satisfies the constraint that state transitions of cells do not depend on the direction of neighboring cells, but gellular automata impose more constraints of different kinds. Before explaining these constraints in detail, let us briefly introduce how to implement gellular automaton (see Sect. 2.4 for details). As shown on the left of Fig. 3.10, one method is to use gel capsules. A reaction solution such as DNA is infiltrated into a capsule made of a gel such as alginic acid. In the method by Arimura et al., a gel capsule is prepared by immersing a droplet of alginic acid in a calcium solution for an appropriate time, and then the reaction solution is encapsulated from the outside. By suspending microbeads in the alginic acid droplets, the microbeads can be inserted into the gel capsule. Among

1 min

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Diffusion between gel capsules

Precision mold

60 min

Gel block made from mold

Scale bar = 1 mm

Solutions in micropores Gel capsules containing microbeads

Gel capsules

Fig. 3.10 Methods for implementing gellular automata

Gel block with micropores

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the molecules in the reaction solution infiltrated from the outside, those fixed to the microbeads stay in the gel capsule. Other molecules or molecules generated by a reaction in the solution can diffuse through the gel wall into adjacent gel capsules. On the other hand, in the method by Murata et al. in Fig. 3.10, a precision mold is used to create an alginate gel block with micropores, and the reaction solution is injected into the micropores. Of the molecules in the reaction solution, those to which anchor molecules (specifically, polyacrylamide) are bound cannot diffuse in the gel wall. Other molecules and generated molecules diffuse through the gel wall and reach adjacent micropores. In either method, the state of an adjacent cell can be known by the molecules arriving from the adjacent cell by diffusion. Such molecules are called signal molecules. That is, each cell constantly generates a signal molecule that represents its state. Signal molecules diffuse through the gel wall and reach adjacent cells. Each cell can know the state of an adjacent cell by receiving a signal molecule. Although not explained in detail here, Hagiya et al. also proposed gellular automata that perform computation, information processing, and pattern formation by opening and closing gel walls [52]. Assuming the above implementation methods, the following constraints can be considered for cellular automata. Asynchrony: Typical cellular automata such as the Game of Life are synchronous, and each cell changes its state all at once, but in the case of gellular automata, it is impossible to completely synchronize all reaction solutions. Therefore, cellular automata that do not assume synchronous state transitions are desired. Totality: In the Game of Life, state transitions of cells depend only on the number of adjacent cells in each state, and do not depend on their directions. This property is also desirable for gellular automata. The states of adjacent cells can be known from signal molecules, but the directions of adjacent cells are not known. The number of adjacent cells in each state can be estimated from the amount of a signal molecule. Boolean totality: This constraint further limits the totality. Since the amount of signal molecules varies depending on various conditions, it is reasonable to assume that it is difficult to accurately estimate the number of adjacent cells based on the amount. Boolean totality requires that the state transition of a cell is determined only by the presence or absence of an adjacent cell in each state. Non-camouflage: Non-camouflage is a further limitation of Boolean totality. Each cell is supposed to generate and diffuse a signal molecule that represents its state. Then, if an adjacent cell of the cell is in the same state as its state, the same signal molecule as that of itself is generated. Since each cell is filled with a signal molecule corresponding to its own state, even if the same molecule is diffused from an adjacent cell, it cannot recognize the diffused one. Therefore, it is required that state transitions of a cell do not depend on an adjacent cell that is in the same state as itself. This request is called non-camouflage. The above constraints define the mathematical model of gellular automata, i.e., cellular automata are called gellular automata if they satisfy the above conditions. We can then investigate theoretical properties of gellular automata. In the following subsections, some theoretical results are explained.

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In gellular automata, a cell state is realized by a state of the reaction solution of the cell. Therefore, the solution is assumed to contain a chemical reaction system with multiple stable states. Molecules produced at high concentrations in each stable state can be used as a signal molecule. Needless to say, it is not easy to implement such a reaction system. For example, it is possible to use the PEN DNA toolbox [53]. Using the PEN DNA toolbox, reaction systems with multiple stable states have been implemented, including a bistable switch. No matter what reaction system is used, the smaller the number of cell states, the higher the feasibility of cellular automata. Also, transitions between cells should be simple. More specifically, cellular automata with fewer branching transitions are easier to implement.

3.3.3 Computational Universality of Gellular Automata Isokawa et al. showed that asynchronous cellular automata that satisfy totality are Turing complete, i.e., computationally universal [54]. In other words, any Turing machine can be simulated by setting an appropriate initial pattern under asynchronous cellular automata that satisfy totality. Furthermore, Yamashita et al. showed that even cellular automata that satisfy Boolean totality and further satisfy non-camouflage are computationally universal [55]. In these results, the lattice space of cells is two dimensional as in the Game of Life, but the Neumann neighborhood is assumed.

3.3.4 Gellular Automata and Distributed Algorithms Although the results on computational universality mentioned above characterize the theoretical possibilities of computation and information processing by gellular automata, they do not suggest the realistic futures of gellular automata. In the first place, cellular automata were inspired by various phenomena in multicellular organisms, especially ontogeny, morphogenesis, self-renewal and self-healing, and were proposed as a discrete model that can reproduce such phenomena. Therefore, it is appropriate to explore the possibilities of gellular automata in that direction. The gellular automata that solve a maze problem in Fig. 3.11 are a typical example. A cell in the gellular automata takes two states, blue and red. A red state transitions to blue when there are three blue states in its Neumann neighborhood. Therefore, the red states spread in the shape of a tree to solve the maze, and eventually only one path consisting of red states will remain to connect the entrance and the exit. Although this example is very simple, it is a concrete example of pattern formation by gellular automata. In general, in the field of parallel and distributed computation, various distributed algorithms have been studied in which spatially distributed computational agents perform collective computation while exchanging information with neighboring

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State transition rule Red

blue if three or more blue neighbors

concentration [nM]

An example of gellular automata that sole a maze problem

b

a

time [min]

Simulation by Ready with 26-var. partial diff. eq. DNA 20 nM, diam. 1600 μm, gel thickness 200 μm

Fig. 3.11 Gellular automata that solve a maze problem

agents. Many of those distributed algorithms, such as shortest path and minimum tree, eventually form some pattern in the space consisting of computational agents. Therefore, it is reasonable to develop gellular automata while referring to the models of those distributed algorithms. Asynchrony is especially important for research in that direction. Although synchrony enriched the mathematical theory of cellular automata, the existence of a synchronous clock is extremely artificial. Even in various parallel and distributed processing models, synchronization is usually not assumed. In models of distributed algorithms, synchronous processing is performed as needed in asynchronous distributed computation. Population protocols are one of the most basic models of such parallel and distributed computation. There is a graph (called an interaction graph) with computational agents as nodes, and two adjacent nodes on the graph simultaneously change their states according to a transition rule. While state transitions of the two nodes are synchronous, which two nodes are selected is asynchronous. Yamashita et al. showed that gellular automata can be used to simulate population protocols [56]. An important property called self-stability is defined for distributed algorithms [57]. A distributed algorithm is defined self-stable if no matter what initial state it starts with, the whole system will eventually settle into stable states. A stable state is not necessary a stationary state, but a state that satisfies the desired condition and continues to be a stable state that may be different from itself. It is guaranteed that such a distributed algorithm will eventually reach a stable state again even if the stability is lost due to external disturbance. How to realize self-stable distributed algorithms by gellular automata is a very interesting question. Hongu et al. have developed the distributed algorithms by gellular automata for solving maze problems, two-distance coloring, constructing spanning trees, and finding Hamiltonian circuits [58].

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3.3.5 From Gellular Automata to Self-Healing Materials Self-stability is closely related to self-organization and self-healing. If computational agents dispersed in space are realized as molecular robots, they can observe the physical conditions at their positions in space by their sensor devices. By observing various physical quantities and concentrations of various molecules and exchanging such information among themselves, they can self-organize various patterns in space. Also, if the conditions change due to external disturbance, the pattern can be reconstructed. A cell in gellular automata is assumed to be a molecular robot. In addition to realizing a computational agent and observing with a sensor device, it can exert various effects as an actuator. By changing its own physical characteristics, it may be possible to transform the morphology of the entire aggregate of cells. Therefore, the aggregate of cells self-organizes while observing the environment, and self-heals according to changes in the environment and itself. At first glance, it is just a block of gel, but it will become an intelligent material in which various kinds of information are propagated, and the conversion of physical properties and the production of substances are constantly repeated.

3.4 Implementation of Gel Automata Takashi Arimura, Ibuki Kawamata and Satoshi Murata T. Arimura AIST-University of Tsukuba, Open Innovation laboratory, Tsukuba, Japan e-mail: [email protected]; [email protected] I. Kawamata · S. Murata Department of Robotics, Tohoku University, Sendai, Japan e-mail: [email protected] S. Murata e-mail: [email protected]

3.4.1 Introduction In addition to theoretical research on the computational capacity of gel automata, research on the implementation of gel automata has been conducted. In the gel automaton, molecular calculations are performed in a reaction solution isolated by gel walls, and the state transitions of cellular automata are figured by molecular diffusion through the gel walls for cell-to-cell communication. Therefore, in order

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to implement this, the following specific terms are required: fabrication of cells, molding of cell arrays with gels, design of DNA molecular calculation solutions, and coordination of molecular diffusion through the gel walls. In the following, we introduce two models of the gel automaton, one using hollow gel beads that can be picked up with tweezers, and the other using a millimeter-sized cell array designed with a mold.

3.4.2 Implementation of a Hollow Gel Bead Model Discretizing micro- or millimeter-order gel capsules as a single space and controlling the communication between capsules, which will make it possible to create a reaction field of gel automata. For example, the self-organization of gel capsules with 0 (red) or 1 (white) in discrete states is shown (the synthesis of neutral white gel capsules (neutral-white) from alginic acid, see Sect. 2.5, “Molecules to condition the diffusion coefficient”). Anionic red capsules (anionic-red) are prepared by immersing neutral-white capsules of about 1 mm in diameter in 9 mM aqueous solution of anionic red dye (New Coccine, red #102) for 2 days. Neutral-white is immersed in 9 mM solution of cationic ammonium chloride to make cationic white capsules (cationic-white). Twenty-three anionic-red and twenty-three neutral-white are slowly agitated in a Petri dish containing an appropriate amount of solvent, and even after repeated attempts for self-association, only random aggregates of capsules are obtained (left picture in Fig. 3.12). On the other hand, when six anionic-red and one cationic-white are slowly agitated in a Petri dish, a hexagonal lattice aggregate of six anionic-red with one cationic-white in the center is obtained (red frame in Fig. 3.12). This is due to the electrostatic interaction between the capsules. The electrostatic interaction, which is essentially a weak intermolecular attraction, does not show sufficient aggregation force in the equilibrium system in the bulk, but it

Fig. 3.12 Hexagonal lattice self-assembly of gel capsules via electrostatic interaction

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is shown to be enhanced in a very cooperative manner at the special gel interface formed by the gel reaction field [59, 60]. This hexagonal self-aggregate can further self-organize to form multiple hexagonal mesh structures, which is effective as a method of discretizing the reaction space. For the communication between gel capsules, it is necessary to establish a state transition law with a threshold and to control the diffusion coefficient through time. The branch migration of DNA aptamer is considered to be a promising technique for state transition law. For precise control of the diffusion coefficient, a diffusion path that can be opened and closed at will is necessary. This could be achieved by employing photo-responsive molecules that change their properties in response to light. Here, we describe the full control of stop-and-flow diffusion of signal molecules between gel capsules by light irradiation. One photo-responsive gel capsule containing the fluorescent signal molecule 5-FAM and two empty photoresponsive gel capsules are placed side by side. By fluorescence microscopy observation, the 5-FAM is not released from the gel capsule even if it is left at room temperature for more than one hour before irradiation. When the gel capsule is irradiated with UV light, the photo-responsive molecules in the gel capsule isomerize from non-polar to polar molecules, creating a diffusion pathway between the capsules. After 20 min, the 5-FAM has diffused into the second capsule, and it diffuses into the third capsule and saturates it in 60 min. On the other hand, when the diffusion pathway is isomerized to non-polar by irradiating with visible light after 20 min, the diffusion of the 5-FAM stops and no release of the 5-FAM occurs even after another 40 min. It has now also been demonstrated that the diffusion pathways between the six gel capsules can be opened and closed and controlled by photo-responsive molecules (Fig. 3.13).

Fig. 3.13 Diffusion of signal molecules in a light-responsive gel capsule

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3.5 Molecules to Condition the Diffusion Coefficient Takashi Arimura T. Arimura AIST-University of Tsukuba, Open Innovation Laboratory, Tsukuba, Japan e-mail: [email protected]; [email protected] In general, when intercellular communication is characterized as a reaction–diffusion model, it is formulated as reaction dynamics within a cell, where only concern signaling molecules diffuse and cross the threshold between cells [61]. Water normally passes between cells by simple diffusion, but in highly permeable cells, channels called aquaporins are formed that allow water to pass through. When the channel is phosphorylated and polar, lots of water molecules can diffuse through it, and when the phosphate group is removed and the channel becomes non-polar, the channel is closed and no water molecules can diffuse through it. Therefore, it could be considered that the diffusion coefficient of polar molecules can be freely changed by controlling the diffusion path to be polar or non-polar, even for communication between capsules. In fact, chemically functionalizing alginic acid with photoresponsive molecules change to polar molecules when irradiated with ultraviolet light and to non-polar molecules when irradiated with visible light. Microgel capsules are constructed with photo-responsive molecules, which can be possibly used as a discretized model of cells. The protocol for the syntheses of photo-responsive microgel capsules is as follows (Fig. 3.14). (1)

(2)

(3)

To a solution of a calcium chloride solution (1 wt%) is added dropwise 1 wt% sodium alginate (80–120) covalently modified with photo-responsive molecules. The mixture is left for 20 min at room temperature to afford gel capsules with each diameter of about 500 μm and a shell thickness of 40–50 μm. The thickness of the capsule is about 10% of the diameter, and the inside of the capsule is in a sol state with no viscosity. Immerse the gel capsule in water for a short time.

Fig. 3.14 Protocol for gel capsule synthesis

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Fig. 3.15 Diffusion control of fluorescent molecule 5-FAM by light irradiation

(4)

Photo-responsive gel capsules containing dye molecules can be prepared by immersing the gel capsules in a solution containing the dye molecules to be diffused for 2 h.

For example, preparing the gel capsules containing 5-carboxyfluorescein (5-FAM) whose diffusion can be monitored by fluorescence microscopy, the diffusion of the molecule by light irradiation can be controlled (Fig. 3.15). (1)

(2) (3) (4)

A non-polar gel capsule without the molecule inside is placed next to a nonpolar gel capsule containing 5-FAM. The non-polar gel capsule is a black circle and the polar gel capsule is a yellow circle. 5-FAM is not released from the non-polar gel capsule even after 2 h without UV light. 2 non-polar gel capsules easily isomerize to polar gel capsules after irradiation of UV light. After 30 min, 5-FAM diffused into the vacant gel capsule, and the diffusion reached saturation in about 50 min. 5-FAM diffusion rate is about 0.5 mm in 30 min.

3.6 1.1 “(Column) Moving Gel” We will introduce Tohoku University’s attempt to implement a hydrogel that moves like a slime mold using programmed DNA reactions. Figure 3.16 is a schematic diagram of the moving gel. When the space is divided into three sections and the sol and gel are placed, the gel region apparently moves from left to right with the

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Fig. 3.16 Schematic diagram of a moving gel. Sols are in a liquid-like state, while gels are in a solid-like state. As time passes from top to bottom, the gel region apparently moves from left to right

passage of time. The principle of the movement is based on the gelation and solation of the polymer. At the boundary between sol 1 and gel, the reaction of solation occurs, and the gel region becomes smaller and smaller every moment. On the other hand, the gelation reaction occurs at the boundary between gel and sol 2, and the gel region becomes larger every second. The apparent movement is realized when the two reactions occur simultaneously. Polyacrylamide [62] with DNA as a side chain is used as a polymer, and the formation and dissociation reactions of the DNA double helix are programmed to correspond to gelation and solation, respectively. In the future, it is expected to become more complex by incorporating sensors and molecular calculators, and to function as an actuator for molecular robots.

3.7 Molecular Robotics Based on Droplet Microfluidics Masahiro Takinoue M. Takinoue Tokyo Institute of Technology, Tokyo, Japan e-mail: [email protected] In recent years, the construction and control of soft-matter molecular systems such as gels, vesicles, and emulsions have been actively studied using biopolymers and chemically synthesized polymers such as DNA, proteins, and lipids. The research ranges from chemical reactors and environmentally responsive/adaptive intelligent materials to molecular robots and artificial cells. In particular, molecular robotics and artificial cell engineering require control of the structure and function of soft matter at the cellular scale, that is, a micrometer scale. DNA nanotechnology has been developed to control nanostructures in a bottom-up manner based on DNA base sequence design. However, micrometer-scale structures are too large for DNA nanotechnology to control their properties completely. Therefore, in addition to DNA nanotechnology, a top-down technique called droplet microfluidics [63] has been used to

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control micrometer-scale structures such as microdroplets, microgels, and microcapsules. This section will explain the application of droplet microfluidics to molecular robotics in constructing microstructures and micro-scale chemical reactions. Droplet microfluidics is a technique to generate and control droplets with diameters ranging from a few micrometers to several hundred micrometers by flowing liquid through a microchannel (Fig. 3.17a), which is then applied to chemical reactions and the construction of gels and vesicles. The flow-focusing method (using a crossshaped channel) (Fig. 3.17b) and the T-junction channel (Fig. 3.17c) are often used to generate water-in-oil (W/O) microdroplets. Another method to generate microdroplets in the air has also been developed; this method generates microdroplets by applying a centrifugal force to a glass capillary containing a solution (Fig. 3.17d). Another application of water droplets in the molecular robotics field is microreactors for nonlinear chemical reactions, including DNA computing reactions [64–66]. The use of W/O microdroplets has been proposed as a method for large-scale analysis and computer control of reactions. Figure 3.18 shows a method to realize highresolution bifurcation analysis by large-scale mapping of the states of DNA oscillatory reactions [64]. In the microfluidic channel, W/O microdroplets with various molecular concentration conditions are automatically generated (Fig. 3.18a). Since fluorescent molecules reflecting the molecular concentrations are introduced into each droplet during the droplet generation, the molecular concentration conditions in each droplet can be determined by analyzing the fluorescent color under a microscope. After observing the reaction in the droplet, the relationship between the conditions and reaction states can be determined and mapped to a bifurcation diagram (Fig. 3.18b). The use of W/O microdroplets allows us to simultaneously observe the order of 10,000 droplets with different molecular concentration conditions, which enables us to create bifurcation diagrams with high resolution at 10,000 points. Figure 3.19 shows a non-equilibrium open-system reactor that utilizes the fusion and fission of W/O microdroplets to computer-control nonlinear chemical reactions

Fig. 3.17 Droplet microfluidic devices. a PDMS-glass microfluidic device. b Flow-focused (crossshaped) microfluidic channel. c T-junction microfluidic channel. d Water droplet generation by the centrifugal capillary microfluidic device

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Fig. 3.18 High-resolution bifurcation analysis by large-scale mapping of DNA oscillatory reaction using W/O microdroplets. a Generation of W/O microdroplets with fluorescent molecules reflecting the molecular concentration conditions. b Mapping reaction states to bifurcation diagram after the reaction based on the concentration of the fluorescent molecules introduced to the W/O microdroplets (Reproduced from Ref. [64] with permission from Springer Nature)

Fig. 3.19 Computer-controlled non-equilibrium open reactor based on fusion and fission of W/O microdroplets. a Microfluidic device. b Fusion and fission of oil droplets for artificial cells and chemical transport. c Principle of pulse density modulation control. A pulse wave p is used to realize a time-varying inflow and outflow velocity q of a substance. T is the period of the pulse, and w is the width of the pulse. d Overview of the feedback control of chemical reactions in the artificial cell reactor. e Automatic search for and maintenance of experimental conditions in which pH oscillation (rhythmic reaction) occurs by feedback control (Reproduced from Ref. [66])

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and bacterial culture [66, 67]. As formulated in Prigogine’s dissipative structure theory, the dissipation of reactants, in addition to the supply of energy by the reacting substrate, prevents the increase of entropy and allows dynamic order to form. The system shown in Fig. 3.19 consists of a W/O droplet-based microreactor used for an artificial cell and a W/O droplet for a transporter; the transporter droplets are used to supply and discharge chemicals to/from the artificial cell droplet. The artificial cell reactor is fixed in the microfluidic channel (Fig. 3.19a); the transport droplets are generated upstream of the microfluidic channel and reach the artificial cell through the microfluidic channel. Surfactants stabilize the droplets in the oil phase; thus, the transporter droplets do not fuse even when they contact the artificial cell. In the flow channel, electrodes are placed between the artificial cell; thus, when an alternate current (AC) voltage is applied, the droplets fuse when the transporter droplets come into contact with the artificial cell (Fig. 3.19b). Due to the oil flow in the channel, the fused transporter droplets are torn apart from the artificial cell droplet by the shear stress of the flow (Fig. 3.19b). In order to achieve a high degree of control over the rate of chemical inflow and outflow, a theoretical formulation was developed. In this model, the chemical inflow and outflow can be considered in the manner of pulse density modulation control (Fig. 3.19c). To achieve a chemical inflow/outflow rate by following a time-dependent function q(t) in Fig. 3.19c (dotted line), we can apply a voltage by following the pulse wave function p(t; T, w). p(t; T, w) take only the values of ‘0’ or ‘1’; ‘0’ indicates the unfused state, and ‘1’ indicates the fused state. This method achieves the feedback control of chemical reaction (Fig. 3.19d); the actual reaction state can be changed by the precisely controlled frequency of fusion and fission to match an expected set reaction state (e.g., oscillation state and its period). Figure 3.19e shows an actual experiment of feedback control, in which the bromate-sulfite-ferrocyanide (BSF) pH oscillation reaction [68] was used. Initially, the reaction solution did not show the oscillation of pH, but by varying the frequency of fusion and fission, the oscillation frequency was changed to a set period of 15 min and maintained for a long time after the set value was reached. Compared to simple diffusion, the droplet-based transport method offers significant advantages such as material concentration, reduction of reaction crosstalk, and parallelization of reactions. Although not using droplets, there are other microfluidics researches in this aspect. For example, there is a microchip-based artificial cell in which DNA is immobilized in a microfluidic channel formed on a silicon substrate, and the inflow and outflow of chemicals can be realized through the channel [69]. Since the inflow and outflow of chemicals are realized by material diffusion in the narrow channels connecting the main channel to the artificial cell reactor, the rate of chemical inflow and outflow is controlled by the thickness of the channels. Moreover, cooperative biochemical reaction dynamics such as wave propagation have been achieved by connecting the artificial cells on a silicon substrate through narrow channels [70]. The dynamics can be controlled by changing the length and width of narrow channels.

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Next, we introduce the technology to construct complex-shaped asymmetric microgel particles, microgel fibers, liposomes, and microcapsules using a microdroplet surface as a template. In general, microdroplets made by microfluidic technology can be applied to the formation of water-in-oil-in-water (W/O/W) double emulsions (Fig. 3.20a) [71], liposomes (Fig. 3.20b) [72–75], and the extraction as gel particles after polymer gelation (Fig. 3.21) [76, 77]. These formed microstructures have been used to construct the bodies of molecular robots and artificial cells. For example, DNA microcapsules have been developed using W/O microdroplets as a template (Fig. 3.20c) [78]. The DNA origami nanoplates were amphiphilized by hybridization of DNA-tagged cholesterol molecules on only one side of the DNA nanoplates and self-assembled on a W/O microdroplet surface. Therefore, the DNA microcapsule is a kind of Pickering emulsion stabilized with DNA nanoplate colloids. Since the DNA nanoplates have a nanopore at the center of the plate, the formed DNA microcapsules have a nanochannel function to transport ions between DNA microcapsules. Figure 3.21 shows the fabrication of microgels and liposomes using a centrifugal capillary microfluidic device (Fig. 3.17d). As shown in Fig. 3.21a, the capillary is

Fig. 3.20 Application of microdroplets generated by droplet microfluidics technology. a Double emulsion. Reproduced from Ref. [71] with permission from ACS Publications. b Liposome formation by jet flow. Reproduced from Ref. [75] with permission from Springer Nature and courtesy of Prof. Koki Kamiya (Gunma University). c DNA-origami-based microcapsules. Reproduced from Ref. [78] with permission, Copyright (2019) Wiley [78]

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Fig. 3.21 Formation of microgels and liposomes by a centrifugal capillary microfluidic device. a Photograph of the centrifugal capillary microdevice. b Multi-compartment gel particles (left) and non-spherical propeller-shaped microgel particles (right) produced with the centrifugal capillary microdevice (a). c Multi-helix microgel fibers produced with a planetary centrifuge with (a). d Liposomes generated with (a). a and b Reproduced from Ref. [77]; c Reproduced from Ref. [79] with permission from the Royal Society of Chemistry; d Reproduced from Ref. [74] with permission, Copyright (2015) Wiley

fixed to a commercially available 1.5-mL microtube by an acrylic holder, the capillary contains an aqueous sol solution (sodium alginate solution), and the bottom of the microtube contains a gelation reagent (calcium chloride). When centrifugal force is applied, droplets of the sol solution are discharged from the tip of the capillary and become gel particles when they reach the gelation reagent (Fig. 3.17d). Glass tubes with multiple inner walls are commercially available and can be used to make microgel particles with multiple compartments, as shown in the left side of Fig. 3.21b [77]. If the compartments are made of several gels, and only some of the gels are dissolved, complex-shaped asymmetric microgel particles can be made, as shown on the right side of Fig. 3.21b [77]. When we use a planetary centrifuge, an orbital rotation and an axial spin around the capillary axis can be achieved when centrifugal force is applied. Thus, if the capillary tip is dipped in the gelation reagent, microfibers with multiple helical structures can be generated (Fig. 3.21c) [79]. In addition, if the gelation reagent solution at the bottom of the microtube is changed to an oil phase with lipids on an aqueous phase, liposomes can be generated by a W/O droplet transfer method (Fig. 3.21d) [74]. As in these examples, droplet microfluidics makes it possible to generate complex microstructures and vesicles available to construct the functional body of molecular robots.

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In conclusion, droplet microfluidics has high compatibility with molecular robotics, and various interdisciplinary researches are possible, such as the construction of autonomous molecular robot bodies [80] and reactors for molecular computing, including gel automata [81], DNA computing [82], and construction of DNA gels and capsules [78, 83, 84]. Further developments of droplet microfluidics for molecular robotics applications are expected in the future.

3.8 Nanopores for Single Molecule Measurement and Their Potential as Membrane Gates Nanami Takeuchi, Ping Liu, Sotaro Takiguchi and Ryuji Kawano N. Takeuchi · P. Liu · S. Takiguchi · R. Kawano Tokyo University of Agriculture and Technology, Tokyo, Japan e-mail: [email protected] P. Liu e-mail: [email protected] S. Takiguchi e-mail: [email protected] R. Kawano e-mail: [email protected]

3.8.1 Introduction Three elements are required for the construction of a molecular robot: sensors, intelligence, and actuators. Liposome-based molecular robots, which employ liposomal membranes as the body, utilize biological membrane proteins or artificial membrane gates as their sensors. Several different types of membrane gate systems exist; G protein-coupled receptors that bind to specific substances and transmit signals downstream, voltage-dependent ion channels that sense the membrane potential, and nanopore proteins that simply form pores in the membrane to transport molecules across the membrane. Although some transporting systems require ATP as the driving energy, nanopore proteins enable concentration or potential gradient-driven transport of substances with size-selectivity. An additional benefit of nanopores in molecular robotics applications is that they are highly stable and easily reconstituted in a lipid bilayer. Owing to their beneficial properties, nanopores are expected to be a potent candidate as a membrane gate in molecular robots. In addition to acting as a robotics element, nanopores have also attracted attention as a single-molecule

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analysis method. α-Hemolysin (αHL), a nanopore protein, is especially used for this technology, and studies on nanopore-based single-molecule DNA detection and DNA sequencing have been reported. Thanks to the size compatibility between DNA molecules and nanopores, we believe that integrating DNA computing technology or DNA nanotechnology with nanopore technology shows great promise in the field of molecular robotics. This chapter outlines the nanopore proteins, basic principles of a nanopore measurement, and recent advances in DNA computing technology.

3.8.2 Principle of Nanopore Measurement 3.8.2.1

Nanopore Measurement

The Coulter principle is based on measuring changes in electrical resistance produced by a particle or cell passing through a small aperture in a conductive solution. Nanopore measurement can be considered as an advanced application of the Coulter counter, shrinking the detection hole from the microscale (single-cell level) to the nanoscale (single-molecule level). Pore-forming proteins reconstituted into a lipid bilayer membrane are utilized as the nanoscale pore (i.e., nanopore). A voltage applied across the membrane enables the observation of an ion current through the nanopore and detection of the target molecules electrically at a single-molecule level, as they cross the membrane through the nanopore. αHL, a pore-forming membrane protein toxin excreted by the bacterium Staphylococcus aureus, has been widely used in nanopore measurements. αHL can form a stable 1.4 nm diameter nanopore, making it suitable for detecting single-stranded DNA (ssDNA) which has a diameter of 1 nm. Other transmembrane channels with a diameter in the 1–2 nm range, such as Mycobacterium smegmatis porin A (MspA) and aerolysin, have also been used for nanopore-based analysis (Fig. 3.22). Besides membrane proteins, solid-state nanopores fabricated from synthetic materials [85], DNA origami nanopores [86], and peptide nanopores [87] have been reported and shown to be applicable in molecular sensing.

Fig. 3.22 The structure of α-Hemolysin (αHL), Mycobacterium smegmatis porin A (MspA), and Aerolysin

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Set-Up of Nanopore Measurement

In a nanopore measurement, high stability is required from the lipid bilayer membrane. Therefore, 1,2-diphytanoyl-sn-glycero-3-phosphatidylcholine (DPhPC), an artificial lipid exhibiting high stability at room temperature, is often used. The lipid bilayer is formed at the boundary surface of two liquid reservoirs filled with an electrolyte solution surrounded by an organic phase comprising the lipids. For the current measurement, electrodes are placed in both of the reservoirs. Ag/AgCl is widely used as the recording electrode because a redox reaction occurs between Ag+ and AgCl on its surface, resulting in an equilibrium potential. An arbitrary potential gradient can be applied between the two Ag/AgCl electrodes across the membrane. In terms of the electrolyte, potassium chloride is generally used because the mobility of chloride and potassium ions in the solution is substantially equal, resulting in negligible differences in positive and negative conductance. In Montal-Mueller (MM) method reported by Montal and Mueller in 1972 [88], the aqueous solution is dropped into the two wells separated by a thin Teflon film with a small aperture. After a lipid monolayer is applied at the air–water interface, the lipid bilayer membrane is formed at the aperture by rising the water surfaces vertically (Fig. 3.23a). Another method for forming planar lipid bilayers is the socalled painting method, in which a lipid bilayer is formed by directly applying a lipid solution to an aperture. However, these methods take time to form the lipid bilayer and the stability of the formed bilayer is limited. As an alternative for these methods, Funakoshi et al. reported the droplet contact method in 2006. In the droplet contact method, two aqueous droplets surrounded by an organic phase containing lipid molecules are brought together, resulting in a bilayer forming on the contact surface (Fig. 3.23b). By reducing the contact area of the droplets, the membrane stability was dramatically improved up to two weeks, making the droplet contact method a highly useful tool for electrophysiological research [89].

Fig. 3.23 Different methods for artificial lipid bilayer formation. a The Montal-Mueller method. The lipid bilayer is formed by vertical movement of the gas–liquid interface (Reprinted from The Biophysical Society of Japan 2015 [90]). b The droplet contact method. A lipid bilayer membrane is formed on the interface of two aqueous droplets surrounded by an organic phase with lipids by contacting (Reprinted from Sci. Rep. 2013 [89])

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Electrophysiological Characterizations of Nanopores

When a voltage is applied over a lipid bilayer comprising a biological nanopore, the ionic current passing through the nanopore can be monitored. A typical current signal generated by a nanopore is shown in Fig. 3.24a. Molecules larger than ions can also be monitored: when a molecule passes through the nanopore, the flow of ions is temporarily inhibited, resulting in a decrease in the observed current (Fig. 3.24b). The distribution of the duration of the blocking event vs. current blocking amplitude reflects the size and properties of the passing molecules, which can be used to identify target molecules statistically (Fig. 3.24c) [91]. Furthermore, the properties of nanopores themselves can be evaluated by current measurements. Hille’s equation [92] can provide the diameter of the nanopore by using the measured conductance, the length of the pore, and the resistivity of the used electrolyte. The I-V curve, a plot reflecting the relationship between the applied voltage and the measured current, provides information on the morphology [93] and ion selectivity of the nanopore [94].

Fig. 3.24 Electrophysiological characterization of nanopores. a The formation of a nanopore is observed as an increase in the measured current. b Molecules translocating through the nanopore result in transient drops in the measured current, when the flux of ions is temporarily blocked (Reprinted from Anal. Chem. 2017 [91]). c Scatter plot showing the relationship between the current inhibition amplitude and the inhibition event duration, and the differences in passing molecules (Reprinted from ACS Nano 2013 [95])

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3.8.3 Application of Nanopore Technology: DNA Sequencing 3.8.3.1

The History of DNA Sequencing

The development of DNA sequencing techniques has a diverse history. Here, we briefly review the first, second, and third-generation DNA sequencing technologies and their scaling to sequence the human genome. First-generation DNA sequencing techniques are based on the Sanger method, first developed by Frederick Sanger and his coworkers in 1977 [96]. In the Sanger method, various lengths of DNA fragments are synthesized using the target DNA as a template, and a certain amount of fluorescently labeled chain-terminating dideoxynucleotides (ddNTP) are included in the reaction to terminate the chain extension reaction at different parts of the sequence. The terminated fragments with different lengths are separated using highresolution gel electrophoresis, resulting in the determination of the sequence based on nucleobase-specific fluorescent detection. Although Sanger sequencing contributed to the Human Genome Project, the complete sequencing of the human genome with first-generation methods alone would have required enormous amounts of both time and money (about $30,000,000). Sanger sequencing had been superseded by second-generation DNA sequencing techniques, marketed under names such as Genome Analyzer (Illumina), 454 Sequencing System (Roche), and SOLiD System (Applied Biosystems), that can sequence a human genome in a massively parallel fashion for less than $100,000. In these methods, the amplified templates are fragmented and annealed to immobilized adaptor primers in a flow cell. The sequencing is performed in parallel with either synthesis or ligase enzymatic reaction using labeled nucleotides. Although these methods enable relatively fast and affordable DNA sequencing, they still require the amplification steps and either fluorescent or luminescent detection, with the need for the amounts of analysis time and chemically labeling processes. Third-generation DNA sequencing, represented by SMRT Sequencing (Pacific Biosciences), has attracted attention as a real-time, single-molecule sequencing method without the need for amplification steps. Zero-mode waveguide, a nano-scale photonic confinement structure, provides a high-density fluorescence array with an illumination volume reduction, resulting in the ability to perform the analysis at the single-molecule level. The single-molecule sequencing data are obtained from a DNA polymerase performing template-directed synthesis using four distinguishable fluorescently labeled deoxyribonucleoside triphosphates. Even though this method still requires enzymatic reaction and fluorescent detection, it drastically improves analysis speed and cost-effectiveness compared to second-generation techniques. Subsequently, a promising strategy for direct and electrical sequencing of DNA with enzyme-free, nanopore technology had been developed, which allows human genome sequencing within $1,000 (Fig. 3.25).

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Fig. 3.25 The evolution of the cost of sequencing one human genome (approximately 3 billion base pairs) (Reprinted from National Human Genome Research Institute [97])

3.8.3.2

The Development of Nanopore-Based DNA Sequencing

The research field of nanopore-based DNA sequencing was launched by Kasianowics et al. in 1996 with the paper on DNA translocation through a biological nanopore [98]. They were successful in the electrical and label-free detection of individual nucleic acid molecules using an αHL nanopore owing to its pore size comparable to that of single-stranded DNA (ssDNA) or RNA (ssRNA). The nucleobase composition of an ssDNA was reflected in current blocking signals with each base having a characteristic blocking amplitude, indicating that nanopore technology has the potential for determining the polynucleotide sequence at a single-molecule level. Following this study, extensive studies have been reported using biological nanopores such as αHL and MspA for sequencing DNA [99]. To identify the individual nucleobases, the motion of DNA has to be controlled at a single-base resolution. The experimental conditions, including applied voltage, solution viscosity, and electrolyte need to be carefully optimized to slow down the translocation speed of DNA through the nanopore. In addition to such optimizations, the use of motor proteins notably slowed down DNA translocation (Fig. 3.26). To that end, nanopores combined with machine-learning analysis realized the identification of nucleobases at a single-base resolution, allowing high-quality DNA sequencing. In 2014, the company Oxford Nanopore Technology introduced MinION, the first commercially available longread nanopore sequencer (Fig. 3.27). By now, MinION has shown its applicability in large-scale DNA sequencing [100] and has decreased the cost for sequencing a human genome down to $1,000. The sequencing accuracy was gradually increased from 66 to 97% by improving the bioinformatics tools and equipped biological nanopore

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Fig. 3.26 Nanopore-based DNA sequencing using motor protein (Reprinted from F1000Res. 2017 [102])

Fig. 3.27 MinION as a portable device for nanopore-based DNA sequencing (Reprinted from Biotechnol. J. 2018 [103])

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[101]. Currently, additional studies have been undertaken, such as further improvement of the accuracy, the read of short oligonucleotides, and the direct sequencing of RNA.

3.8.4 Application of Nanopore Technology: The Wide Variety of Molecular Sensing 3.8.4.1

Low Molecular Weight Compounds

In nanopore sensing applications, the compatibility between the diameter of the nanopore and the target molecule size is a crucial factor. If the target molecule is too small for the nanopore, the molecule would pass through the pore too quickly for an inhibitory current to occur (Fig. 3.24b). Therefore, to detect molecules other than nucleic acids, especially low molecular weight compounds, the pore has to be modified in order to adjust the diameter of the nanopore. In 1999, Gu et al. inserted and placed cyclodextrin into a mutant αHL from the trans side in low-pH solution to narrow the pore diameter and successfully detected organic molecules [104]. The same approach has also been successful in identifying two or more types of metal ions [105] and the dielectric isomer of a drug [106]. In these studies, the modifications of the amino acid sequence of αHL were investigated to adjust the interaction with the target molecule, resulting in facilitating the identification of the target molecule. In addition, we have reported the detection of cocaine using αHL and a cocaine-specific binding DNA aptamer [107]. Using this technique, we succeeded in selective single-molecule detection (Fig. 3.28a). In the absence of cocaine, aptamers translocate freely through the nanopore. In the presence of cocaine, large cocaineDNA complexes are retained in the pore, causing an observable decrease in the measured current. This method could detect a 300 ng/ml concentration of cocaine within 60 s. Thus, modification and mutation-based modification of the internal environment of the nanopore or using aptamers allowed more specific and sensitive molecular sensing.

3.8.4.2

Polymer Compounds

In 2007, Robertson et al. inserted polyethylene glycol with different sizes into αHL nanopore and reported that by analyzing the molecular weight-specific current blocking ratios, the size distribution of the PEG sample could be elucidated similarly to a conventional mass spectrometry-based analysis (Fig. 3.28b) [109]. In addition, the detection of polymeric nanoparticles such as polystyrene using solid-state

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Fig. 3.28 Nanopore detection on single-molecular level. a Schematic overview of detection using the cocaine-binding aptamer and the typical current–time traces with cocaine (Reprinted from J. Am. Chem. Soc. 2011 [107]). b Molecular weight distribution analysis of PEG using αHL. Molecular weight-specific inhibition current values (upper right) could be obtained with a nanopore analysis, revealing the size distribution of the PEG sample, similar to conventional mass spectrometric analysis (lower right) (Reprinted from Proc. Natl. Acad. Sci. 2007 [108])

nanopores has also been reported [110]. It has been suggested that nanopore measurements could be helpful for non-destructive characterization of polymer compounds in real-time.

3.8.4.3

Proteins and Peptides: Toward Amino Acid Sequencing

To detect water-soluble proteins in the size range of 3–4 nm, solid-state nanopores are mainly used because membrane protein nanopores are usually too small to sense such proteins. Silicon nitride or graphene is usually used for fabricating the nanopores, and the pore is formed by an electron beam using transmission electron microscopy (TEM). Using these solid-state nanopores, detection of various proteins such as bovine serum albumin (BSA) [111], avidin [112], and protease [113] has been reported. In addition to solid-state nanopores, we have attempted to reconstitute large biological nanopores (with a diameter of >3 nm) and succeeded in the detection of GranzymeB using Perforin as the nanopore [91]. Nanopore technology has attracted attention as the next generation of not only protein detection but amino acid sequencing. To this end, the size compatibility between nanopores and peptides is one of the key points to address. We have

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succeeded in using the transmembrane protein of a translocon from malaria parasites as the sensing pore to distinguish the difference in molecular weight between two individual poly-L-lysine (PLL), long PLL (Mw: 30,000–70,000) and short PLL (Mw: 10,000) [114]. Although some trials to detect polypeptides using biological nanopores [115] or to determine the 20 amino acids [116] have been reported, nanopore-based amino acid sequencing is still a tremendous challenge.

3.8.5 Application of Nanopore Technology: Diagnostic Tool as a Liquid Biopsy 3.8.5.1

MicroRNA as a Biomarker

MicroRNAs (miRNAs) are a class of non-coding RNA with 18–25 nucleotides involved in eukaryotic gene regulation. As shown in Fig. 3.29, miRNAs are produced by endonucleases Pasha and Drosha, and further processed by Dicer to maturation in the cytoplasm. The mature miRNA forms an RNA-induced silencing complex (RISC), resulting in transcript degradation and translational repression [117]. In addition to their role as post-transcriptional gene expression regulators, miRNAs have recently attracted attention as a promising biomarker. In the past decades, it has been revealed that the aberrant expression profile of circulating miRNAs in bodily fluids is linked to many diseases, including cancer [118]. Therefore, miRNAs are expected to be a biomarker in liquid biopsy, which is a non-invasive approach for diagnosis, with great potential for point-of-care testing (POCT).

Fig. 3.29 Synthesis and gene regulating mechanism of microRNA (Reprinted from Circ. Heart. Fail. 2014 [117])

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General Methods for MiRNA Detection

In general, quantitative reverse transcription real-time polymerase chain reaction (RT-qPCR) and microarray are popular methods for detecting miRNA. Although these methods are broadly used, they suffer from several shortcomings such as errorprone amplification, cross-hybridization, poor sensitivity, time-consuming process, and expensive reagents or instruments. Other techniques have been proposed for miRNA detection, including colorimetry, bioluminescence, and electrochemistry [119]. However, these methods still require the labeling or chemical modification of oligonucleotides. As an alternative to the discussed methods, a rapid, easy, and cost-effective diagnostic tool is required for the needs of POCT technology.

3.8.5.3

Nanopore-Based MiRNA Detection and DNA Computing-Assisted Pattern Recognition

The detection of oligonucleotides with nanopores is based on the size difference of single-stranded (ssDNA) and double-stranded DNA (dsDNA). ssDNA can pass through the nanopore, whereas the larger dsDNA cannot pass or clogs the pore owing to the size mismatch. Under electrophoretic conditions, dsDNA moving through nanopore undergoes so-called “unzipping” with the hybridized DNA strands detaching. The unzipping results in a long translocation time. Using this phenomenon, Wang et al. successfully detected target miRNA as unzipping current signals using a programmable DNA probe, which partially hybridized with target miRNA [120]. This system enabled the detection of target miRNA at the picomolar level, with a practical demonstration of a cancer diagnosis. The authors stated that the accuracy of the nanopore measurement was higher than that of RT-qPCR. Subsequent studies reported that modifications of the probe, including polyethylene glycol (PEG) [121] or locked nucleic acid (LNA) [122], improved detection specificity. In addition to single miRNA detection, the simultaneous detection of multiple miRNAs is important because tumor cells show complicated miRNA expression patterns. One way to realize this is to utilize DNA computing. We have previously reported the DNA computing-assisted pattern recognition of multiple miRNAs using nanopore technology. The research field of DNA computing has been developed as a tool for solving mathematical problems [123]. The output information is conventionally decoded from nucleic acid molecules to a human-recognizable signal by gel electrophoresis or fluorescent detection. To employ rapid and label-free decoding, we have applied this nanopore technology to detect output molecules in mathematical DNA computation [103, 124, 125]. As a real-life application of this integration of DNA computing and nanopore decoding, we successfully recognized the expression pattern of 2 miRNAs simultaneously using a DNA logic operation (AND gate) (Fig. 3.30) [126]. In this method, the output information is reflected in the structural change of nucleic acid molecules, which is electrically decoded as current blocking duration. Utilizing enzymatic reactions, we also succeeded in detecting miRNA at a femtomolar level [127]

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Fig. 3.30 DNA computing-assisted microRNA expression pattern recognition (Reprinted from Anal. Chem. 2018 [126])

and constructing an autonomous drug releasing system, triggered by the presence of target miRNA [128]. These methodologies have the potential for acting as in vitro diagnostic tools and intelligence elements in molecular robots [129].

3.8.6 Conclusion Nanopore proteins form nanoscale pores in lipid membranes and transport substances with size-selectivity. With the use of nanoscale pores, nanopore measurement allows the rapid, electrical, and label-free detection of target molecules at a single-molecule level. Nanopore measurement technology also enables functional evaluation of membrane proteins and artificial membrane gates, including pore size and molecular selectivity, for molecular robotics applications. Besides, combining with DNA computing technology, we believe that molecular robots with sensors and intelligence can be constructed, with endless applications in field such as targeted drug delivery and personalized medicine. For example, the following molecular robot that has a theranostics (simultaneous diagnosis and therapy) system could be built. When a liposome-based molecular robot equipped with a theranostics system described above approaches a tumor cell, miRNAs secreted in high concentrations enter the liposome through nanopores. Then, an antisense drug is synthesized inside the liposome by a DNA computing system, subsequently automatically rapturing the liposome to release the drug.

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3.9 Synthetic Biology Yutetsu Kuruma Y. Kuruma Japan Agency for Marin-Earth Science and Technology, Yokosuka, Japan e-mail: [email protected] Synthetic biology is a rapidly growing research field since 2000, based on extensive knowledge of biochemistry and genetics [130]. Synthetic biology aims to build up advanced systems for material production utilizing cell functions and to design microorganisms earning de novo biological functions [131]. Although it is similar to conventional metabolic engineering, synthetic biology offers much larger scale analyses and technology. For example, genome synthesis technology, which emerged from the human genome analysis project,2 and genome editing technology, represented by CRISPR/Cas9, have enabled it possible to modify living systems of cells. In contrast to these in vivo synthetic biology, the attempts to construct cellular functions in vitro have accelerated rapidly in the last decade [132]. Building living cells by assembling molecules and genes is important to understand how a cell organized its vital activity in micro space. This is now becoming a global trend and being promoted as a consortium-type research, e.g., Build-a-Cell3 (U.S.), fabriCell (U.K.), BaSyC4 (Netherlands), and MaxSynBio5 (Germany). Also, in Japan, there is a society of artificial cell research and the related fields,6 Japan Society for Cell Synthesis Research (JSCRS). Artificial construction of cells is meant to challenge us to cross the boundary between living and non-living things. Although it is not feasible to construct highly complicated cellular systems like mammalian cells from the beginning, the construction of a simple cell composed of only the minimum necessary functions for sustaining cell alive is becoming more and more of a reality every year. This ambitious research has a great implication for the study of the origins of life because the organisms that emerged in the early Earth environment is thought to have started out as the simplest form of life [133]. Recent developments in cell-free technology and microfluidics devices have made it possible to partially or fully reproduce some basic cellular functions. Furthermore, chemically designed and constructed molecules that do not exist in cells can mimic the behavior of living cells. This chapter introduces the recent challenges of artificial cell construction by synthetic biological approaches [134].

2

https://engineeringbiologycenter.org/about/. https://www.buildacell.org/. 4 https://www.basyc.nl/. 5 https://www.maxsynbio.mpg.de/13480/maxsynbio. 6 http://jscsr.org/. 3

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3.9.1 Artificial Photosynthetic Cells Without exception, every machines need energy to work including living organisms. In cells, a chemical energy is stored in adenosine triphosphate (ATP) and cells generate it by themselves through metabolism. Thus, the machinery converting physical and/or chemical energy source from the outer environment to ATP molecule is important for life system. ATP is produced through phosphorylation of adenosine diphosphate (ADP) by ATP synthase that is driven by proton (or sodium) motive force generated by respiration chain complexes or photosystems. These machineries are composed of a dozen of membrane proteins, therefore, reconstruction of them from each component on artificial membrane is technically difficult. On the other hand, rhodopsin that is one of the most studied membrane proteins can pump monovalent cations from the outside to inside of cell membrane using a photo energy. For example, bacteriorhodopsin can pump proton based on the photoisomerization of retinal molecule that integrated in the bacteriorhodopsin. Using these two membrane proteins, ATP synthase and bacteriorhodopsin, we rationally designed and constructed an artificial organelle that composed them on the liposome membrane. In the artificial organelle, first the bacteriorhodopsins pump protons from the outside to inside of liposome membrane stimulated by light, then generated proton gradient drives ATP synthase that produces ATP from ADP and phosphate at the exterior of the organelle. This unique system is actually found in haloarchaea, which equipped them on the cell membrane not as organelle. So constructed artificial organelle have been applied into artificial cells. Lee et al. encapsulated the artificial organelle inside micron-scale membrane vesicles (GUVs: giant unilamellar vesicles) and performed ATP photosynthesis within the GUV lumen [135]. The photosynthesized ATPs were consumed as energy for internal actin polymerization, resulting in the generation of force that changes the shape of artificial cell from the inside (Fig. 3.31a). Berhanu et al. have applied the same approach to perform protein synthesis inside GUVs using the energy of photosynthesized ATP [136]. They showed that 1–3 mM ATP, which is comparable to intracellular concentrations, was synthesized inside GUVs when the artificial organelles were illuminated. The photosynthesized ATP was consumed as energy for aminoacylation of tRNAs or/and for guanin triphosphate (GTP) generation from guanin diphosphate and phosphate. Moreover, the photosynthesized ATP was employed as substrates of transcription that is subsequently translated as a part of green fluorescent protein (GFP). These results indicate that all roles of ATP in the cell were reproduced in the scheme of artificial cell. Furthermore, Berhanu et al. succeeded to synthesize the parts of the artificial organelle, bacteriorhodopsin and the parts of ATP synthase, using the photosynthesized ATP [136]. This indicates that the constructed artificial cells are energetically self-sustaining and self-growing, just looks like real cells. The artificial organelle can be substituted with a biomaterial derived from living cells. Chromatophore is a small membrane vesicle that can be obtained by disrupting photosynthetic bacteria. Because all the necessary components for photosynthesis,

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Fig. 3.31 Photosynthetic artificial cells. a Artificial organelle consists of proteorhodopsin, photosystem II (PSII), and ATP synthase. b and c Actin polymerization in the artificial cell producing ATP by light (Reprinted from [135]). d Artificial organelle consists of bacteriorhodopsin and ATP synthase encapsulated in artificial cell. e Protein synthesis by light in the artificial cell (Reprinted from [136])

i.e., reaction center, cytochrome c, and ATP synthase, are located, chromatophore can synthesize ATP by light. Altamura et al. encapsulated these chromatophore inside GUVs to employ as organelle for energy generation in artificial cell, and they showed that the photosynthesized ATP were consumed for transcription [137]. As described above, the light-based energy production system has been reconstructed and implemented in the artificial cells. This is important for the construction of energetically independent artificial cells. Although such organelle does not exist in nature, it showed that we can expand a biological system through logically designing and constructing from molecules.

3.9.2 Cell Division Using Canonical or Non-Canonical Lipids Self- reproduction of cells is the most characteristic feature of living organisms, and the most fundamental role of the life system. Self-reproduction requires the replication of genome, the genetic information, and the growth and division of the envelope, cell membrane. Several attempts to reproduce genome duplication have been reported. However, only few experiments have so far been conducted to reproduce cell membrane growth-and-division. This is because that the mechanism of the membrane growth-and-division is complicated ranging multi-dimensional events,

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i.e., lipid synthesis in the cytosol, insertion into the lipid membrane, and dynamic deformation of the membrane envelop. To address this problem, chemically-modified lipids have been applied by several groups to construct self-dividing artificial cells. Kurihara et al. chemically constructed a precursor of amphiphile, which is similar to natural phospholipid but water solubilized by capping the hydrocarbon chain with hydrophilic compound [17, 138]. This unique molecule was supplied to the prepared giant vesicles and inserted into the vesicle membrane. The amphiphiles integrated into the vesicle membrane were converted to phospholipids, which are the constituents of the membrane, by the catalyst that pre-existed in the vesicle membrane. As a consequence, they observed the deformation and division of the vesicle membrane. Interestingly, the membrane integration of the amphiphile precursor is stimulated by the internal biochemical reactions that amplify short DNA fragments. This indicates that vesicle division and DNA replication are linked. Devaraj ‘s group chemically modified a phospholipid that was introduced an amine at the part of acyl-chain [139]. They reacted this synthetic lipid with the acyl-AMP supplied via the synthesis by FadD10 from free fatty acids and ATP, then produced intact de novo phospholipid on the vesicle membrane. Using this mechanism, they showed that the constructed vesicles grew and divided like cell proliferation. Although these chemically modified lipids do not exist in cells, they proved that membrane growth-and-division can be reproduced as a consequence of physicochemical reaction when phospholipids were newly synthesized on the lipid bilayer. Interestingly, the same principle can be found in L-form type bacteria, which lacks a cell wall but can propagate in FtsZ-independent manner through overproducing phospholipids [140]. In more biological approaches, several researchers have attempted to synthesize natural phospholipids using enzymes inside vesicles. Kuruma et al. demonstrated the synthesis of phospholipids inside liposomes by synthesizing the enzymes, acyltransferases, and allowing them to function on the liposome membrane. The acyltransferases were synthesized from the corresponding genes by an encapsulated cell-free protein synthesis system [141]. Using the same approach, Danelon’s group has synthesized various phospholipids found in living cell membranes by cell-free synthesized phospholipid synthases [142, 143]. However, in both studies, the amount of synthesized phospholipid was not sufficient for the deformation of the vesicle membrane. Synthesis of fatty acid, which corresponds to the volume of the lipid bilayer of vesicle membrane, was also reconstructed by Yu et al., but the yields arrested again after reaching the plateau at 200–300 μM (100–150 μM as diacyl-phospholipid) [144]. While these results clearly show that phospholipids can be synthesized if the membrane is equipped with functional enzymes, these clarified that the difficulty in increasing the yield of phospholipid synthesis is the core of the problems in the construction of growing artificial cells. In order to double the surface area of a 30 μm diameter vesicle, for example, 1 mM phospholipids must be synthesized inside as new lipids, moreover all of which must be inserted into the membrane. Feeding free fatty acids to the vesicle membrane from the outside is possible; however, those must eventually be converted to more stable phospholipids. To overcome this problem, it

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Fig. 3.32 Lipid synthesis in artificial cells. a, b Growth and division of giant vesicle containing the chemically modified lipids which triggered by polymerase chain reaction inside (Reprinted from [138]). c Non-canonical phospholipid synthesis by FadD10 and chemically modified lipids. d Structure of the modified phospholipid and the enzymatically synthesized acyl-AMP. e Lipid-synthesizing artificial cell encapsulating FadD10 (Reprinted from [139]). The artificial cells are expressing FadD10 at the inside. f Artificial cells synthesizing phosphatidylserine by multi-enzymes, and g microscopy image of them using LactC2-eGFP (green color) and a fluorescent lipid (purple) (Reprinted from [143])

is important to build an in vitro system that can synthesize phospholipids more efficiently and sustainably (Fig. 3.32).

3.9.3 CO2 Fixatioin by Artificial Cells The application and development of useful cellular functions is an important aspect of synthetic biology. It is also important to generate new biological tools as possible solution for social problems we face now or in the future. Among them, reduction of atmospheric CO2 concentration is one of the global issues, thus the mechanism of plants and photosynthetic bacteria is attracting a great deal of attention. Artificial cell systems or in vitro (cell-free) systems are also expected on this line, and recently impressive works have been reported.

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Erb’s group has reconstructed a CO2 fixation system by assembling 17 purified enzymes within an in vitro to build up CETCH (crotonyl–coenzyme A (CoA)/ethylmalonyl-CoA/hydroxybutyryl-CoA) cycle [145]. Furthermore, this artificial system has been coupled with thylakoid membrane extracted from spinach, to generate the driving energy of the CO2 fixation. The thylakoid membrane allows the photosynthesis of ATP and the reduction of NADP+ , thus was used as the energy generation system to drive the reconstructed CO2 fixation reaction cycle and eventually produces glycolate. This cascading reaction was performed not only in vitro but also in water-in-oil droplets mimicking inside of cells. Attempts to construct such cell mimicking systems are becoming more complex year by year. Cai et al. have succeeded to construct an artificial CO2 -fixing system that efficiently produces starch more than plants [146]. They engineered key enzymes that have introduced the mutations that avoid the inhibitory effect of ATP/ADP binding, resulting in a high level of starch production. In more developed system, they combined with a chemical reaction unit using an inorganic catalyst that produces methanol from carbon dioxides. So developed chemoenzymatic cascade achieved a high starch productivity ~410 mg l−1 h−1 from CO2 . This is much higher than the natural Calvin cycle of maize. The phrase “no plants required” in the article is impressive to synthetic biology researchers.

3.9.4 Conclusion and Challenges As described above, many cell functions have been reconstructed in vitro or/and in vesicles (artificial cells) even highly complicated systems consisting of multielements. Most of these are essential subsystems for sustaining cell alive. Therefore, when we combine the reconstructed each system as one unit, we have a much chance to create living artificial cells that are almost synonymous with living cells. To date, gene expression, genome replication, energy production, and membrane synthesis systems were partially or entirely reconstructed from the molecules, and some of them were performed inside membrane vesicles. Such reconstruction experiments may accelerate further in the future. Toward the creation of truly “living” artificial cells, however, we are remaining a serious challenge, i.e., self-reproduction of cell. Self-reproduction is the most characteristic property of living organisms that consists of a highly complicated system. Although replication of genetic information has been mostly reproduced, the reproduction of cell membrane growth-and-division has not been achieved yet. The difficulty of cell membrane growth and division is that it is a multidimensional event that spans the cytoplasmic and intramembrane reactions and involves many membrane protein machineries. Perhaps researches of membrane protein synthesis and function will become more important in the future. When this problem is resolved, the reconstruction of cell self-reproduction will become a reality. That will be an important first step for artificial cells to proliferation.

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Chapter 4

Molecular Nanotechnology for Molecular Robots Masayuki Endo

Abstract To realize the molecular robots, it is required to build robust nanostructures and flexible dynamic nanostructures, introduce molecular switches for sensing and manipulation, and incorporate dynamic functions for actuation. These individual systems are assembled as modules for creation of integrated molecular robots. For these purposes, design and construction of nanoscale structures using biomolecules has made great strides. Biomolecules have unique properties such as sequencedependent assembly of molecular components to be a predesigned structure. DNA origami allows design and construction of various nanoscale structures and nanomachines. RNA nanostructures can be designed and constructed using specific RNA motifs and their interactions. Peptide structures can also be rationally designed and used as building blocks for assembling them into multidimensional nanostructures, such as a virus capsid, using characteristic structural motifs and interactions. In addition, novel molecular nanomachines with controllable functions have been created by chemical synthesis. Furthermore, researches on artificial cells have been progressing, in which lipid-bilayer membranes are used to mimic cells to perform complex biochemical reactions inside. The outer shells of a molecular robot are made of lipid membranes (liposomes). To make them work stably as a molecular robot, researches are being conducted to stabilize liposomes using DNA-based frameworks.

For creating molecular robots, key technologies have been rapidly developed in recent years. Molecular robots for expressing intelligence need integration of sensing, computing, and actuating systems [1]. To realize the molecular robots, it is required to build robust nanostructures and flexible dynamic nanostructures, introduce molecular switches for sensing and manipulation, and incorporate dynamic functions for actuation. These individual systems are assembled as modules for the creation of integrated molecular robots. For these purposes, the design and construction of nanoscale structures using biomolecules has made great strides in the field of nanotechnology. Biomolecules have unique properties such as sequence-dependent assembly M. Endo (B) Kansai University, Osaka, Japan e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2022 S. Murata (ed.), Molecular Robotics, https://doi.org/10.1007/978-981-19-3987-7_4

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of molecular components to be a predesigned structure. Especially, nucleic acids and peptides have been used to construct nanostructures using their sequences and structural motifs in a programmed fashion. In addition, the distance between target molecules can be precisely defined in the nanostructures. This allows the distancedependent switching for the expression of functions and control of mechanical movement. In terms of individual molecular technology, DNA origami allows the design and construction of various nanoscale structures and nanomachines. RNA nanostructures can be designed and constructed using specific RNA motifs and their interactions. Peptide structures can also be rationally designed and used as building blocks for assembling them into multi-dimensional nanostructures, such as a virus capsid, using characteristic structural motifs and interactions. In addition, novel molecular nanomachines with controllable functions have been created by chemical synthesis. Furthermore, researches on artificial cells have been progressing, in which lipidbilayer membranes are used to mimic cells to perform complex biochemical reactions inside. The outer shells of a molecular robot are made of lipid membranes (liposomes). To make them work stably as a molecular robot, researches are being conducted to stabilize liposomes using DNA-based frameworks. In this chapter, we will discuss the basic technologies to design and construct molecular robots. These individual molecular technologies are rationally combined to create integrated molecular robots.

4.1 Basics of DNA Origami Design and Construction Yuki Suzuki and Masayuki Endo Y. Suzuki Mie University, Tsu, Japan e-mail: [email protected] M. Endo Kansai University, Osaka, Japan e-mail: [email protected] Since its development by Rothemund in 2006, the DNA origami technique has been widely used in many research fields and has become a standard approach for constructing DNA nanostructures with user-defined shapes. Here, we discuss the trends and characteristics in DNA origami while overviewing its design principle.

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4.1.1 Two-Dimensional (2D) DNA Origami DNA origami developed by Rothemund in 2006 originated from the self-assembly of DNA motifs, such as double crossover (DX) tiles, and it represents a powerful approach to obtaining DNA nanostructures with designed, finite-sized shapes. In this technique, a long single-stranded DNA (ssDNA), called a ‘scaffold’ strand, is folded into a prescribed shape with the aid of a large number of short ssDNAs (‘staple’ strands) (Fig. 4.1) [2]. DNA origami design starts by drawing the target shape using the path of the scaffold strand. In general, circular ssDNA is folded into a designed shape in a unicursal manner. Staple strands are applied as complementary strands to the scaffold to make all strands shape the designed double-helix structure and stabilize it. The path of each staple strand is designed so that the three distant scaffold regions were held together and aligned parallel to each other. In 2D DNA origami, crossovers must be arranged at regular intervals, so that all the double helices in the origami structure are aligned on the same plane. Specifically, the paths of the scaffold strand and staple strand are adjusted so that the crossover is located on the opposite side of a DNA helix every 1.5 turns (Fig. 4.1b). This 1.5 turn corresponds to approximately 16 base pairs (bp) of the well-defined B-DNA

Fig. 4.1 Construction of DNA origami. a Long circular single-stranded DNA (scaffold strand) is folded into a desired shape using a large number of short complementary strands (staple strands), whose sequences are designed according to the target shape. Practically, the DNA origami structure is constructed (self-assembled) by heating and slowly cooling (annealing) a mixed solution of scaffold strands and a set of staple strands. b In the two-dimensional DNA origami, the interval between adjacent crossovers is adjusted so that the all double helices are aligned in the same plane. The schematic is inspired by original work by Rothemund [2]

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structure (1 turn = 10.5 bp). The staple strands are normally designed to cross the adjacent DNA helices every 16 bp and form a double strand of 8 bp in each of the previous and the next helices on the opposite side. Therefore, the standard length of each staple strand in conventional 2D DNA origami is 32 (=8 + 16 + 8) bases. The number of staple strands required to produce a DNA origami depends on the overall size of the target shape. For example, when a full length of 7249 nucleotides (nt) of M13 mp18 ssDNA (details will be described later) is used as a scaffold strand to produce DNA origami of about 100 nm × 100 nm, approximately 230 staple strands with different sequences are required. It should be mentioned that the sequence of each staple strand is designed depending on the target shape; in turn, even though the same scaffold strand is used, other shapes can be constructed by changing the set of staple strands.

4.1.2 Three-Dimensional (3D) DNA Origami A 3D structure can be created by extending the design approach for 2D DNA origami. In 2009, various 3D DNA origamis were reported one after another by different research groups [3–7]. The used methods can be divided into two types. One is folding a 2D DNA origami into a hollow polyhedral nanostructure (Fig. 4.2) [5–7]. In this method, a 2D DNA origami was first designed for the development of a polyhedron. The target polyhedron can be obtained by assembly using DNA hybridization. The DNA origami box created by Anderson et al. was designed according to this design approach, but one side could be opened via a DNA strand displacement reaction

Fig. 4.2 Three-dimensional DNA origami structure constructed by folding two-dimensional DNA origami. a Development view of DNA origami box. Reproduced from Ref. [6] with permission. b The DNA origami box can be opened via a strand displacement reaction. Reproduced from Ref. [6] with permission. c Development view of octahedron DNA origami capsule. d DNA origami capsules with gold nanoparticles. Capsules can be opened and closed via photo irradiation, and gold nanoparticles can be released by a strand displacement reaction

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(Fig. 4.2a, b). Such a hollow nanostructure with an opening and/or closing mechanism can be used to encapsulate various molecules (e.g., proteins and gold nanoparticles) into its interior. It is also possible to implement the stimuli-responsive release of encapsulated molecules [8] (Fig. 4.2c, d). Another method for creating a 3D DNA origami structure was developed by Douglas et al. [3]. Since B-DNA makes two turns at every 21 bp, 2/3 (240°) turns to occur every 7 bp. By arranging crossovers at these intervals and bundling the DNA double helices, it is possible to design a pleated sheet, where DNA double helices are arranged at an angle of 120° to each other (Fig. 4.3a). Folding this sheet into multiple layers results in a 3D structure, where double helices are arranged in a honeycomb shape on its cross-section. This type of 3D DNA origami is often called ‘honeycomb DNA origami’ because of its cross-sectional characteristics. A method for introducing twists and/or bends into honeycomb DNA origami structures has also been developed [4]. If only a part of the DNA strand that forms the honeycomb-shaped DNA bundle is shorter or longer than 7 bp, which is the standard distance for the crossover interval, a torque is generated in the direction of mitigating this change. Using this method, the bending and twisting of the overall shape can be tuned by adjusting the number of bases for each DNA helix (Fig. 4.3b). It can be said that this technique is affected by the balance between the flexibility of the DNA duplex itself and the robustness of the honeycomb DNA bundle. This

Fig. 4.3 Various three-dimensional DNA origami. a Schematic diagram of honeycomb DNA origami. The schematic is inspired by original work by Douglas et al. [3]. b How to introduce bending and twisting. Divide the DNA bundle arranged in a honeycomb shape into sections of 7 bp, and change a part of the section from the standard length (7 bp). Reproduced from Ref. [4] with permission. c Example of honeycomb DNA origami with bending and twisting. Reproduced from Ref. [4] with permission. d Two- and three-dimensional DNA origami with curvature. The schematics are inspired by the original work by Han et al. [9]

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technique realizes a variety of complex-shaped 3D DNA origamis that do not appear to be made of DNA at first sight (Fig. 4.3c). The same idea can be applied to conventional DNA origami to produce a wide variety of shapes with curvatures. This includes the successful construction of 2D DNA origami, where DNA strands are arranged concentrically, and hollow 3D DNA origami with curved surfaces, such as spheres and pots [9, 10] (Fig. 4.3d). In each case of DNA origami developed so far, the basic design concept involves bundling the DNA double helices in parallel via crossovers. In recent years, methods of folding a scaffold strand into a wire-frame-like nanostructure have also been developed [11, 12], further expanding the achievable DNA nanostructures and their applications.

4.1.3 Selection of Scaffold Strands DNA origami requires a long ssDNA with a known sequence as the scaffold DNA. Generally, a phage-derived circular M13mp18 ssDNA (7249 nt) is used. It is known that M13mp18ss DNA has a relatively random base sequence and is less likely to form an intramolecular secondary structure. These properties are favorable for the specific binding of staple strands to specific positions, enabling high-yield production of DNA origami. Other types of ssDNA, such as p8064 (8064 nt) and p7560 (7560 nt), which are modified and lengthened M13 mp18 ssDNA, are also commercially available as scaffold strands for DNA origami. Note that both 8064 and 7560 are common multiples of 7 and 8, respectively, and 8064 is also a common multiple of 21 and 32. In practical DNA origami production, the folding of all the scaffold strand bases is relatively rare. In many cases, only the region required for the desired shape is folded into an origami, and the extra region remains as an ssDNA loop. However, when the loop structure is unfavorable, a scaffold strand of appropriate length is prepared by cutting M13mp18 ssDNA with a restriction enzyme [13]. The excised ssDNA may be used as it is (i.e., as a linear ssDNA), but it is often circularized via ligation to prevent its degradation from the 3' -or 5' -end (Fig. 4.4a). Preparation of scaffold DNAs of different lengths enables the systematic construction of a series of similar DNA origami structures of different sizes [14] (Fig. 4.4b).

4.1.4 Addressability of Staple Strands Each staple strand prepared for a certain DNA origami structure has a different sequence from all others and is incorporated into different positions in the origami structure. Therefore, the assembled DNA origami becomes an “addressable” structure through the staple strand. It can also be determined whether the end of the staple strand comes to the front or back side of the origami structure. This “addressability” is

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Fig. 4.4 Preparations of a smaller circular single-stranded DNA (ssDNA) template and DNA origamis with different sizes. a M13mp18 ssDNA is annealed using two oligonucleotides to create restriction sites. The complex is then double-digested using restriction enzymes. The strand with the target length can be used as a linear ssDNA or ligated into a circular ssDNA with the aid of a splint DNA. b Series of DNA origamis with different sizes

a notable feature of DNA origami, enabling specific placement of various molecules at “(almost) arbitrary positions” of “desired sides” of DNA origami through the 3' and/or 5' -modification of staple strands. Many studies on DNA origami have been based on the above-described addressability. The most popular application is as a platform for nano-reaction systems [15, 16]. Attempts have been made to investigate the positional relationship-dependent efficiency of biochemical reactions by arranging enzymes on a DNA origami in a specified positional relationship and reconstructing a multistep reaction (cascade reaction). It is also possible to create a pattern of DNA strands via the 5' - or 3' extension of staples. Extended staples are not only used for drawing patterns, but also used as “tracks” (scaffolding for strand substitution reactions) for molecular machines, such as DNA walkers and DNA motors [17, 18] (see Sects. 3.6 and 3.7 for details).

4.1.5 Toward Larger DNA Origami Structures The practically achievable size of DNA origami is limited by the length of the scaffold strand. One of the straightforward approaches to creating a larger DNA origami structure is the use of a longer ssDNA strand as the scaffold. There are successful examples of folding long scaffold strands of approximately 50,000 nt into DNA origami structures [19, 20]; however, this type of approach requires an enormous

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amount of staple strands for a single structure and often results in low target product yields. The common approach toward larger DNA origami structures is multimerization of the pre-assembled origami structures, where sticky-ended cohesions are often used (Fig. 4.5a, b). A part of the staple strands located at the end of the DNA origami is extended, and their sequences are designed so that two (or more) origami structures are connected to each other in the prescribed orientation. There are two popular design approaches for the connection: (a)

(b)

The extended strands work as staple strands of the partner DNA origami. That is, the extended staple sequence forms a base pair with the scaffold strand of the partner DNA origami [2]. The extended strands make duplexes with their complementary strands that are also extended from the partner origami [21].

In either case, the sequences of the extended strands should be carefully designed, so that they are orthogonal to other staples. As an advanced technique, a photoresponsive, ion-responsive, or pH-responsive sequence could be introduced into the extended staples to realize stimuli-responsive assembly/disassembly [22–24].

Fig. 4.5 Various DNA origami assemblies. a Example of a dimer obtained by base pairing between extended staple staples and the scaffold strand of a binding partner. b Example of a dimer obtained by base pairing between extended staple staples. c Three-dimensional DNA origami nanostructure that self-assembles into multimers based on shape complementarity and blunt-ended stacking interactions. Reproduced from Ref. [27] with permission. d “Mona Lisa” drawn by assembling a large number of DNA origami pieces in a programmed manner. Reproduced from Ref. [28] with permission

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In addition to sticky-ended cohesion, stacking interactions between blunt ends are often used for multimerisation [25]. Because a conventional DNA origami is constructed by arranging double helices in parallel (or multiple layers), the blunt ends of the double helix can be densely arranged at the ends of the origami. Based on this structural feature, multiple DNA origami can be connected via blunt-ended stacking. The specificity of the connection can be further improved by designing shape complementarity of the contacting sides/faces [25–27] (Fig. 4.5c, d). This technique is reminiscent of jigsaw puzzles and Lego blocks, where pieces are connected to each other based on shape matching. In the example shown in Fig. 4.5d, 64 different pieces of origami are hierarchically assembled in a prescribed positional relationship to draw “Mona Lisa” by skillfully designing sticky ends and blunt ends while considering the shape complementarity of each contacting side [28].

4.1.6 Summary and Future Prospects Rapid progress in DNA origami technology has opened up a new era, where 2D and 3D DNA nanostructures with almost arbitrary shapes are created. In addition to the complementary base-pairing of DNA molecules, the shape complementarity between/among DNA origami nanostructures has also been utilized to construct higher-order structures in a programmable manner. Although methodologies to utilize various DNA origami nanostructures with different shapes/functions as “parts” and organize them into the desired macroscopic “systems” have not been fully established, the achievements in the past decades show that we are getting closer to this end. In addition to structural design based on molecular programmability, controlling the reaction field and space using top-down technology will be key for the future development of DNA-origami-based molecular robotics.

4.2 Lipid-Bilayer-Assisted Two-Dimensional Self-assembly of DNA Origami Nanostructures into Higher-Order Architecture Yuki Suzuki Y. Suzuki Frontier Research Institute for Interdisciplinary Sciences, Tohoku University, Aramaki aza Aoba 6-3, Aoba-ku, Sendai 980-8578, Japan e-mail: [email protected] The primary limitation of DNA origami is that the size of the constructed DNA nanostructure is limited by the length of the scaffold strand. However, if DNA

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origami structures are used as building blocks for further assembly into higherorder structures, DNA structures with much larger surface areas can be constructed. Among a variety of approaches, surface-assisted self-assembly (SAS) is a promising method for obtaining one- or two-dimensionally ordered DNA origami arrays. SAS is a popular approach in the field of DNA nanotechnology, where a variety of twodimensional patterns are self-assembled from DNA motifs (DNA tiles, Y-motifs, T-motifs, etc.) on a solid surface, such as a mica surface (Fig. 4.6). One key to the success of SAS is the realization of appropriate (i.e., not too weak and not too strong) adsorption conditions that ensure two-dimensional diffusion of component molecules (such as DNA motifs) on the substrate surface. DNA origami nanostructures are generally prepared in a buffer solution (pH 7.5–8.3) containing 10–20 mM Mg2+ . However, in such a solution, they are strongly adsorbed onto the mica surface and do not exhibit two-dimensional diffusion. This problem can be solved by adjusting the buffer composition, for example, by adding several hundred mMs of NaCl to weaken the electrostatic interaction between DNA and the mica surface [29–31]. An alternative approach for SAS is changing the surface property while maintaining the same solution conditions. For this approach, not only solid surfaces, but soft surfaces can also be used [32]. Lipid bilayers, which are biocompatible interfaces, are a good option. In particular, glass- or mica-supported lipid bilayer membranes can provide a flat surface with desired physicochemical properties (fluidity, surface charge, etc.) by optimizing their lipid compositions. It is also advantageous that the surface of the origami in contact with the membrane surface can be pre-determined via hydrophobic modification of the staple strands. Considering these advantages, lipid membrane surfaces have been utilized to self-assemble DNA origami nanostructures into various two-dimensional crystalline structures, such as a lattice structure and a close-packed structure [33, 34]. These ordered structures can be functionalized into more complex structures or patterns by periodically arranging protein molecules, metal nanoparticles, and other nucleic acid nanostructures on or inside their surfaces [35]. Multimerizing DNA origami structures on three-dimensional curved membrane surfaces, such as liposome surfaces, are being attempted. In particular, in the context of synthetic biology, DNA origami nanotechnology is employed to mimic the structure and function of proteins involved in membrane deformation and fusion. Indeed, various DNA origami structures, whose multimerization induces liposome deformation have been reported [36–38]. In the not-too-distant future, the transformation of liposome shapes due to being arbitrarily manipulated by the self-assembly of membrane-adsorbed DNA nanostructures using stimuli-responsive DNA might occur [39].

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Fig. 4.6 Lipid-bilayer-assisted two-dimensional self-assembly. a Schematic illustration of the process of DNA origami assembly on supported lipid bilayers. b Topographic atomic force microscopy images of representative two-dimensional DNA origami arrays

4.3 Trends in DNA Tile and DNA Brick Technology Satoshi Murata S. Murata Department of Robotics, Tohoku University, Sendai, Japan e-mail: [email protected] Introduction The term “crystal” refers to a solid material composed of a periodic arrangement of units such as atoms, molecules, and colloidal particles. DNA nanocrystals can be defined as periodic arrangements of molecular units composed of DNA. There are also variations in the arrangement of the units. For example, consider a jigsaw puzzle in which all the pieces have the same shape. If there are no colors or patterns on the pieces, the entire structure is homogeneous, and the same pieces are repeated everywhere. However, if the pieces are black and white, there are many ways to arrange them, such as alternating rows of black and white stripes or a checkerboard pattern. The width of the stripes, size of the checker, and other properties of repetition can be varied so that an infinite number of periodic patterns can be formed. Furthermore, as we will see later, there are ways in which the units follow certain rules, and the

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resulting patterns are nonperiodic. This is called algorithmic self-assembly, which is a unique crystallization process of DNA nanostructures that cannot be found in natural crystals. In the following section, we discuss DNA nanocrystals, focusing on DNA tiles and DNA bricks. DNA Tiles One of the typical motifs of DNA nanocrystals is the DNA tiles, which are nanoscale pieces of a jigsaw puzzle. Each piece of an ordinary jigsaw puzzle contains bumps and bruises, and their shape complementarity determines how the pieces fit together. In the case of DNA tiles, the complementarity of the base sequence of the singlestranded part, called the sticky end, plays the role in fitting together these tiles. The planar nanocrystal is assembled by hybridization of the complementary sticky ends in the process of the random collision of countless DNA tiles floating in the solution. This process, in which tiles assemble and crystallize by themselves, is called self-assembly (Fig. 4.7). Self-assembly is a transition process toward a thermodynamically stable state, which is, a state in which as many hydrogen bonds as possible are formed between the bases of single-stranded DNAs. To produce DNA crystals with fewer defects, annealing (gradual cooling) of the solution is often applied (Fig. 4.8). When the water temperature is raised to 95 °C, even the longest DNA double helix cannot maintain its structure and denatures into two single strands. When several single-stranded DNA molecules, which make up a DNA tile, are mixed in a reaction tube, and annealed from 95 °C to room temperature, the single-stranded DNA molecules first form DNA tiles, and then the tiles join at their sticky ends to form two-dimensional (2D)

Fig. 4.7 Jigsaw puzzle and DNA tiles

Fig. 4.8 DNA tile fabrication process (annealing)

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Fig. 4.9 DNA tiles (DX molecule)

crystals. This process is common to all DNA nanostructures, including DNA origami and DNA bricks, and will be described in detail later. A DNA tile is a supramolecule comprising several single-stranded DNA strands. It consists of branching structures to make four arms and sticky ends available at the end of each arm. In the DX (double crossover) molecule, two antiparallel crossover branches are used as the branching structure [40]. In a buffer solution containing Mg2+ ions, to suppress the electrostatic repulsion between negatively charged DNA, the crossover branches of DNA prefer a closed conformation. However, with only one crossover, the angle between the helices could not be uniquely determined because they are allowed to rotate around the hinge (Fig. 4.9a). By placing two crossovers in a row, as shown in Fig. 4.9b, the two double helices can be fixed in parallel. In the DX molecule, the length (number of bases between the crossovers) is adjusted to avoid inconsistency in the phase of the double-helix geometry. By connecting the sticky ends, a planar structure is created in which the tiles are arranged in two dimensions. Several variations of DNA tiles have been proposed, including triple-crossover (TX) tiles (Fig. 4.10a) [41] with three helices arranged in a bundle, cross tiles (Fig. 4.10b) [42] with four helices arranged in a junction, and n-point stars (Fig. 4.10c) [43] with rotational symmetry. The T-motif developed by the authors (Fig. 4.11) [44] is based on a T-shaped junction structure that is different from crossover junctions. The T-motif has the property of self-assembling while weakly adsorbing onto the substrate surface, such as a mica surface, and thus can produce planar crystals with a large area and few defects. Algorithmic Self-assembly If the sticky end of a DNA tile consists of only one type of complementarity, we have a 2D crystal that simply repeats the same tile. If we have sticky ends with multiple complementarities, we can arrange tiles in diverse ways. For example, if there are two different sequences of sticky ends labeled 1 and 0, the right end of tile A is connected to the left end of tile B by sequence 1, and the right end of tile B is connected to the left end of tile A by sequence 0, the self-assembled structure is …A-1-B-0-A-1-B-0…, where A and B alternate in the stripe.

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Fig. 4.10 Various DNA motifs. a TX tile [41], b cross tile [42], and c 3-point star tile [43]

Fig. 4.11 T-motif tile (top) and its crystalline form (bottom) (Reprinted from Angewandte Chemie Int. Ed. 2009 [44])

We can create more complex patterns by increasing the type of sticky end (label type) and the tiles that use them. For example, we can assign two types of sticky end sequences, 1 and 0, to four different tiles, as shown in Fig. 4.12. If we consider the label on the left side of the tile as the input signal to a cell and the label on the right side of the tile as the output signal from the cell, an exclusive OR calculation is performed every time the tiles are joined. This is a visualization of the time transition of a one-dimensional (1D) cellular automaton. If only one label 1 cell exists in the seed column, a fractal pattern called the Sierpinski gasket is obtained [45, 46]. Because the connection rules can be designed arbitrarily, 2D crystals with various patterns can be created. The initial bit sequence at the edge can be set in the DNA origami structure. As shown in this example, the self-assembly process of DNA tiles is equivalent to that of the 1D cellular automaton. Because 1D cellular automata are known to be computationally universal, if we can make defect-free crystals of DNA tiles, then

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Fig. 4.12 Principle of algorithmic self-assembly

DNA tiles are also computationally universal. However, as shown in Fig. 4.6, if tiles with mismatched ends are embedded during the assembly, the subsequent calculation results will be incorrect. Such a mismatch is inevitable with a certain probability. Therefore, research has been conducted on reducing defects in crystals created by DNA tiles [47, 48]. DNA Brick The simplest type of DNA tile is called a single-strand tile (SST). This tile is made up of a single strand of DNA. A hairpin-like single strand is divided into four segments, which corresponds to the four sticky ends of the DNA tile. By arranging SSTs in a periodic fashion, ribbon-like or tube-like structures can be created [49]. The relative positions of SST tiles can be uniquely determined by assigning different sequences to the sticky ends of each pair. This property is fully exploited in “DNA bricks.” One DNA brick is a 32 nt long single-stranded DNA, divided into four domains of 8 nt each, so that each brick joins the other four. The first 2D version of the DNA brick, the “DNA Canvas,” was developed to assemble bricks into a 2D rectangular area [50]. This was extended to a three-dimensional structure, where a box of 10 × 10 × 10 bricks could be built, and the desired shape could be created by removing specific bricks from the box [51]. Subsequent studies showed that the size of the structure could be further increased, and the yield was further improved by increasing the length of the bricks to 52 nt (13 nt domains × 4) or 74 nt (18 nt domains × 4) without changing the basic design [52]. Rectangular structures (molecular weight 500 MDa) consisting of up to 30,000 bricks were obtained using this method (Fig. 4.13). The advantage of DNA bricks is that if a library of all brick sequences is synthesized in advance, any desired shape can be created by simply selecting and mixing appropriate sequences from the library. Although the preparation of a large number of brick sequences is much costlier than that of DNA origami, it is expected to

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Fig. 4.13 Concept of DNA brick

be a highly versatile technology when combined with software design support and automation of experiments using pipetting robots. Step-by-step assembly of DNA origami tiles As mentioned in the DNA origami section, a method has been developed to make 2D crystals of arbitrary patterns using square DNA origami as tiles (motifs). In normal DNA tiles or DNA bricks, all strands are combined in one reaction tube and one-pot annealing is performed. However, in this method, to create any pattern with a small number of strands, the assembly proceeds in stages using separate reaction tubes. For example, to create a square assembly of an 8 × 8 array of DNA origami, the first step is to anneal 64 different tiles (DNA origamis) in 64 reaction tubes. In the second stage, the tile assembled in the first stage is used to anneal 2 × 2 tiles in 16 reaction tubes. In the third stage, four of these tiles are collected and annealed in 4 reaction tubes, and in the fourth stage, they are mixed in one test tube. Therefore, the aggregate is enlarged by a factor of four to 8 × 8 tiles. It is important to note that the arrangement and number of adhesive ends on the DNA origami are designed in such a way that the next stage requires annealing at a temperature lower than that used in the previous stage. By attaching appropriate adhesive edges to tiles, it is possible to draw complex pictures such as the Mona Lisa on a collection of origami tiles [28]. Summary Research on DNA tiles and DNA bricks has gone in many different directions such as to make homogeneous giant crystals, increase the number of tiles to make larger

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and more complex shapes, and to make the tiles themselves more complex. The reason this kind of research continues in the world of DNA nanotechnology is that it is the most direct way of achieving “self-assembly,” in which molecules assemble themselves into the desired shape.

4.4 Liposomes Mechanically Supported by the Cytoskeletal Structure of DNA Miho Yanagisawa and Kei Fujiwara M. Yanagisawa The University of Tokyo, Tokyo, Japan e-mail: [email protected] K. Fujiwara Keio University, Kanagawa, Japan e-mail: [email protected] The basic structure of a cell membrane is a phospholipid bilayer. Artificially prepared phospholipid bilayer vesicles, called liposomes, have been used as materials to understand the physicochemical properties of cell membranes and as capsules for drug delivery systems (DDS). However, the use of liposomes as DDS is still a major challenge. Liposomes are semipermeable membranes that easily collapse by rapid dehydration and swelling owing to osmotic pressure changes that can occur in vivo. This suggests that liposomes can unintentionally break down and leak out before the drug reaches the affected area. Several attempts have been made to address this problem. For example, previous studies focused on the mechanical reinforcement of the soft membrane structure, either by filling the internal space of liposomes with a polymer gel [53], or by covering the liposome surface with a polymer [54]. However, these approaches pose additional problems. In the former case, the drug may remain trapped in the network of the polymer gel, and the smooth release of the drug by membrane disruption may be inhibited. In the latter case, the polymer on the liposome surface may lead to unintended interactions with surrounding cells. Therefore, different solutions have been desired. For example, we use DNA nanotechnology to mechanically stabilize liposomes by mimicking the cytoskeletal structure that strengthens the cell membrane. Three different single-stranded DNAs are used to achieve this (Fig. 4.14a). At high temperatures, the DNAs exist as a single strand. However, when the temperature is lowered, DNAs with complementary sequences form a double helix structure, forming a Y-shaped motif (Fig. 4.14b). With a further decrease in the temperature, the non-binding sites at both ends (i.e., the sticky ends) connect to each other, eventually forming a hexagonal network structure. As DNA is negatively charged, the use of a positively charged surfactant in the inner layer of the bilayer liposome allows the DNA network structure to be localized just below the membrane. We named this artificial cytoskeleton structure as the

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Fig. 4.14 a Three different single-stranded DNA sequences, b schematic diagram of the DNA network that constitutes the DNA cytoskeleton [55]. When the temperature is lowered, the three single-stranded DNAs form a Y-motif DNA. The three unconnected parts, called sticky ends, connect to each other to form a network structure

“DNA cytoskeleton.” Although the DNA cytoskeleton is very thin (less than 1 μm), it greatly increases the elasticity of liposomes, resulting in flexibilities comparable to those of human red blood cells. For example, under highly osmotic conditions where almost all liposomes without a DNA cytoskeleton collapse, approximately 80% of the liposomes with a DNA cytoskeleton survive. Therefore, the DNA cytoskeleton formed under the membrane prevents the collapse of the soft membrane, similar to an actual cytoskeleton in living cells [55]. Because the reinforcement derived from the DNA cytoskeleton depends on the double-helix network, the strength of the reinforcement can be adjusted by changing the network geometry and enthalpy of the double-helix structure. The DNA cytoskeleton can easily collapse in the presence of enzymes or by changes in the temperature or ionic strength. It is also possible to design DNA sequences that can collapse in response to external environmental changes. For example, if the DNA structure is designed to collapse by exposure to UV light, the drug trapped inside the liposome can be released at the desired time (Fig. 4.15). Therefore, the DNA cytoskeleton constructed in this study not only improves the mechanical durability of liposomes, but also increases their value as functional materials in pharmaceuticals and cosmetics by expanding the possibility of imparting various functions to liposomes. Fig. 4.15 Example of application of liposome with DNA cytoskeleton for DDS. Schematic illustration of how the DNA cytoskeleton collapses upon UV irradiation and the drug inside the liposome is released

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4.5 Molecular Nanomachines Constructed Using DNA Origami Masayuki Endo M. Endo Organization for Research and Development of Innovative Science and Technology, Kansai University, Osaka, Japan Institute for Integrated Cell-Material Sciences, Kyoto University, Kyoto, Japan Abstract DNA presents a powerful tool in the fields of nanotechnology and biology, allowing the assembly of various molecules and nanomaterials in a programmed manner. DNA also enables the construction of desired nanoscale structures through the manipulation of its sequences. Structural nanotechnology, especially DNA origami, is widely used to design and create functionalized nanostructures and devices. In addition, DNA molecular machines have been created and manipulated using specific DNA strands and external stimuli to perform linear, rotational, and reciprocating motions. In addition, complex molecular systems have been created in the DNA nanostructures by precisely arranging multiple molecules and molecular machines to mimic biological systems. Currently, DNA nanomachines, such as molecular motors, operate on DNA nanostructures. Dynamic DNA nanostructures with mechanically controllable systems have also been developed. This chapter describes recent researches surrounding DNA nanomachines and nanosystems designed using DNA origami nanostructures.

4.5.1 Introduction The use of DNA nanotechnology is growing rapidly and is widely accepted as a tool for interdisciplinary research. The formation of double-stranded DNA (dsDNA) molecules can be controlled by selective sequence-dependent base pairing, and the expected structures are formed based on a periodic double-helical geometry. This technology allows the construction of various self-assembled structures through the placement of functional molecules and nanomaterials to build complex molecular devices. DNA origami is a programmed DNA assembly system based on established DNA nanotechnology, which enables the design of two- and three-dimensional (2D and 3D) nanostructures of various shapes and defined sizes [2, 56]. In addition to structural design, DNA is used to create molecular machines with controllable molecular systems that allow complex movements. Since the double-helical structure is formed through base pairing, the dissociation and re-association of complementary DNA strands can be reversibly controlled by heating and cooling, respectively. This means that the molecular assembly of DNA strands can be manipulated through

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dynamic control of the binding and dissociation of DNA strands. The thermodynamic parameters of base pairing between DNA strands are pre-determined, therefore appropriate DNA sequences can be designed to generate molecular switches to control the movement of DNA nanomachines [57]. DNA molecular machines can be combined with DNA nanostructures, and mechanical nanodevices are currently being created with nanoscale accuracy. The mechanical components of these nanodevices are manipulated by specific molecules, metal ions, and external stimuli such as light, pH, and temperature [58]. In addition, DNA nanostructures can be customized using functional molecules and nanomachines, which offers great advantages in creating devices that combine each function as a distinct module. This chapter reviews recent advances in the study of DNA nanomachines constructed using designed DNA origami nanostructures. It also describes the application of DNA origami nanomachines to optical and biological devices.

4.5.2 Controllable DNA Nanomachines and Designable DNA Nanostructures DNA nanomachines DNA nanomachines are inspired by biological molecular machines that can be observed in biological systems. Several nanomachines have been created to achieve rotational, reciprocating, and walking motions [57]. The DNA molecular machine is primarily operated by the addition and removal of specific DNA strands to induce hybridization and dehybridization of DNA strands. In particular, DNA strands with an additional DNA sequence called a “toehold” are used to achieve diverse and complex movements. The addition of a complementary strand with a toehold component enables isothermal induction of DNA strand exchange from preformed dsDNA (strand displacement reaction; Fig. 4.16a). Therefore, differences in the stabilization energy of hybridization can be used to replace DNA strands in a thermodynamically stable direction. Using this method, DNA tweezers that can reversibly switch between a closed and open structure were created by controlling the binding and dissociation of two DNA strands (set and unset strands; Fig. 4.16b) [59]. A DNA walker system that is fully controlled by multiple strand displacement, and a DNA motor system that moves autonomously through the reaction of nicking enzymes have been built to achieve directional movement [57]. The operation of nanomachines uses hybridization and dehybridization of DNA strands containing the toehold part. Thus, the operational speed is affected by the toehold length and sequence [60]. Therefore, the order of the operational speed depends on the kinetics of hybridization and dehybridization of the DNA strands. In addition, nucleic acid switches have been developed to control the association and dissociation of DNA strands [39]. Metal ions and pH conditions induce switching; for example, K+ induces G-quadruplex formation, acidic conditions induce triple helix formation, and metal ions induce base-pairing formation.

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Fig. 4.16 Operation of DNA machines and creation of nanostructures by DNA origami method. a DNA strand displacement reaction via toehold sequence used for the operation of DNA nanomachine. b DNA tweezers using strand displacement reaction [59]. Set strand controls the open to the closed state of the green DNA strand, and the unset strand removes the set strand to establish an open state. c Preparation of nanometer-scale structures using DNA origami method. Long ssDNA (M13mp18) and short complementary DNA strands (staple DNA) are self-assembled by annealing [2]. When the target molecules (circles) are bound to the staple DNA, the molecules can be placed at the predesigned positions on the DNA origami structure

Photochemical switches such as DNA strands containing photoisomerization units, including azobenzene derivatives, can be used for the reversible control of hybridization and dehybridization of DNA strands [61, 62]. Such switches are also available for the regulation of nanomachines movements. Construction of DNA nanostructures DNA is also an excellent material for building accurate nanoscale structures. Various methods have been developed based on the complementarity of DNA sequences and the periodicity of the DNA double-helical structure to create nanoscale structures [63]. DNA origami, which enables the construction of various designed 2D nanostructures, has been widely investigated to create novel functionalities [63]. DNA origami was initially developed to construct planar nanostructures with defined sizes and shapes (ca. 100 nm) that are formed by the self-assembly of sequencedesigned DNA molecules [2, 4, 56]. In this method, long single-stranded DNA (ssDNA; M13mp18) and complementary DNA strands (staple DNAs) with designed sequences according to the target structure are mixed together, then annealed to form a pre-designed structure by self-assembly (Fig. 4.16c). The formed structures can be confirmed by atomic force microscope (AFM). In addition, a method for the design

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and construction of 3D origami structures has been developed, and the computerassisted design software caDNAno was developed [4, 56, 64]. DNA origami 3D structures are routinely confirmed by transmission electron microscope (TEM). Since different staple DNA strands are used at every position in the formed DNA origami structure, functionalized molecules can be placed at the desired positions in the DNA nanostructure. Various biomolecules, functional molecules, and nanomaterials are attached to synthetic DNA strands, such that these molecules and materials can be placed into the DNA nanostructure in a position-specific manner, to create new functions. We have developed a method for directly observing the behavior of enzymes and DNA structural changes using high-speed AFM, and controlling the reaction by fixing substrate DNA strands on the DNA origami [65, 66]. In addition, various manipulations of the molecules can be performed in the volume of nanoscale space constructed in the DNA origami structure. We used this method to directly visualize the movement of biomolecules and synthetic molecules in a defined DNA origami nanostructure using high-speed AFM to characterize the properties of target molecules at the single-molecule level.

4.5.3 Direct Observation of Mobile DNA Nanomachines on DNA Origami Surface DNA molecular machine on DNA nanostructure The complex movements of DNA nanomachines require a controllable molecular system in which the nanomachines are operated by specific DNA strands in a programmed manner. Seaman et al. conducted a pioneering study combining molecular machines and DNA nanostructures. Using the dsDNA conformational change called B–Z transition, where the dsDNA conformation changes from being righthanded (B-form) to left-handed (Z-form), reciprocating motion of the DNA nanostructure was observed [67]. In addition, they developed a molecular machine that can rotate 180° at the ends of two adjacent dsDNAs, called a PX-JX2 device, by hybridization and removal of DNA strands [68]. They also successfully captured triangular DNA nanostructures using the sequence specificity of the four single-stranded ends by introducing two devices into the DNA origami and rotating each triangle [69]. Using this method, four types of PX-JX2 patterns could be operated with specific DNA strands, and four different types of nanostructures were captured. Furthermore, Seeman et al. created an assembly line in which a DNA walker was operated on the DNA origami and captured multiple gold nanoparticles (AuNPs; Fig. 4.17a) [70]. They deployed three PX-JX2 devices and manipulated a DNA walker moving on a predesigned track (pathway). The movement of all devices and the movement of DNA walkers were controlled by specific DNA strands. AuNPs of different sizes were placed on the PX-JX2 devices and transferred to the DNA walker by rotational motion at specific positions. These results indicate that the DNA walker

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Fig. 4.17 Assembly line with a DNA walker capturing gold particles and DNA spider molecule walking in a track on DNA origami. a The DNA walker binds to the DNA strand on the DNA origami with three legs, and gold particles (AuNPs) are collected with three hands [70]. The DNA walker stops at three places on the DNA origami and receives AuNPs (C1, C2, C3) to be transferred by rotating PX-JX2 DNA devices. Multiple operations on DNA origami and corresponding AFM image. b The DNA spider binds onto the DNA origami using three legs hybridized to ssDNAs (cleavage site is RNA) in the track. The three legs contain DNAzyme for cleavage of RNA site in the ssDNA [71]. DNA strands in the track before and after cleavage are presented by brown and light brown circles and stopping DNA strands are indicated by red circles. The path for walking with instructions (start, follow, turn, and stop) can be programmed on the DNA origami. c AFM image of DNA spider molecule walking on the DNA origami track. Start (top), walking (middle), and stop (bottom)

moved in one direction under the control of three points on the track, the delivery of AuNPs was controlled by the PX-JX2 devices, and the AuNPs were transferred to the DNA walker. After performing each of these steps, DNA walkers with three types of bound AuNPs were obtained with a yield of 43%. Since the PX-JX2 device can use specific DNA strands to control the ON–OFF switching of AuNP transfers, the product can be obtained in high yield (>90%), and the error rate is entirely suppressed (1%). In addition, eight patterns of AuNP capture by the DNA walker were achieved using three PX-JX2 devices by ON–OFF switching operations of these devices. Yan et al. demonstrated that a DNA molecular machine called a “DNA spider” can traverse various path patterns built on DNA origami (Fig. 4.17b) [71]. DNA spiders consist of three DNA strand legs and one captured DNA strand (captured leg). The three legs of the DNA spider contain a DNA enzyme (DNAzyme leg) able to hydrolyze RNA. ssDNAs containing a cleavable RNA site were placed in the DNA origami as a track for the DNA spider to walk along. The DNA spider was immobilized at a specific position in the DNA origami using a trapping DNA strand, then dissociated and began to walk along the track. The DNA spider bound to the

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ssDNA of the DNA origami track and used its DNAzyme moiety to cleave the RNA site in the ssDNA and walked autonomously. The DNA spider moved forward along a predetermined track and eventually stopped at a specific area in the DNA origami track, where the ssDNA did not have an RNA site for cleavage. All of these processes, including initiation, track walking, and stopping, were controlled in a programmed manner. In addition, when the position of the DNA spider on the DNA origami was measured using super-resolution microscopy and it was found that the spider moved at a velocity of 3 nm/min. DNA motor walking on a predesigned track To observe the movement of a DNA motor (complementary DNA strand), we created a DNA motor system on DNA origami with a track (ca.100 nm) consisting of 17 ssDNAs. We expected the DNA motor to move continuously in one direction (Fig. 4.18a) [72]. The operating principle of the DNA motor is as follows. As shown in Fig. 4.18b, ssDNAs are placed on the DNA origami, the DNA motor strand hybridizes at one site, and the nicking enzyme Nt.BbvCI selectively cleaves the duplex of the DNA motor/DNA substrate strand in the track. Subsequently, the shortened cleaved DNA strand dissociates, and the motor strand moves forward to the adjacent DNA strand having the same sequence. This is known as branch migration. Since ssDNAs are placed equidistantly along the track, after enzymatic cleavage and branch migration, the DNA motor proceeds autonomously. After installing the DNA motor at the end of the track, the position of the DNA motor was observed using AFM. The DNA motor moved in one direction after the reaction. In addition, the single-molecule movement of the DNA motor on the track was directly observed during AFM scanning (Fig. 4.18c). Analysis of DNA motor motion revealed that the intermediate state of the DNA motor during branch migration can be visualized by high-speed AFM, and that the DNA motor moves stepwise along the ssDNA in the track. Furthermore, we created a DNA motor system using complex pathways constructed on DNA origami to control the movement of molecules with nanoscale precision (Fig. 4.18d) [18]. We created a branched track on the DNA origami that contained gates on both sides of the three branching points (junctions), to control the walking direction of the DNA motor. The gates were initially closed using blocking strands, and the DNA motor could pass through only when the blocking strands were removed (Fig. 4.18e). The position of the DNA motor after the enzymatic reaction was examined using AFM analysis and fluorescence quenching. The DNA motor moved forward in the direction of the opened gate and finally reached the four designated endpoints (Fig. 4.18f). This DNA motor system was successfully developed by constructing a complex branched pathway on DNA origami with nanoscale control of the direction of the DNA motor movement in a programmed manner. We also constructed a light-driven DNA motor system using photochemical reactions [73]. The movement of a light-driven DNA motor was investigated using DNA origami. Four DNA strands containing a disulfide bond in the first three strands were placed on the DNA origami that functioned as a track. A pyrene-binding DNA motor was introduced and hybridized in the first position [74]. Electron transfer from the pyrene moiety using UV irradiation induced the cleavage of disulfide bonds, resulting

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Fig. 4.18 A DNA motor system constructed in a DNA origami structure [72]. a A traversal rack consisting of 17 ssDNAs (green) constructed on a DNA origami structure. The DNA motor strand (red) moves on the track in one direction using an enzymatic reaction. b Mechanism of DNA motor system using branch migration. After cleavage by nicking enzyme, the DNA motor hybridized to the ssDNA (green) in the track translocates to the adjacent DNA strand with the same sequence via intermediate state (branch migration). c Single-molecule visualization of the DNA motor using high-speed AFM and its analysis. The DNA motor moved stepwise along the ssDNAs in the track, and the intermediate state of the branch migration could also be visualized. d A DNA motor system using a branched track and controllable gates [18]. A track consisting of ssDNAs having three branch points (junctions) and four end points constructed on the DNA origami structure. The DNA motor moves along the ssDNAs in the branched track from the start position. e Gate opening controls constructed on both sides of the branch point by strand displacement. f Branched track controlled by multiple gates and the programmed movement of the DNA motor by following the instructions. AFM images of DNA motor movement directed by the designed instruction

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in a continuous movement of the DNA motor until it reached the last ssDNA along the track. Depending on the duration of irradiation, the position of the DNA motor was determined by the position of the four ssDNA on the DNA origami, and the reaction rate of each step was estimated to be 0.95 × 10–2 to 1.3 × 10–2 s−1 from the distribution of DNA motor positions. In addition, the movement of the DNA motor could be observed directly in real time using high-speed AFM under UV irradiation.

4.5.4 Mechanical DNA Origami for Device Applications Controllable DNA origami nanomachine One of the goals of DNA nanotechnology is the creation of molecular machines, molecular robots, and mechanical devices [75]. Robust mechanical devices have been designed and constructed using a relatively rigid 3D origami structure [56]. Three-dimensional DNA origami, which undergoes structural changes in response to salt concentration and temperature, has also been developed (Fig. 4.19a) [27]. The system uses π–π stacking interactions and shape fitting that occur between base pairs at the ends of dsDNA molecules. In this design, a structure consisting of two pluggable rods can be rotated at the center, which can lead to a change between the open and closed forms. The X-shaped structure (open form) was closed in a fitted shape according to the salt concentration and changed to a rod-shaped structure (closed form). By controlling the temperature, opening and closing operations were detected using changes in fluorescence, and the structure remained intact even when opening and closing actions were repeated more than 1,000 times. This demonstrates the excellent performance of mechanical DNA origami as a component of molecular machines. In addition, this component was further assembled to build a movable nanoscale robot that can open and close arms depending on the salt concentration by incorporating the mechanical parts. This switchable structure was further modified with photoresponsive molecules to control the open/closed conformations upon light irradiation [76]. Using photoswitching DNA strands containing azobenzene moieties [62], a photoresponsive structure forming the open and closed conformations was constructed, which could be distinguished by gel electrophoresis and AFM imaging after UV and visible light irradiation. Moreover, the reversible shape changes during photoirradiation were directly visualized using high-speed AFM. Under UV irradiation, the closed nanostructures were opened during the fluctuations. Subsequently, the opened nanostructures were closed under visible light (Vis) irradiation (Fig. 4.19b). These results show that the opening and closing of nanostructures can be reversibly controlled by simply switching the wavelengths of light. In addition, four photoswitchable nanostructures were assembled into tetramers. Open/closed configurations of scissor-actuator-like higher-order objects can be induced by photoswitches with UV and Vis light irradiation. As seen here, the switching function is maintained even when the structures

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Fig. 4.19 Reconfigurable DNA origami structures that change their conformation in response to physical stimuli and DNA strands. a DNA dynamic nanodevice that responds to temperature and salt concentration. Two domains can rotate around the central axis. The nanodevice reversibly opens and closes in response to temperature [27]. A molecular robot that opens and closes the arms in response to salt concentration. b HS-AFM images of conformational changes of the DNA nanodevice with photoswitching strands and UV/Vis irradiation [76]. c Reconfigurable DNA nanostructure in leftand right-handed locked states controlled by strand displacement with toehold-containing DNA strands [77]. d Plasmonic nanostructure with two gold nanorods (AuNR). The locked and relaxed state can be controlled using photo-responsive DNA strands and UV/Vis irradiation [78]. In response to light, locked and relaxed states occur, which can be spectroscopically read-out according to the plasmonic interaction involving AuNRs. e Reconfigurable DNA origami tripod with AuNRs. Releasing (R) and locking strands (L) are employed for stepwise manipulation of the angle between DNA arms [79]

were attached to surfaces, which may provide interesting possibilities for constructing light-responsive electronic or photonic devices using DNA origami switches. Controllable DNA origami optical device One of the objectives of the development of controllable DNA nanostructures is the generation of optical devices. Synthetic molecular machines typically operate below the nanometer scale. Using 10–100 nm-sized DNA nanostructures and molecular machines, the controlled operation of individual molecular machines with greater dimensions should be achieved and have many practical applications. Two rod-shaped reconfigurable DNA nanostructures connected at the center as a pivot

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were constructed, and their rotational direction and left- and right-handed locked states were controlled by strand displacement with toehold-containing DNA strands (Fig. 4.19c) [77]. An optical switch was attached to the host nanostructure to create a light-driven plasmonic nanosystem that exhibits reversible chiroptical function with large-amplitude modulation [78]. The reconfigurable DNA nanostructures were functionalized with photoswitching DNA strands [62], whose locked and relaxed states were controlled by visible and UV light irradiation, respectively. Two gold nanorods (AuNRs) were assembled onto a reconfigurable DNA origami scaffold to create a plasmon nanostructure (Fig. 4.19d). In the locked state, the expected peaks were observed in circular dichroism (CD) spectra, while no peaks were observed in the relaxed state because of the random orientations of the two AuNRs. Reaction rates for unlocking and locking were identified as 5.0 × 10–3 and 1.3 × 10–2 s−1 , respectively. Reversible switching between the relaxed and locked states of the plasmonic nanostructures was achieved by alternating UV and visible light irradiation, respectively. An optically controlled plasmon nanosystem was constructed on a designed DNA nanostructure that could be read out using optical spectroscopy. Light can reversibly “write” and “erase” the conformational states of the nanostructure. This plasmonic nanosystem provides unique features of optical addressability, reversibility, and modulability, which are essential for the development of all-optical molecular devices with the required functionalities. In addition, using a reconfigurable DNA origami tripod, three AuNRs were attached to the structure, and the angle and distance between the AuNRs were precisely controlled by toehold-mediated strand displacement (Fig. 4.19e) [79]. Reversible conformational changes of the three different structures were detected by shifts in the plasmonic resonance peak and characterized by dark-field scattering spectra. Using reconfigurable DNA origami, various 3D plasmonic nanostructures can be used to construct and study the plasmonic resonance of AuNRs, and their optical properties can be controlled by strand displacement and photoreactions. Rotary motor device Biological rotary motors such as bacterial flagellar motors [80] and F1 F0 –adenosine triphosphate (ATP) synthase [81] play important roles in living systems. To mimic native rotary motors, a rotary apparatus was constructed from three different DNA origami components: a rotor unit and two clamp elements that form an axle bearing (Fig. 4.20a) [82]. The architecture had a bearing cavity and cylindrical envelope of the rotor unit. The three units were assembled in a stepwise fashion using the shapecomplementarity of the components. Finally, a mechanically interlocked architecture was constructed by closing the top. To observe the rotation mechanism, the crank lever of the rotor unit was extended to a length of 550 nm, and fluorescent dyes were attached to the end. Using this apparatus, rotary movements were observed using total internal reflection fluorescence (TIRF) microscopy. Arc-like optical signatures were frequently observed in the individual frames (Fig. 4.20b, left). By summing up 1,500 frames, a donut-like optical signature with a diameter of ~1 μm was observed, which corresponds to the rotational diameter of the crank lever (Fig. 4.20b, right). The rotation behavior can be

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Fig. 4.20 Design of a DNA-based rotary apparatus [82]. a Assembled trimeric rotor apparatus with closed brackets and a docked rotor (top) and assembled trimer with an undocked mobile rotor (bottom). b Single-particle images of fluorescence microscopy acquired in total internal reflection (TIRF) mode. Right: Sum of all 1,500 images acquired during respective single-particle recording. c Single-molecule DNA rotational measurement using DNA origami construct with motor protein. Rotation of dsDNA with motor protein was observed using a DNA origami rotor with fluorescent dye. AFM images of DNA origami rotors. d Trajectory of dye position on a DNA rotor connected to dsDNA which was unwound by RecBCD in the presence of ATP

changed by adjusting the solution conditions and bearing variants in the apparatus. The rotor can dwell in the docking sites or randomly rotate around the central axis of the bearing. These results suggest that mobility of the rotor, dwell positions, and time can be controlled through rational design. This prototype of the rotary apparatus can be used as a platform for creating synthetic rotary motors.

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4.5.5 Mechanical DNA Origami for Biological Applications Detection of molecules using dynamic DNA nanostructures Actuators associated with molecular detection have also been created using DNA origami nanostructures. A scissor-type mechanical DNA origami that responds to specific molecules was designed and constructed [83]. When the nanostructure captured a particular molecule, the two arms closed and pinched the molecules, which was used to detect a single target molecule. In addition, a rhombus-shaped DNA origami nanoactuator whose conformation was changed according to the length of the strands adjusted by specific DNA strands were created [84]. This nanoactuator was further used as a nanosensor that responds to ions, restriction enzymes, or specific RNA strands. Characterization of molecular interactions is one of the applications of mechanical DNA nanostructures. To understand higher-order chromatin configurations that control genomic structures, the energy landscape for nucleosome association is important. Tweezers-type DNA origami was used to integrate two nucleosomes to measure interaction forces between nucleosomes (Fig. 4.21b) [85]. This reconfigurable DNA origami can function as a force spectrometer to measure at subnanometer resolution. The distance between the two nucleosomes was measured using electron microscopy. From such measurements, the relative nucleosome orientation did not affect nucleosome interactions. However, histone H4 acetylation and

Fig. 4.21 Mechanical DNA origami for biological applications. a Measurement of nucleosomenucleosome interactions using tweezers-shaped DNA origami and TEM imaging [85]. b DNA nanorobot that recognizes cells and activates signaling pathways in cells [87]. Nanorobot in the closed state (top). Antibodies are placed inside the cylinder and the barrel-shaped structure is closed by the DNA strands used as “locks”. The target molecule (red circle) binds to the blue DNA strand (aptamer DNA), and then the initial dsDNA dissociates (inset). Nanorobot in the opened state (bottom). When the nanorobot opens, internal antibodies bind to specific molecules on the cell surface and transmit signals inside the cell

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removal of histone tails significantly weakened such interactions. This force spectroscopy provides a powerful tool for studying the physical properties of molecular interactions at high resolution. DNA nanorobots for biological applications One of the goals of the development of DNA molecular machines is to control cellular functions. DNA origami structures are also used as molecular containers, which can be equipped with a dynamic opening and closing system to release or expose encapsulated target molecules. The first example of a dynamic nanostructure with an opening and closing system is a DNA box, whose lid opening is controlled by strand displacement with a toehold-containing DNA [6]. An octahedral structure with a photoresponsive opening and closing system was constructed to capture and release an AuNP [8], and this system functioned in the cell under photoirradiation control [86]. A DNA nanorobot that recognizes target biomolecules on cell surfaces was constructed to selectively control cellular functions by changing its structure. A hexagonal barrel-shaped nanorobot was created, which was equipped with an opening and closing system (Fig. 4.20b) [87]. One end of the structure has a hinge that allows the opening of the structure into two domains. When the target molecule was bound to the connected dsDNA (lock) in the closed structure, the structure was opened by dissociation of the connecting dsDNA (Fig. 4.20c). The barrel structure is designed to open by recognizing and binding to target molecules on the cell surface. When the barrel structure is designed to open with two types of target molecules, the barrel can be opened using two types of target molecules present on the cell surface. A specific antibody was incorporated into the structure. Opening of the tubular structure by recognition of target biomolecules on the cell surface led to the binding of this antibody to receptors on the cell surface and subsequently activated a signaling pathway to induce cell death or cell division, as per the instructions. This system can be used to kill cancer cells based on the recognition of cell-specific markers. Nanorobots have the potential to represent an innovative type of medical molecular robot that can be programmed using a variety of biomolecules.

4.5.6 Summary and Perspectives DNA origami technology has enabled the precise arrangement and manipulation of target molecules for specific reactions. In these studies, the dynamic manipulation of molecules has been combined with DNA origami structures, and molecular nanomachines have been constructed and operated. Techniques have been developed for setting a traversal route on DNA origami structures, to control the movement of DNA nanomachines. Translocation directions and destinations can be controlled using complicated branching pathways with gates constructed on the DNA origami surface. In addition, a technique for controlling the motion of single molecules based on photochemical reactions has been developed. Furthermore, dynamic DNA

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origami devices such as reciprocal and rotary nanodevices have been developed, and their movements can be controlled by incorporated mechanical switches that are responsive to target molecules and external stimuli. More advanced dynamic nanorobots have been developed to control specific cellular functions. A reversible open/closed system is used to initialize the configuration of the nanostructures and to repeatedly change them into specific shapes by photoirradiation with defined wavelengths of light. Photonic modification of DNA-based nanomachines can be used in biological applications, such as cargo transportation and the configurational change of biomolecules in mesoscopic systems. Furthermore, these nano-sized switchable molecular devices could be used as reconfigurable nanomaterials, and may become useful tools for regulating biochemical reactions. These systems enable the transport and release of target molecules in a spatiotemporal and autonomous manner. Technologies aimed at regulating the dynamic movement of molecules with nanoscale precision are still under development, and these have the potential to be intelligent “molecular robots” that can process environmental information with computational programing and transfer this into various actuations. Acknowledgments This work was supported by JSPS KAKENHI (Grant Number 21H02057).

4.6 DNA Origami for Biological Applications Masayuki Endo M. Endo Organization for Research and Development of Innovative Science and Technology, Kansai University, Osaka, Japan Institute for Integrated Cell-Material Sciences, Kyoto University, Kyoto, Japan

4.6.1 Introduction DNA has become an excellent material for the creation of accurate nanoscale structures. DNA origami technology enables the creation of desired nanoscale structures and the construction of molecular devices with specific functions [2, 88]. One of the targets of DNA origami is its application in the control of cellular functions and living organisms. DNA is biocompatible and the sequence information can be used for gene expression and regulation. Drug delivery systems using DNA nanostructures have been developed [89]. Using DNA origami technology, drugs, celltargeting molecules, and other functional molecules, can be assembled into one nanostructure by simple self-assembly. In other words, the DNA origami structure

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has a great advantage in not only the design and construction of nanosized structures but also the incorporation of multiple functions that can be integrated as modules to create molecular devices and customized according to the purpose. Functionalized nanostructures can be used for drug delivery. In addition, by incorporating molecular switches in mechanical DNA origami structures, DNA nanorobots have been created for molecular delivery systems with high target selectivity.

4.6.2 Intracellular Delivery and Control of Cellular Functions Using DNA Origami Structure Cellular delivery and immunostimulation The various applications of DNA origami nanostructures have great biological potential, and have already been extended to cellular studies. A few examples of DNA nanostructures being resistant to various types of nucleases have been reported [90]. DNA origami constructs were able to maintain their integrity without degradation or damage in the cell lysate of a series of cell lines [91]. The high stability of DNA nanostructures in a biological system and the favorable compatibility with functional biomolecules such as proteins and aptamers demonstrate that nanostructured DNAs are promising biomaterials for live cell analysis and safe drug delivery. Prior to DNA origami, studies on the cellular incorporation and control of their functions have been conducted using small DNA assemblies such as a tetrahedral frame-type DNA structure (6 nm). Cytosine-phosphate-guanine (CpG) oligonucleotides can be recognized by endosomal Toll-like receptor 9 (TLR9) to induce immunostimulatory responses in immune cells [92, 93]. The CpG sequence targeting TLR9 was attached to the tetrahedral structure for intracellular delivery and induction of cytokine production (Fig. 4.22a) [94]. In addition, multiple-branched DNA nanos-

Fig. 4.22 DNA nanostructures for immunostimulation. a A CpG sequence was introduced at the apex of the tetrahedral structure and incorporated into cells. Increased production of cytokines was observed [94]. b DNA origami tube with CpG sequence and endocytotic pathway to induce immune responses [96]. Left: Three different types of CpG sequences were attached to the DNA origami tube via a specific handle. Endocytotic pathway of the DNA tube with CpG to immune cells and subsequent stimulation of the immune responses

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tructures bearing CpG sequences have been developed for noninvasive intracellular delivery and enhancement of the immune response [94, 95]. These structured small DNAs can be used for cellular uptake without the use of a commonly used cationic transduction reagent. Furthermore, taking advantage of a triggering mechanism, tubular DNA origami serving as a carrier system has been designed and constructed to investigate immune responses in mammalian cells (Fig. 4.22b) [96]. The DNA origami tube (~80 nm × 20 nm) with binding sites (handle sequences) for CpG and anchor sequences. The delivery performance and immunostimulatory responses of DNA tubes containing CpG have been investigated in mouse splenocytes. The origami tube was taken up by immune cells and then fused with a vesicle containing TLR9 segregated by a Golgi apparatus. The DNA tube with CpG sequences was recognized by TLR9 in a vesicle to induce an immune signaling cascade. Production of cytokines and CD69 was observed to further stimulate the immune response. Introduction of DNA origami structure into cells and functional expression Designed DNA origami structures have been used for the construction of drugs and molecular delivery systems. Initially, a fluorescent dye-labeled DNA origami was added to the cultured cells, and its introduction into the cells was confirmed. It was also found that the efficiency of uptake differs depending on the size and shape of the DNA origami and that a more compact structure (50 nm) was more easily taken up than the long rod-shaped structure (400 nm) [97]. The introduction efficiency also depends on the cell type. The DNA origami structure has also been used as a drug carrier to load the anticancer drug doxorubicin (Dox) (Fig. 4.23a) [98]. Dox was introduced into the DNA origami structure at a high concentration, and efficient introduction into cells and remarkable cytotoxicity were observed. Furthermore, a drug delivery system using DNA origami has been developed for living organisms. DNA origami was used to investigate tumor targeting in mice and drug persistence in tumors [99]. DNA

Fig. 4.23 Delivery and functional control of drug-introduced DNA origami structures. a Preparation of DNA origami structures. Anticancer drug doxorubicin (Dox) was intercalated to the DNA origami, and the DNA origami/Dox complex was introduced into cells [98]. b The amount of Dox binding and the release rate are controlled using DNA origami nanotubes with different pitches (usual 10.5 and loose 12 base pairs) [101]

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origami showed specific tumor accumulation, and by introducing Dox into the DNA origami structure, it showed delivery to the tumor and a remarkable antitumor effect. In addition, small interfering RNA (siRNA), which suppresses protein expression, was incorporated into the DNA origami structure to suppress tumor growth [100]. For instance, siRNA of Bcl2, which suppresses apoptosis, was introduced into the cells through DNA origami, leading to the suppression of Bcl2 expression by RNA interference, thereby suppressing cancer cell growth and tumor growth in mice. The DNA origami drug delivery system has been shown to be effective against cancer cells and is useful for the development of biocompatible drug carriers and delivery systems. Drug release using the characteristics of DNA origami The 3D DNA origami structure can be used to gradually release the anticancer drug into the cells (Fig. 4.23b) [101]. Two types of 3D origami structures, with different double-helical pitches (represented by the number of base pairs per rotation of the helix) and different levels of looseness between the base pairs, have been designed and constructed. The amount of Dox binding to the DNA origami with a pitch of loose 12 base pairs (T-Nano) increased by 33% compared to the standard type containing 10.5 base pairs (S-Nano). The pitch of the DNA origami can be adjusted so that the release rate of Dox can be controlled over time. It is efficiently taken up into cells, confirming that these DNA origami structures form an efficient delivery system for Dox. The Dox-DNA origami accumulates inside the cell, effectively inducing the apoptosis of cancer cells. Thus, the rate of drug release can be controlled and adjusted by designing DNA origami structures with different pitches. This is a good example of a drug release system that utilizes the properties of DNA origami structures. Coating of the DNA origami structure with lipids The instability of the DNA structure and the undesired activation of the immune system in the in vivo environment are obstacles to its application to living organisms. Therefore, it is necessary to suppress the nuclease-mediated degradation of DNA origami structures as well the activation of the immune system in vivo. Natural particulate structures such as viruses have a mechanism to avoid recognition of the immune system during infection by covering the body with lipids. To prevent the decomposition of DNA origami and circulating in the murine blood, a DNA device was created in which an octahedral frame-shaped origami structure (approximately 50 nm in diameter) was covered with a lipid bilayer (Fig. 4.24a) [102]. Due to a PEG-modified lipid coating, the DNA device showed resistance to degradation by nucleases. Immune activation was significantly reduced compared to that of the uncoated structures. When these DNA origami devices were injected into mice, non-lipid-covered devices were rapidly eliminated (half-life, 38 min), whereas the PEG-lipid-covered DNA devices were retained in the blood for a significantly longer time (half-life, 370 min). Therefore, coating with PEG-modified lipids effectively maintains the DNA origami device in living organisms. Furthermore, by covering the 3D DNA origami structure with a cationic polymer, the DNA origami was stably retained at low salt concentrations and in the cell culture

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Fig. 4.24 Stabilization of the DNA origami structure by chemical modification. a A molecular device of a frame-type DNA origami covered with a lipid bilayer membrane and PEG [102]. b Retention and distribution of fluorescence-labeled DNA devices in mice (after 2 h). Left: No coating. Accumulated in the bladder. Right: With coating. Distributed throughout the body. c Coating of cylindrical DNA origami with cationic PEG (polylysine K10-PEG5K) [103]

medium (Fig. 4.24b) [103]. In particular, by covering with a PEG-cationic polymer, it was retained in the body of mice for a long period, such as 24 h. These studies show that DNA origami devices complexed with biocompatible materials are suitable for biological and medical applications.

4.6.3 DNA Nanorobot DNA molecular machine with a photoresponsive system In the field of DNA nanotechnology, DNA machines and mechanical nanostructures have been created to conduct controlled operations. A DNA origami structure with a mechanical system has been developed for sensing biomolecules, and the detection of shape transition of the structures has been performed in response to specific molecules [104]. When DNA origami pliers capture a target molecule, the two arms close to sandwich the molecule, which can be detected by atomic force microscopy (AFM) [83]. In addition, an origami structure capable of various mechanical movements can be constructed from a rigid 3D structure, and a molecular machine that can open and close in response to metal ions, temperature, and light has also been developed [4, 27, 105]. Using DNA origami technology, it is possible to create capsule-shaped structures with an open/closed system. We created an octahedral DNA origami structure in which two pyramids can open and close in response to light for intracellular molecular delivery. We also developed a molecular system that can release included-gold nanoparticles and biomolecules by opening and closing using azobenzene-containing DNAs that respond to UV and visible light irradiation [8]. By introducing these photo-responsive DNA origami capsules into cells and irradiating specific cells with a laser, we succeeded in selectively opening the capsules introduced inside the cells (Fig. 4.25) [86]. Therefore, by manipulating nanoscale capsules from the outside, it is possible to create a system that can release drugs inside the cells. In addition, we

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Fig. 4.25 Introduction into cells of DNA nanocapsules that respond to light and open by light irradiation [86]. The cells into which the photoresponsive nanocapsules have been introduced are selectively irradiated with light. Confocal fluorescence microscope images of cells into which nanocapsules have been introduced (left) and individual cells after light irradiation (right). Aperture of 1-cell selective nanocapsules by laser irradiation at 405 nm

have succeeded in manipulating the morphology of stem cells from the outside by utilizing the expansion and contraction of DNA nanostructures in response to light [106]. DNA nanorobot that responds to molecular signals The opening and closing function of DNA origami nanostructures can be used to control cellular functions. The first example is a DNA nanorobot that can recognize biomolecules, change their 3D structure, and control their functions in a cell-selective manner [87]. The nanorobot is designed as a barrel-shaped structure that is split into two domains, and one end is connected to be opened (Fig. 4.26a). Initially, the tubular structure with dsDNA locks is closed. When the target molecule binds to the dsDNA containing the DNA aptamer at the end of the structure, the dsDNA locks dissociate to open the barrel structure (Fig. 4.26a). Using this DNA nanorobot, antibodies of human leukocyte antigen (HLA) on the leukocyte surface were placed inside the structure and locked with an aptamer-containing dsDNA. Using this unlocking system, DNA nanorobots can selectively unlock the structure in response to cells that express the target proteins on the surface. When the structure is unlocked using two types of target molecules, the nanorobot can bind only to the cell surface with two types of target molecules. Furthermore, using the DNA nanorobot, the cellular functions are controlled via signal transduction inside the cell. An antibody effective for signal transduction is introduced into the nanorobot, which can bind to the cell surface by unlocking in response to cell-specific molecules. When the nanorobot binds to the cell, a signal is transduced inside the cell to control the proliferation and activation of T cells. This shows that the nanorobot system can successfully regulate cellular functions by following programmed instructions.

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Fig. 4.26 DNA nanorobots equipped with a mechanical switch for biological applications. a DNA nanorobot that recognizes cells and activates signaling pathways in the cell. Nanorobot in a closed state (top) [87]. Antibodies are attached inside the barrel-shaped structure and are closed by DNA strands that are used as “locks” (dashed rectangle). Mechanism of structure opening by the “key”. The target molecule (red circle) binds to the blue DNA strand (aptamer DNA), and the initial dsDNA dissociates. Nanorobot in the open state (bottom). Internal antibodies were incorporated inside to bind to cell-specific molecules. b DNA nanorobot targeting the specific tumor [107]. Thrombin is attached to the sheet, and the structure is closed in a tubular shape by aptamers. When a tumorassociated target protein is attached to the aptamers, the tubular nanorobot opens to expose the thrombin, which performs blood coagulation at the tumor site

Nanorobots targeting tumors Mechanical DNA nanorobots also have the potential to be highly efficient drug delivery systems that respond to target molecules. A mechanical DNA origami structure effective for tumors has been designed, which responds to a target molecule in a programmed fashion (Fig. 4.26b) [107]. Tubular-shaped DNA origami nanorobots are functionalized with a DNA aptamer that binds to a protein specifically expressed on tumor-associated endothelial cells on the outside, and a blood coagulation protease thrombin inside the nanorobot. The aptamer that responds to a target protein in tumor cells acts as a lock for the DNA nanorobot to induce mechanical opening. When the structure opens, the thrombin introduced inside is exposed and promotes blood coagulation at the tumor site. Using a mouse model with a tumor, the DNA nanorobot introduced into the blood can selectively deliver thrombin to tumor-related blood vessels, induce intravascular thrombosis and tumor necrosis, and inhibit tumor growth. In addition, the nanorobots are immunologically inactive in vivo. This demonstrates that DNA nanorobots with multiple functions can be a promising strategy for accurate drug delivery in cancer treatment.

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4.6.4 Conclusion Advances in DNA origami technology have made it possible to freely design and create nanoscale devices, and to construct DNA molecular devices that incorporate the desired functions. Taking advantage of the characteristics of the DNA molecules, drug transport, control of cell function, and treatment of tumors have been performed using DNA nanodevices integrated with multiple functions. Furthermore, studies that are close to practical use, such as in vivo stability and avoidance of immune response, have been conducted. Since dynamic nanostructures can be constructed using DNA origami, DNA molecular devices have also been created, which contain specific molecules and unlock in response to the target molecules or light stimulus. Furthermore, nanorobots that find and treat tumors in vivo have also been developed. In the future, I believe that these DNA molecular devices can be novel drug delivery agents, in which customized functional modules can be integrated according to the purpose, and programmed medical treatments can be provided. Novel nanomedicine with DNA molecular devices and nanorobots has the potential to drastically change the present diagnosis and therapy.

4.7 Single-Molecule Imaging of Enzymatic Reactions on DNA Origami-Based Nanochip Hisashi Tadakuma H. Tadakuma ShanghaiTech University, Shanghai, Japan Future molecular robots would be composed of various parts made of different materials, including biological- and electronic materials. The low energy consumption of biological materials, such as enzymes (including proteins and nucleic acids), is one of their distinguishing features. For example, the bandgap of silicon, which is the basis of semiconductors and used for most electric devices, is approximately 1.1 eV (45 kBT at 300 K; kB is the Boltzmann constant). In stark contrast, the thermal fluctuation of biomolecules is approximately 0.025 eV (about 1 kBT). Therefore, understanding the detailed mechanism of the enzyme, an efficient molecular part, could be beneficial for developing artificial material-based parts. Single-molecule imaging is a powerful method for elucidating the mechanism of an enzyme. This is partially due to inactive molecules contaminated in molecule ensembles, hindering the bulk measurements. In contrast, researchers can trance the entire reaction process in single-molecule imaging. Therefore, they can evaluate and/or exclude inactive molecules from the population, making the measurement accurate. Enzymes sometimes work collectively in groups of molecules. For example, motor proteins, such as kinesin and dynein walking along microtubules, work collectively

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with other types of motor proteins in cellular transport. Therefore, the number of molecules and molecular layout are critical. However, it is difficult to rationally control the number of molecules and molecular layout with conventional methods (e.g., adsorbing motor molecules on plastic beads). In contrast, DNA origami allows the molecular layout at nanometer resolution because the pitch of the DNA, the base material of DNA origami, is 0.34 nm. Therefore, the molecular mechanism of the enzymes can be elucidated [108, 109]. It is possible to integrate biological and versatile materials, enabling the implementation of a new function that would be difficult to integrate using only biological materials (for example, optical regulation was achieved in [109]). The application of DNA origami and DNA nanotechnology is not limited to motor proteins. Interestingly, recent studies show that enzyme activities on DNA origami have distinct characteristics. Although further studies are required, this is hypothesized because the negative charge of the DNA origami causes a different water status and/or pH than normal water [110, 111], indicating that we would be able to achieve new functions beyond the original biological ones. The medical field is an important sector for the application of molecular robots. Depending on the situation, the cell produces the required enzyme using information from the cell genome, which serves as a blueprint (hereafter, gene expression). If we can fully regulate gene expression, we can control the cell fate (please also refer to Prof. Saito’s section). Recently, we completed the construction of a transcription nanochip with integrated RNA polymerase (RNAP) and DNA and measured activity at the single-chip level (Fig. 4.27, [112]). We measured the single nanochip activity using a water-in-oil droplet. We found that the activity of a single nanochip, containing an enzyme, was comparable to the activity of a bulk reaction–diffusion system containing many enzymes. These results could be explained by the high effective concentration of RNAP and gene by proximalization. Our estimate showed that the effective gene concentration is higher than 2 μM at a distance of 50 nm between RNAP and genes. We integrated sensor function into the nanochip, resulting in an autonomous nanochip capable of sensing, computing, and producing output at a single-chip level. Moreover, the integrated material of the nanochip has no material constraint, allowing for the implementation of photo-responsive elements and Fig. 4.27 Single-molecule measurement of nanochip enzymatic activity using water-in-oil droplet. Measuring at 0.4 pM concentration of nanochip in 20 μm size water-in-oil droplet, we can measure single nanochip activity

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harnessing the rewriting of logic functions (photo converting from the 3-input AND switch to Majority switch). Future advancements in these technologies may aid in the development of high-performance molecular robots.

4.8 RNA Nanotechnology Hirohisa Ohno, Chisato Sawanobori and Hirohide Saito H. Ohno · C. Sawanobori · H. Saito Kyoto University, Kyoto, Japan e-mail: [email protected] C. Sawanobori e-mail: [email protected] H. Saito e-mail: [email protected]

4.8.1 What Is RNA? Ribonucleic acid (RNA) is a molecule similar to DNA with the same basic unit, a nucleotide, which consists of a sugar molecule attached to a phosphate group and a nucleobase. In both DNA and RNA, nucleotides connect like a string. The main differences between DNA and RNA are (1) the sugar-phosphate of DNA contains deoxyribose and RNA contains ribose, which has a hydroxyl group attached to the pentose ring in the 2' position, and (2) RNA uses uracil instead of thymine as the complementary base to adenine (Table 4.1). While DNA is used to store genetic information in vivo, RNA plays a central role in translating the genetic information of DNA into proteins. RNA has been divided into several types including messenger RNA (mRNA), which carries a copy of the genetic information in DNA, and transfer RNA (tRNA), which works as an adaptor that matches base sequences to amino acids based on the genetic code, and ribosomal RNA (rRNA), which is the major component of the enzymes that synthesize proteins. Other types are microRNA, which regulates the amount of protein expression by degrading mRNA with complementary base sequences, and ribozymes (RNA enzymes), which have an enzymic activity to degrade and link RNA strands. The diverse function of RNA in vivo and its ability to self-assemble like either DNA or proteins are attractive features for the field of nanotechnology, which aims to create functional molecules using RNA [113–115]. In this article, we introduce major aspects of RNA nanotechnology, focusing on the construction of nanoscale structures.

Protein synthesis (translation), regulation of gene expression Relatively unstable In vitro transcription, chemical synthesis (low yield, high cost)

Watson-Crick type

B-form

Storage of genetic information

Relatively stable

PCR, chemical synthesis

Bases pairing

Duplex

Biological function(s)

Stability

Synthesis

A-form

Watson-Crick type + non-Watson-Crick type

A (adenine) C (cytosine) G (guanine) U (uracil)

A (adenine) C (cytosine) G (guanine) T (thymine)

Bases

Ribose

RNA

2’-deoxyribose

Ribose

DNA

Table 4.1 The differences between DNA and RNA

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4.8.2 RNA Nanotechnology Based on RNA-Specific Structural Motifs Progress in RNA structural biology has led to an ever-growing library of threedimensional (3D) structures consisting of RNA molecules, from the ribozymes group I introns to RNA–protein complexes, such as ribosomes and spliceosomes. These studies have shown that RNA molecules often fold to form intricate structures in a manner resembling proteins. The basis of these structures is Watson–Crick base paring, but various other forms of nucleotide interactions based on base–base and base–ribose interactions also exist. Overall, a large number of characteristic conformational patterns, called RNA structural motifs and different from the usual (A-form) double helix, have been found in a variety of RNA molecules. Typical examples are GNAR tetra-loop, UNCG tetra-loop, kink-turn [116], A-minor motif, and Ribose Zipper [117]. Since natural RNA molecules with complex 3D structures are formed by combining RNA structural motifs, it should be possible to apply the same design to create artificial RNA molecules with novel 3D structures. The first example of constructing RNA nanostructures using RNA structural motifs as structural modules is tectoRNA [118]. The RNA structural motif used here is a loop-receptor (LR) motif found in group I intron ribozymes (Fig. 4.28a, i). In this motif, the nucleotides positioned at the terminal loop (“loop”) interact with the nucleotides in the internal loop (“receptor”) through the non-Watson–Crick interactions. Jaeger et al. designed tectoRNAs, which were rod-shaped RNAs with two of the elements of LR motifs (loop and loop, loop and receptor, or receptor and receptor). Fig. 4.28 RNA nanostructures made of RNA structural motifs. a Various RNA structural motifs. b Various nanostructures constructed using RNA structural motifs

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The tectoRNA formed a dimer with another tectoRNA molecule bearing the corresponding elements (loop and/or receptor) (Fig. 4.28b, I). The dissociation constant of the LR motifs alone is several hundred nM, but the dissociation constant between tectoRNA molecules in which LR motifs are inserted in the appropriate conformational orientation is several nM, indicating an approximately 100-fold stronger binding affinity [119]. The same research group introduced four LR motifs into an H-shaped RNA molecule and linked the motifs in a planar sequence to create a long string-like structure of micrometer scale [118, 120] and also constructed tectoRNA that form a tubular complex by bundling multiple molecules arbitrarily [121]. Around the same time as tectoRNA was introduced, a structure using a 180° kissing loop (KL) motif was published [122, 123]. This motif is present at the initiation site of dimerization in the genomic RNA of the human immunodeficiency virus, with two duplexes linked at 180° via a terminal loop site (Fig. 4.28a, ii). Since the loops are interacted by the complementary base pairing of six nucleotides, it is possible to change the binding pair freely by changing the sequence of the loop region. Harada et al. made what they call LEGO by controlling the multimer formation pattern of RNA to create cyclic dimers, trimers, tetramers, and linear multimers by connecting two 180° KL motifs with a single-stranded linker and an appropriately designed base sequence at the loop site. In both tectoRNA and RNA LEGO, the formation of the desired structure has only been confirmed by gel electrophoresis, and the shape of the molecule cannot be observed directly. RNA structures were first directly observed by atomic force microscopy (AFM) [124]. The RNA structural motif used in that study was derived from a pRNA (prohead RNA or packaging RNA) derived from ϕ29 bacteriophage (Fig. 4.28a, iii). This pRNA constitutes a packaging device to pack genomic DNA into the capsid during phage replication. pRNA has two loops involved in interactions with other molecules and can form multimers using complementary sequences at the loop sites. This loop– loop interaction was used to associate multiple molecules to form dimers [124, 125], trimers [124, 126], and structures consisting of many pRNAs linearly connected [124]. In addition, the three-way junction (3WJ) motif of pRNAs has been used to create trigeminal structures [127–129], dendrimers with dendritic branches [130], triangles with the 3WJ motif at the apex [131], quadrilaterals [132], and pentagonal structures [133] (Fig. 4.28b, II–IV). In addition to planar structures, tetrahedral wireframe structures using 3WJ motifs in pRNA as the vertices have been reported [134]. Another 3D structure used the loop–loop interaction of pRNA to create triangular and quadrilateral trimers and tetramers [135]. The same study produced triangular and quadrangular wireframe structures by complementary associating them at the duplexes extending from the 3WJ. However, these initial studies made little modification to the pRNA, and the designs were based on the primary structure level of the base sequence of the loop site rather than the 3D molecular structure of the pRNA. The first example of designing a structure that combines multiple types of RNA structural motifs extracted from natural RNA molecules at the 3D structure level and actual direct observation is the RNA jigsaw puzzle [136] (Fig. 4.28b, V). Square-shaped structures were constructed by aggregating four RNA molecules with an rRNA-derived right angle (RA) motif

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(Fig. 4.28a, iv) at the apex and a 180° KL motif at both ends of the RNA duplex bent at 90°. The RNA jigsaw puzzle was groundbreaking in that it presented a wide range of possibilities of RNA as a material for nanotechnology, such as the ability to form arbitrarily shaped molecules by combining RNA structural motifs as blocks in three dimensions. Further, by changing the length of the edges, the size can be changed easily while maintaining the shape, and large size-scale structures with arbitrary 2D patterns can be formed by connecting squares. Severcan et al. created a square-shaped structure (Fig. 4.28b, VI and VII) similar to the RNA jigsaw puzzle using the 3WJ motif (Fig. 4.28a, v) derived from rRNA and the tRNA motif (Fig. 4.28a, vi) derived from tRNA and showed that these motifs could also be used as 90° structural components [137]. Since tRNA motifs are branched in three directions, they can be the vertices of a polyhedron. Eight tRNA motifs were used to create a prismatic wireframe structure [138]. Because the angles between the three duplexes in the tRNA motifs are 70°, 90°, and 120°, the prism has a twisted shape. Another structural motif with a 90° bend is the domain IIa bulge in the IRES of the hepatitis C virus (Fig. 4.28a, vii). This motif was used to form a square-shaped structure [139] (Fig. 4.28b, VIII). A 120° KL motif (RNA I/IIi kissing-loop from the transcript encoded in the ColE1 plasmid of E. coli) (Fig. 4.28a, viii), which joins the loops at an angle of 120°, was used to create a hexagonal structure [140] (Fig. 4.28b, IX). It is possible to create hexagons with this motif at its axes by associating six RNAs with loops at both ends. Due to the physical flexibility of RNA molecules, not only hexamers but also pentamers and heptamers can be formed. Since loop–loop interactions in this motif are based on Watson–Crick-type complementary base pairing, the binding pattern can be freely programmed by changing the sequence of the loop sites, making it possible to form only hexamers or only pentamers. The RNA structures described so far are composed of multiple units of relatively short RNA that self-assemble. On the other hand, Cody et al. created a tiled structure consisting of RNA strands longer than 6000 nt [141] they called single-stranded RNA (ssRNA) origami, because it looks similar to DNA origami consisting of double strands arranged in parallel at first glance. However, ssRNA origami are fabricated using a different method than DNA origami. ssRNA origami is made by folding only a single, long RNA strand without the use of a staple strand. To make this possible, a secondary structure in which the RNA strands do not knot or cross each other is prepared by arranging branching and loop motifs called “dovetail seams” that connect parallel duplexes. Using the 180° KL motif for some of the loop motifs, the local intermolecular folding structures are linked together to form a single trapezoidal or parallelogram-like structure. This design allows the long RNA strands to fold into the designed structure while being transcribed in vitro without the need for heat denaturation or annealing despite their length exceeding 600 nt. This design and folding mode is thought useful for the future construction of RNA structures in cells.

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Fig. 4.29 RNP nanostructures constructed using RNA–protein interaction motifs. a Triangular structures made of BoxC/D-L7 motif. b Square-shaped structures made of rRNA-L1 motif

Natural functional RNA molecules often form a complex with proteins (RNA– protein (RNP) complexes), which have more complex structures and advanced functions. In some complexes, the protein recognizes and binds to a specific sequence or 3D structure of the RNA. Sites where RNA and protein specifically interact as one structural motif (RNP structural motif) can, like RNA structural motifs, be used to construct nanoscale structures. Moreover, RNP structural motifs allow for complex structures that cannot be formed by RNA alone. Accordingly, we constructed nanostructures using the box C/D-L7 motif [142] (Fig. 4.29a). Inside this RNP structural motif are a box C/D RNA, a kind of kink-turn motif [116], in which RNA duplexes are bent at the internal loop, and L7 protein, a ribosomal protein that bends the angle of the RNA to about 60° by binding to it [143] (Fig. 4.29a left). Taking advantage of this characteristic 3D structure, we designed an equilateral triangular structure (Fig. 4.29a right). When this complex was observed by AFM, amorphous particles were observed in the absence of L7 protein, but equilateral triangular structures were observed in the presence of L7 protein. Real-time observation by high-speed AFM showed that the RNA shape changes from a circle to an equilateral triangle by the sequential binding of three L7 proteins [144]. We also created the rRNA-L1 motif [145] (Fig. 4.29b), which consists of the ribosome protein L1 and the binding RNA motif [146]. Here too, the bending angle of RNA is fixed by the protein binding [146]. Since the angle was about 90°, a square-shaped structure was constructed. In this way, it has been shown that RNA structures of various shapes can be constructed using naturally occurring RNA or RNP structural motifs. At the same time, there are many more structural motifs that have yet to be used to construct

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artificial molecules. Using them will expand the number of molecules with complex shapes.

4.8.3 RNA Nanotechnology Based on the Same Methodology as DNA RNA, like DNA, forms Watson–Crick base pairs, and RNA strands with complementary base sequences can associate with each other to form a stable double-helix structure. Therefore, DNA nanotechnology based on Watson–Crick base pairing can be applied to RNA. However, it is important to note that there is a difference in the 3D structure between the DNA double helix and the RNA double helix (Fig. 4.30a). DNA usually forms a B-form double helix with 10.5 base pairs in one cycle (Fig. 4.30a left). On the other hand, the double helix formed by RNA is A-type, with 11 base pairs in one cycle (Fig. 4.30a right). Biomolecules such as RNA are physically flexible, so misalignment may be tolerated to some extent, but for precise structures, it is necessary to adjust the length of continuous duplexes and the position of junctions to match the differences in 3D structures from DNA. The first RNA nanostructures created based on DNA nanotechnology are cubic wireframe structures [147]. Similar to polyhedral wireframe structures [148] (Fig. 4.30b-i) and tetrahedral wireframe structures [149] (Fig. 4.30b-ii), a cube is formed by hybridizing multiple RNA strands (Fig. 4.30b-iii). In this example, the cube has a length of 10 base pairs per side and a grain size of about 13 nm. Hoiberg et al. have also created an octahedral wireframe structure with one side 12-nm long using a similar method [150]. Although they are planar structures, polygons were also made using the same method [151] (Fig. 4.30b-iv). RNA strands were efficiently folded by connecting the duplexes that make up each side of the polygon with four “UUUU” nucleotides to create triangles, squares, pentagons, and hexagons with 22 base pairs per side. An application of DNA origami methodology to RNA, in which a single long scaffold strand is hybridized with a number of short staple strands to create arbitrary shapes [2] (Fig. 4.30b-v), was reported by two groups in 2013 [152, 153]. Those studies used RNA for the long strand (717 nt and 1,071 nt, respectively) and DNA for the staple strand to produce rectangular tile tubes [152] (Fig. 4.30b-vi), ribbons, rectangles, and triangular tiles [153] (Fig. 4.30b-iii). Later, fabricated rectangular tiles and tubes using RNA for staple strands were fabricated, demonstrating pure RNA origami [154]. Yu et al. combined the T-junction used to create planar DNA tiles [155] in three dimensions to create a square columnar wireframe structure consisting of eight units [156], while Stewart et al. applied a double crossover (DX) tile [40] (Fig. 4.30b-viii) to RNA to fabricate RNA tiles [157] (Fig. 4.30b-ix). The group later connected the tiles to other tiles at the ends to create giant tubular structures that resemble twisted ribbons [158].

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Fig. 4.30 RNA nanotechnology based on the methodology of DNA nanotechnology. a DNA and RNA show different duplex conformation. b DNA nanotechnology (left) and its application to RNA (right)

Han et al. recently presented a new type of DNA origami technology consisting of long single-stranded DNA [159]. Using PX motifs (parallel crossover or paranemic crossover) [160, 161] for the branching and linking of DNA duplexes, they succeeded in folding long DNA strands into arbitrary shapes without creating a knot (Fig. 4.30bx). In the same paper, they applied the technique to RNA and produced rhombic tiles consisting of more than 6,000 nt of ssRNA (Fig. 4.30b-xi). The above examples show how DNA nanotechnologies have been used to create RNA nanostructures, but many others, such as DNA bricks [162] and polyhedral wireframe structures consisting of single strands [163], have yet to make the transition. Other DNA nanotechnologies, such as polyhedral wireframe structures [164], convert some strands to RNA to form DNA/RNA hybrids [165]. These same

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construction principles should also be applicable to RNA, which would expand RNA nanotechnology.

4.8.4 Other RNA Nanotechnology The RNA nanostructures introduced above are designed to have well-defined certain 3D shapes at an atomic scale. However, there are also reports of some microscopic RNA structures which do not have specific shapes on an atomic scale. Lee et al. used cyclized DNA as a template to synthesize long RNA strands with a continuous hairpin structure by in vitro transcription [166]. This RNA spontaneously aggregated in a solution into a fibrous structure. Fibrous substances gathered to form a sheet that aggregated while folding, resulting in a spherical structure rich in folds. They called this structure, which is a few micrometers in diameter, a microsponge [166]. They also produced membranous structures with sizes of several millimeters by drying RNA at 37 °C [167] and sonicated the membranes to produce nanosheets of less than one micrometer [168]. Hung et al. prepared hydrogels from RNA [169]. They were initially developing a treatment for dementia using aptamers, which are RNAs that bind to specific molecules. While obtaining aptamers targeting AMPA receptor by in vitro evolution, they observed that the aptamers formed gels. The gelation was caused by the formation of aptamer intermolecular networks, through the specific sequence motifs. The next example is a gel-like structure in a cell. Eukaryotic cells possess granules, which are aggregated structures of RNA and proteins unseparated by a membrane. Various types of granules, such as stress granules and P-bodies, are thought to be involved in the transport and storage of mRNA and regulate gene expression. Nakamura et al. introduced three polyadenine RNA-binding motifs in tandem into a protein with an interprotein binding domain that is capable of forming gels. By expressing this protein in mammalian cells, they succeeded in creating an artificial RNP granule consisting of RNA and protein [170]. This accomplishment may help us understand the physicochemical properties, construction principles, and biological roles of natural RNP granules and also clues in the development of new gene expression control tools. Other topological structures have been presented such as trefoil knots, made of cyclic single-stranded RNA, and Borromean rings, where cyclic DNA and cyclic RNA are intertwined [171].

4.8.5 Application of RNA Nanostructures Until now, the RNA nanostructures we have described are mostly used as carriers to hold functional molecules. The most common cargo is the siRNA, which is about 20 nt short dsRNA with complemental sequence to the target mRNA sequence for

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RNAi (RNA interference) to suppress the expression of a target gene. A simple way to unite nanostructures with siRNA is to extend the double-stranded portion of the 3WJ motif or the end of the nanostructures. The part corresponding to the introduced siRNA is cut out by the RNA processing enzyme called Dicer. The DX tiles [157], polygons [151], and pRNA-based structures [125–127, 129, 132] introduced above along with others have been connected to siRNAs in this way to suppress target genes in human cells or in mice. In the case of structures without such duplex ends, a nick or junction is established on the duplex and the siRNA sequence is linked to it. These structures include the 120° KL hexagon [140, 147], cubic wireframes [147], and RNP triangles [144]. Hoiberg et al. used siRNAs for edges to form octahedral wireframe structures [150]. They placed the 2-nt long 3' protruding end required for Dicer recognition on the edges of the octahedron to cut out the edges. This octahedron-shaped structure was as effective as ordinary siRNA at suppressing the expression of the target gene in human cells. In the above examples, siRNA was generated by cutting out double-stranded RNA from the nanostructure by Dicer, but other mechanisms for siRNA release, such as strand exchange reactions, have also been created [172]. Aptamers, which are RNA molecules that bind to specific molecules, can also be easily introduced into a nanostructure by inserting them at the end or middle of RNA molecules. For example, aptamers that emit fluorescence when bound to dye molecules can be introduced into cubes [147], 120° KL hexagons [173], pRNA structures [131, 132], and RNP quadrilaterals [145]. Aptamers with the ability to bind to specific receptors have also been delivered in nanostructures to target cells expressing those receptors [129, 173]. Other examples include peptides that bind to certain breast cancer cell-specific cell surface antigens [144], folate for cancer cellspecific delivery [127], CpG oligo DNA for immune activation [133], and ribozymes [127, 131, 132]. Using nanostructures as carriers, it is easy to create complexes that combine the functions of various molecules [132, 173]. Generally, RNA is considered unstable in vivo. However, the nanostructures above improve its thermal stability [131] and resistance to degrading enzymes [144, 145]. The different shapes and sizes of nanostructures change the RNA kinetics in vivo, which can be exploited to develop effective drug carriers that exhibit favorable pharmacokinetic profiles.

4.8.6 Future Challenges for RNA Nanostructures Although RNA structures of various shapes have been produced, most are small in size and have very simple shapes compared to natural RNA molecules. To expand RNA nanotechnology, it is necessary to increase the diversity of the size and shape of the fabricated molecules. To do so, it is important to adopt the methodology of DNA nanotechnology to produce larger structures and to increase the library of RNA structural motifs.

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In order to improve the functionality of RNA structures, we recommend hybridization with materials other than RNA. Although RNA structures have higher stability compared to linear ssRNA, they are often inefficient as drug carriers in vivo. In addition, depending on the cell type, the introduction of RNA may elicit an immune response as a defense mechanism against foreign nucleic acids, resulting in strong cytotoxicity. This response may be controlled by hybridization with DNA or by chemical modifications of the RNA [151, 172, 174, 175]. In addition, the function of proteins is much more diverse than that of nucleic acids. Since RNP motifs can be used to directly link RNA and proteins [142, 144, 176], protein functions can be easily introduced into RNA structures. Omabegho et al. have utilized the Kt-L7 motif to integrate a protein system consisting of actin-myosin and an RNA–DNA system that undergoes secondary structural changes by strand exchange to create a molecular motor with two independent operating systems [177]. In this way, by combining RNA with other biomolecules, it is possible to achieve functions that are difficult if using RNA alone. Solving these issues will advance the creation of molecular robots using biomolecules and applications in the fields of the life sciences and medicine, such as the control of cell functions.

4.9 Trends in the Peptide/protein Design Technology Kazunori Matsuura K. Matsuura Tottori University, Tottori, Japan e-mail: [email protected] When creating molecular robots, methods are used to spontaneously construct nanostructures using biomolecule self-assemblies. The DNA nanotechnology represented by “DNA origami” has made it possible to freely design and construct various nanostructures. Nano-architectures consisting of peptides/proteins are also constructed by rationally designing their self-assembly [178] This chapter outlines the trends in these design technologies.

4.9.1 Nano-architectures Based on Protein Self-assembly In many cases, nano-architectures self-assembled from proteins are constructed using protein–ligand interactions or protein–protein interactions, e.g., Hayashi and coworkers constructed one- and two-dimensional assemblies of heme proteins, such as cytochrome b562 , using the heme and heme pocket interaction (Fig. 4.31a) [179]. Wagner and coworkers succeeded in creating a protein nanoring with an 8–20nm diameter by interacting an artificially dimerized dihydrofolate reductase with

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Fig. 4.31 Examples of nano-architectures constructed using a protein self-assembly

the dimerized inhibitor (Fig. 4.31b) [180]. Aida and coworkers demonstrated that protein nanotubes are self-assembled from spiropyran/merocyanine-modified 14mer protein GroEL known as a molecular chaperone and the formation and dissociation can be controlled using photo-isomerization (Fig. 4.31c) [181]. Protein nanotubes are formed by the interaction between the phenoxide anion of merocyanine and the Mg2+ ion, but are dissociated by the photo-isomerization of an uncharged spiropyran. They also demonstrated that green fluorescent proteins (GFPs) were encapsulated in protein nanotubes and released using photo-isomerization. Protein nanocapsules possessing C 3 -symmetry axes can be constructed using the self-assembly of a fusion protein. In this self-assembly, a dimer-forming unit is linked with a trimer-forming unit. Yeates and coworkers succeeded in constructing 15-nm tetrahedral protein cages using a fusion protein self-assembly of a bromoperoxidase trimer unit together with the M1 matrix dimer protein unit of the influenza virus (Fig. 4.32a) [182, 183]. Recently, Baker and coworkers succeeded in constructing an icosahedral nanocapsule of 25 nm diameter using an artificially designed trimeric

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Fig. 4.32 Examples of protein nanocapsules constructed using a symmetric protein unit selfassembly

protein self-assembly (Fig. 4.32b) [184]. They also constructed a 120-subunit icosahedral protein nanocapsule by using a designed trimeric and pentameric protein co-assembly (Fig. 4.32c) [185]. These nanocapsules are interesting because they are similar to the assembling principle of spherical viral capsids.

4.9.2 Nano-architectures Based on a Peptide Self-assembly Construction of nano-architectures using rationally designed peptides that are partial structures of proteins has also been actively investigated. Pioneering work by Ghadiri on creating peptide nanotubes using cyclic peptide hydrogen bonding has been reported in the 1990s (Fig. 4.33a) [186]. A peptide nanotube formed using the dipeptide Phe–Phe self-assembly in an aqueous solution, which was reported by Gazit and coworkers, is also well known (Fig. 4.33b) [187]. By designing secondary structures, such as a α-helical coiled coil and a β-sheet, various nano-architectures can be constructed. The coiled-coil structure can be designed by choosing a proper arrangement of hydrophobic, electrostatic, and hydrogen-bonding amino acid residues on

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Fig. 4.33 Examples of self-assembled peptide nanotubes

a helical wheel (Fig. 4.34a). The β-sheet structure can be designed by alternating hydrophilic and hydrophobic amino acids and arranging complementary charges (Fig. 4.34b). Jerala and coworkers demonstrated that tetrahedral structures with a

Fig. 4.34 Design of a coiled-coil peptide (a) and β-sheet-forming peptide (b)

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side of 7 nm were constructed by the intramolecular folding of a single polypeptide chain composed of 12 concatenated coiled coil-forming segments (Fig. 4.35) [188]. They call this strategy the “peptide origami”. A three-way junction arrangement of self-assembling β-sheets or coiled-coil peptides helps construct peptide nanocapsules. In 2005, we succeeded in creating a virus-like nanocapsule for the first time by forming a self-assembly of three-way junction peptide conjugates (Fig. 4.36a) [189]. We synthesized a trigonal peptide conjugate containing three anti-parallel β-sheet-forming peptides (Trigonal-(FKFE)2 ) and showed that it was self-assembled into nanocapsules with a diameter of 20 nm in

Fig. 4.35 Peptide origami formed through an intramolecular coiled-coil

Fig. 4.36 Examples of trigonal peptide conjugates that self-assemble into spherical nanocapsules

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water. The diameter is comparable to the estimated diameter of a dodecahedral structure self-assembled by forming an anti-parallel β-sheet. We also demonstrated that a trigonal peptide conjugate bearing three tryptophan zipper-forming peptides CKTWTWTE self-assembled into nanocapsules with a size of about 30 nm at neutral pH, but into irregular aggregates in an acidic environment [190]. Furthermore, we found that a trigonal conjugate of glutathione formed spherical assemblies with sizes of 100–250 nm in water (Fig. 4.36c) [191, 192]. Two years after our first report, Gazit and coworkers reported that a trigonal conjugate of dipeptide Trp–Trp self-assembled into nanospheres with sizes of 200–500 nm in a methanol–water solution (Fig. 4.36b) [193]. By adopting a similar strategy for coiled-coil peptides, Ryadnov and coworkers designed a trigonal conjugate of three antimicrobial peptides with positive charges, which selectively formed a coiled-coil structure with a complementary antagonist peptide with negative charges. The conjugates are self-assembled into antimicrobial peptide nanocapsules with a size of about 20 nm (Fig. 4.36d) [194]. Woolfson and coworkers demonstrated that two complementary trigonal hubs composed of coiledcoil bundles (Hub A and Hub B) co-assembled into nanocapsules with a size of about 100 nm (Fig. 4.36e) [195]. In 2010, we were the first to create an artificial viral capsid in the world by forming a self-assembly of C 3 -symmetric β-annulus motif, which participates in forming a dodecahedral internal skeleton of the tomato bushy stunt virus (Fig. 4.37a) [196]. The 24-mer β-annulus peptide fragment was synthesized using the Fmoc-solid-phase method. The dynamic light scattering and transmission electron microscopy of the aqueous solution of a β-annulus peptide showed the formation of spherical structures of sizes 30–50 nm. Synchrotron small-angle X-ray scattering measurements revealed the existence of a hollow inside the assembly. Various molecules can be encapsulated in an artificial viral capsid. The pH dependence of the β-potentials of the artificial viral capsids indicates that the C-terminal is directed toward the exterior, whereas the N-terminal is directed toward the interior of the capsids [197]. As the capsid interior should be cationic at pH 7, the anionic M13 phage DNA and CdTe quantum dots were encapsulated into the capsid through an electrostatic interaction [197, 198]. By modifying the interior-directed N-terminal of a β-annulus peptide with a Ni–NTA complex, it is also possible to encapsulate His-tagged green fluorescent protein into the capsid (Fig. 4.37b) [199]. In contrast, by modifying the C-terminals of the β-annulus peptide, it is possible to create artificial viral capsids decorated with functional molecules at the outer surface. As shown in Fig. 4.37c, we have succeeded in creating artificial viral capsids decorated with gold nanoparticles [200], singlestrand DNA [201], coiled-coil spike [202], human serum albumin [203], ribonuclease S [204], horseradish peroxidase [205] and lipid bilayer (envelope) [206]. As described in this chapter, molecular techniques for free design and construction of various functional nano-architectures such as protein/peptide nanocapsules have been developed. In the future, it is expected that various molecular robots will be developed using protein/peptide nano-architectures.

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Fig. 4.37 a Construction of an artificial viral capsid using a self-assembly of β-annulus peptides derived from the tomato bushy stunt virus, b Encapsulation of the His-tag GFP into an artificial viral capsid, c Surface decoration of an artificial viral capsid

4.10 Peptide Design and Molecular Robotics Naoto Nemoto N. Nemoto Saitama University, Saitama, Japan DNA may be the best masterpiece of all molecules. Studying life sciences leads to a greater understanding of biomolecules, which, in turn, leads to greater admiration of their multifunctionalities. Using DNA to build molecular robots represents a rational approach. DNA was identified approximately 80 years ago to play the crucial function of carrying genetic information. In 1992, single-stranded DNA was discovered to form various tertiary structures based on its sequence, as observed for RNA and proteins, and bind target proteins and other molecules (i.e., DNA aptamers) [207]. Thus, the functions of biopolymers are not limited by their components (nucleotides or amino acids). Enzymes were initially thought to be a proprietary of proteins; however, enzymatic functions of RNA [208] (termed ribozyme) were discovered by

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Cech et al. in 1983. Subsequently, Breaker et al. showed in 1994 [209] that DNA can also function as an enzyme, called DNAzyme. Thus, functional differences between DNA or RNA and proteins have been redefined over the past 40 years, revealing the general functional universality of biopolymers. Nonetheless, DNA is primarily characterized by its double helix structure. In contrast to proteins, DNA does not acquire a higher function by forming quaternary structures. However, this dogma may be changing through the development of DNA origami [210] by Rothemund and colleagues. Indeed, research into molecular robotics combines DNA with various materials to create interesting nano-devices [211]. For these reasons, many believe that it is too late to use peptides for molecular robotics research. Moreover, many readers believe that DNA alone is sufficient for the development of molecular robots. That may be correct, but when we think about the evolution of life, the following questions may come to mind: why did life move from an RNA world to an RNP world (a world composed of RNA and protein) by developing a complex system, the translation machinery? The question is, as we have seen, RNA and DNA can work as enzymes and bind with various targets as seen, for example, with antibodies. Does nature require the coding of polypeptides from RNA using amino acids? DNA and RNA are composed of only four types of bases, whereas proteins are composed of twenty amino acids. Thus, greater diversity can exist with proteins. Furthermore, from the study of the origin of life, amino acids were postulated to be readily produced during the first stage of the formation of Earth whereas conditions were unfavorable, which made nucleic acid synthesis challenging. Now take into consideration cells. Lipids used as materials for cell membranes were probably present when the first cell emerged Thus, peptides and lipids may have interacted before cells existed. These interactions may have played essential roles in leading to the formation of cells. In fact, many kinds of membrane proteins are embedded in cell membranes, and without these membrane proteins, it is unlikely that the cell can function as a cell. Therefore, a detailed understanding of the relationship (interaction) between the membrane lipid bilayer and peptides is important for understanding of the emergency of cell. We have been examining molecular evolution by in vitro evolution technology, in which peptides and proteins of various sequences are synthesized in vitro using a cell-free translation system, and specific functional molecules are selected from these vast sequences (called libraries) and evolved (molecular design is performed by evolutionary molecular engineering). Therefore, as shown in Fig. 4.38, a selection system was devised using a peptide library with random sequences against a liposome, an artificial lipid membrane, to obtain a peptide that binds the liposome. Interestingly, experiments have identified peptides that bind strongly to liposomes with sequences not found in nature [212]. We have identified functional peptides by conducting experiments under simplified conditions not found in nature. New functionalities of novel peptides not found in nature are possible using different combinations of libraries and selection criteria.

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Fig. 4.38 a In vitro selection system for liposome-binding peptides b imaging of liposome-binding peptide (modified with fluorescein) on a liposome

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Combining these novel molecules may yield a molecular robot that functions as a living organism, thus yielding another “nature” different from life on Earth.

4.11 Molecular Machine and Nanocar Katsuhiko Ariga K. Ariga WPI Research Center for Materials Nanoarchitectonics (MANA), National Institute for Materials Science (NIMS), Tsukuba, Japan e-mail: [email protected] Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Japan In this chapter, the basic concept, designs, and operation modes of molecular machines and nanocars (molecular cars) are discussed on the basis of supramolecular chemistry and surface sciences.

4.11.1 Molecular Machine with Supramolecular Chemistry The concept of molecular machines is that a single molecule works like a machine. Since a molecule is a unit of matter such as an organic compound, if it can move and perform various functions, it would be the ultimate small machine. If we take the word in its broadest sense, molecular machines have been developed long ago. In fact, our bodies are full of machines that could be called molecular machines [213]. Proteins cut molecules like scissors, or conversely, create new molecules from several molecular parts, and DNA replicates itself with the help of proteins. They could be what we call molecular machines. What is needed now is the development of technology that will allow humans to synthesize such molecular machines from organic compounds and other materials, and produce what we want, not what is readymade. Without the help of living organisms, it could be difficult to create complex protein-like machine structures. It is generally difficult for molecules of limited complexity that can be synthesized from organic synthesis to work like machines. If one molecule cannot satisfy these aims, a reasonable solution is to try to combine molecules to achieve the goal. This would be assigned to supramolecular chemistry [214]. A supermolecule is defined as an assembly of two or more molecules through noncovalent interactions that exhibit properties and functions that cannot be obtained from the components alone. The approach taken by supramolecular chemistry is also said to create something that exhibits superior functions by simply collecting molecular parts without attaching them. Molecules can also be combined to form supramolecules that are useful as molecular machines. Those attempts led to the 2016

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Nobel Prize in Chemistry as a pioneering example of molecular machines. Examples are rotaxane [215] and catenane [216]. These supermolecules play a very important role in the design of molecular machines. Rotaxane is a word coined from a word meaning “wheel” and a word meaning “axis”, and is a molecule in which the axis of a molecule such as a polymer is attached to a cyclic molecule such as a cyclodextrin. If both ends of the axis are blocked with a larger functional group, the ring will not come off the axis. On the other hand, catenane is a structure of cyclic molecules passing through each other, meaning that the molecules are connected in a chain. The molecular machines that were the subject of the Nobel Prize in Chemistry is shown in Fig. 4.39 The example of Sauvage et al. uses a catenane structure (Fig. 4.39a) [217]. In this molecular machine, two integrated molecular rings interact strongly with copper ions at specific sites. Depending on the electronic state of the copper ion, the site of interaction changes, causing the rings to rotate relative to each other, which can be regarded as a molecular motor. As seen in the molecular machine example, catenanes are physically intertwined supramolecules that are never separated but are flexible in their movements. These characteristics make them suitable for the design of molecular machines that move in response to stimuli. The development of molecular machines that skillfully use the rotaxane structure has been led by leading groups in supramolecular chemistry such as Stoddart et al. [218]. Figure 4.39b shows an example of a molecular shuttle, which is also regarded as a symbolic molecular machine. A cyclic molecule of viologen dimer stays at the electron-donating benzidine moiety when the axial molecule is neutral, but moves to the biphenol moiety when this benzidine moiety becomes cationic upon oxidation. This is called a molecular shuttle because it looks like a cyclic molecule moving between the two stations. In the case of molecular motor developed by Feringa (Fig. 4.39c), two aromatic parts are connected through a double bond [219]. One part can be step-wisely rotated relative to the remaining half upon inputs of external stimuli. These molecular machines may be regarded as masterpiece molecules, achieved through wise designs well-considered with organic chemistry and stimulus-responsive strategies in supramolecular chemistry. Recently, challenging research on the use of molecular machines in biomedical fields was reported [220]. In the reported system, molecular motors are adsorbed to cell membranes, and external stimuli such as UV radiation can cause disturbances that result in structural changes in molecular motors and the opening of holes in the cell membrane. Just a small amount of molecular motor can diffuse into the living cell, causing necrosis or diffusion of external chemicals into the living cell. By introducing short peptide segments into the arms of the molecular motor, the mechanical action of the molecular motor can also selectively occur in target cells like tumor cells with corresponding recognition sites. Because the size of molecular motors is at the 1 nm level, the rupture of thick cell membranes due to nanomechanical effects is not always immediate; the concerted movement of 1-nm-sized motors can cause significant dislocation of membrane components.

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Fig. 4.39 Examples of molecular machines: a catenane-type molecular rotor; b molecular shuttle; c molecular motor. These structures are given by courtesy of Dr. Masayuki Takeuchi (NIMS)

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4.11.2 Molecular Machine at the Air–Water Interface Molecular machines are molecules synthesized by an excellent molecular design, and by applying various stimuli to them step by step, the molecules are working like rotors or shuttles. Placing molecular machines at flexible interfacial media as described below allows us to manipulate the molecular machine with simple mechanical actions as applied stimuli [221]. This is done at a macroscopic interface such as a surface monolayer spread at an air–water interface. The molecular machine used in this proposal is a steroidal cyclophane (Fig. 4.40) [222, 223]. This molecule consists of a central cyclophane structure with four soft arms with connected cholic acid plate structure, which are connected to the central cyclophane ring via flexible arm structures. The hydrophilic and hydrophobic surfaces of the cholic acid plate structure are inextricably linked. When the steroid cyclophane is spread on the water surface, the hydrophilic surface of the cholic acid opens up to contact the water surface at low pressure. Upon application of pressures laterally to this monolayer of steroidal cyclophane, the arms can be bent to form a more compact structure, creating a molecular cavity. The steroidal cyclophane molecules are used as a monolayer on the aqueous phase containing the guest fluorescent naphthalene-type dye molecules, and the monolayer is mechanically compressed and expanded. As the monolayer is mechanically compressed, the conformation of the steroid cyclophane changed from flat to cavity-shaped, and at the same time, the guest molecules in the aqueous phase can be captured. Furthermore, the monolayer of steroidal cyclophane traps and releases guest molecules in response to repeated compression and expansion. This coupling of macroscopic mechanical manipulation, i.e., compression and expansion, to the function of the molecular machine is largely dependent on the structure of the interface. The interface has a macroscopic scale in the in-plane (transverse) direction and a nano-size (molecular size) in the thickness direction. Because of this anisotropic structure, macroscopic motions and variations in the transverse direction can be reflected in the molecular functions in the thickness direction. Most discussions of interface science have been concerned with how the properties of surfaces and interfaces differ with respect to the bulk state. However, as shown in this example, the interface has a role that has not received much attention so far, namely, to connect the macroscopic and nano-scales. The functions (recognition capabilities of guest molecules) of molecular receptors can also be tuned by mechanical manipulation at the air–water interface. A monolayer of cholesterol-armed cyclen, a molecular receptor, spread at the air–water interface, and is continuously deformed by compressing the monolayer from the lateral direction [224]. The molecular deformation changes the twist of the molecular receptor, and the asymmetric environment facing the aqueous phase can be adjusted. For example, when valine was recognized from the aqueous phase, the D-isomer is preferentially recognized at low surface pressures, while the L-isomer is selected at high surface pressure. In other words, the enathioselective recognition ability toward

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Fig. 4.40 Guest capture through mechanical conformational changes of steroid cyclophane molecular machine

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amino acids can be artificially controlled by applying mechanical pressure to the identical molecular receptor. As a similar example, the control of nucleobase recognition ability was demonstrated upon applying lateral pressures [225]. In the latter example, a receptor molecule called armed cyclononane is used to identify nucleobases. This molecular receptor has multiple carbonyl groups and tertiary amino groups, and when they are assembled as a monolayer at the air–water interface and compressed, the two-dimensional arrangement of these hydrogen-bonding functional groups changes continuously. The binding behavior of thymine and uracil derivatives to this surface was investigated, and found that the difference in binding strength varied with lateral pressure, achieving a 70–80 fold difference in binding between thymine and uracil derivatives under optimized conditions. Thymine and uracil have only a small structural difference of one methyl group and the hydrogen-bonding pattern is the same. Natural nucleic acids (DNA and RNA) cannot distinguish between them. In the above system, molecular recognition ability through receptor tuning by artificial molecular deformation exceeds those of natural nucleic acids. Figure 4.41 shows an overall summary of the modes of molecular recognition [226]. The most basic type of molecular recognition is represented by traditional host molecules such as crown ethers and cyclodextrins, which considers the single most stable structure between host and guest molecules (single stable structure mechanism). Most of the molecular recognition considered in synthetic molecules is in this style. Shinkai and coworkers have added control of molecular recognition by external stimuli [227]. For example, they connected two crown ethers with azobenzene and switched the molecular recognition ability by isomerization (cis and trans) of the azobenzene by light irradiation (switching mechanism). The molecular recognition system by external stimuli and most of the current molecular machines are based on the switching mechanism that transitions between multiple stable states (It can be said that Shinkai’s system is the real origin of molecular machines). On the other hand, what has been proposed here is a tuning mechanism, in which the host and receptor structures are continuously changed to accomplish the intended purpose. This can be thought of as a tuning mechanism, which exploits the innumerable conformational and sequence changes that soft organic molecules can undergo [228]. It can be said that this is an approach that draws out the potential of organic molecules including molecular machines and molecular receptors, which has not been explored so far.

4.11.3 Nanocar (Molecular Car) and Nanocar Race In the pioneering research on molecular machines that was the subject of the Nobel Prize, they did not directly observe the movement of each molecule. Structural changes such as rotating and shuttling of molecular machines were estimated with spectral data of countless molecular machines dissolved in solution. Later, with the advancement of nanotechnology, scanning tunneling microscopy (STM) and other techniques made it possible to directly observe and manipulate the target objects even at atomic and molecular levels. Based on these historical backgrounds, James M.

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Fig. 4.41 Working modes of molecular receptor for molecular recognition: a single stable state mode; b switching mode; c tuning mode

Tour and his colleagues proposed to use molecules like cars (so-called nanocars and molecular cars) and demonstrate their motional behaviors [229]. This was also part of an educational program to introduce children to the wonders of nanotechnology, and was the beginning of history of the nanocar. In other words, there was the NobelPrize-winning concept of molecular machines, and the progress of nanotechnology made it possible to observe molecules and directly demonstrate that molecules themselves could be moved like cars, and that molecules could work as car-like machines.

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Furthermore, visualization of car-like molecular machines in race-like competition attracts world top researchers. It becomes strong motivation for the nanocar race came to be held [230]. The Nobel Prize for molecular machines was awarded in 2016, and the first nanocar race was held in the spring of the following year (2017). This timeline was completely coincidental, but the timing was so perfect that it seemed as if it had been prepared. The operation of the nanocar race is as follows. First, nanocar molecules are vacuum-deposited on a gold substrate that is cooled to cryogenic temperatures in an ultra-high vacuum. A single nanocar is dragged with an atom-sized tip to a specific position (starting point of the race) on the gold surface with well-defined structures. Participant teams compete to drive their nanocar through interaction with the tip along a predetermined course without touching the nanocar. The outline of the rules of the nanocar race can be summarized as follows. (1) (2) (3) (4) (5) (6) (7)

Total course length: 20 nm + curve + 50 nm + curve + 20 nm = total length of approx. 100 nm Time limit: 36 h In the event of an accident, the nanocar to be operated on can be changed. One team will race in one sector on the gold surface. Six hours of course maintenance (cleaning) can be done before the race starts. During the race, it is not possible to change the operating probe. It is prohibited to use the probe to push the nanocar.

In other words, it is a race to see how fast the nanocar can run 100 nm along a predetermined zigzag course using an ultrasmall probe tip placed close to the nanocar molecule in nanometer size without directly touching the nanocar and with applying electrical stimuli to the nanocar. The worldwide six teams participating in the race came up with their own car designs. As for the car design, the French and American teams designed a car with wheels that move as a car runs. In their car design, molecular tires are connected to the main body through a linear unit (axle) that can rotate. It is referred to as a real car design. The rational design of windmills is used for nanocars of the Swiss and German teams. The strategy is to reach the goal as the small car body spins lightly around and around. The Japanese team took a completely different strategy, a butterfly or sneeze worm type strategy, utilizing the deformation of nanocar molecules (Fig. 4.42) [231]. This is an idea that has not been seen in real-world cars or transportation systems, and it takes advantage of the fact that molecules can be freely deformed and move. In technical terms, this strategy uses inelastic activation (molecular vibration excitation by inelastic tunneling) to vibrate the nanocar and propel it forward. The strategy is to experimentally determine which direction the nanocar will move in when stimulated, and then to combine the results to move the nanocar in the desired direction. The aim of the Japanese team is to prove that even ordinary molecules can move like a car if they are well stimulated, without synthesizing complicated car-like molecules. In other words, it was purposed to show the versatility of so many molecules to perform nanocar-like functions for the future development of technology.

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Fig. 4.42 Stimulation of nanocar (Japanese team version) by sharp tip

The remaining team, a joint Australian-American team led by Tour, the founder of nanocars, had an extraordinary obsession with winning. They changed their car design on the day of the race. The nanocar announced in advance was a legitimate car shape, but it was a ruse, and on the day of the race, the team went into the race with a secret weapon, a rocket-shaped nanocar. The race was full of accidents, such as the wheels of the nanocar falling off and the control computer crashing. In the end, the rocket nanocar was crowned champion on the silver surface, while the Swiss team’s windmill was the winner on the gold surface.

4.11.4 Short Perspectives The above is a brief summary of molecular machines and nanocars. Although there is a romance of science in them, they may just be science for fun. Isn’t the molecular machine just a toy? Isn’t nanocar racing just a hobby? Isn’t it research that could be subject to sorting? Are nanocars and molecular machines really useful to the world? These are questions that have been actively asked even before the Nobel Prize was awarded. Recently, some clues to these questions have emerged as mentioned above. For example, it has been reported that turning a molecular motor around in the cell membrane of a cancer cell opens a hole in the cancer cell and kills the tumor. It has

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also been shown that the interfacial environment can be used to manipulate molecular machines with macroscopic scale mechanical actions. Molecular machines can be operative in daily life actions such as our hand motions. With the advancement of such technologies, the practical use of molecular machines may become a reality. If you think about it, there are many naturally occurring molecular machines, called biomolecular machines, working in living organisms. In other words, living things operate by the action of their molecular machines. Biological systems are made up of very rational mechanisms, and are considered to be something that artificial functional systems should learn from. Molecular machines that contribute to medical treatment and molecular machines that mimic the functions of living organisms have ample potential to be useful.

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Chapter 5

Molecular Actuator for Molecular Robots Akira Kakugo

Abstract Recent progress in molecular machines or molecular devices, as exemplified by the 2016 Nobel Prize in Chemistry, has greatly accelerated the development of molecular robots through the fusion of various fields. An actuator is a critical component of molecular robots as an actuator is required to generate motion by the molecular robots by transferring the energy obtained from an internal or external source. To date, various attempts have been undertaken, based on bioengineering or synthetic chemistry, to design and fabricate actuators for molecular robots. In this chapter, the application of various natural and synthetic molecules as the actuator of molecular robots is described. The topics covered in this chapter will be the fabrication of molecular robots using reconstructed linear biomolecular motors, application of peptides as the molecular actuator, cell-sized liposomes containing acting, rotary biomolecular motors, bacterial flagellar motor, F1 FO ATP synthase and their prospects as molecular actuators. Synthetic or supramolecular actuators for molecular robots such as water-soluble gels and inorganic layered crystals will be also discussed.

Recent progress in molecular machines or molecular devices, as exemplified by the 2016 Nobel Prize in Chemistry, has greatly accelerated the development of molecular robots through the fusion of various fields. Like any machine, molecular robots require several molecular parts namely sensor, processor and actuator. Autonomous, and concerted functioning of these parts critical to mandate operation of molecular robots. An actuator is a critical component of molecular robots as an actuator is required to generate motion by the molecular robots by transferring the energy obtained from an internal or external source. To date, various attempts have been undertaken, based on bioengineering or synthetic chemistry, to design and fabricate actuators for molecular robots. In this chapter, application of various natural and synthetic molecules as the actuator of molecular robots is described.

A. Kakugo (B) Department of Chemistry, Hokkaido University Graduate School of Science, Sapporo, Japan e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2022 S. Murata (ed.), Molecular Robotics, https://doi.org/10.1007/978-981-19-3987-7_5

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In Sect. 5.1, the fabrication of molecular robots using reconstructed linear biomolecular motors as actuators and the regulation of group behavior of the robots are discussed. The fusion of bioengineering with DNA technology has enabled precise regulation of the collective motion of the robots. Peptides and proteins have been attracting much attention as components of molecular robots due to their advantages in self-assembly and biological recognition. The application of peptides as the molecular actuator, along with their spatiotemporal regulation using light, is discussed in Sect. 5.2. In recent years, synthetic biology has played a crucial role in the advancement of molecular robotics. Encapsulating cellular components in cell-sized liposomes has been a fascinating approach to unravel the working principles of living beings which in turn has appeared beneficial for designing molecular robots. In Sect. 5.3, the regulation of cell-sized liposomes containing actins has been described, which shows the promise to employ this synthetic system as a driving unit for molecular robots. Rotary biomolecular motors in living organisms are fascinating examples of molecular actuators with the capability of producing rotational motion. The rotary motors have also appeared as potential candidates as the actuators of molecular robots. Section 5.4 describes various features of the bacterial flagellar motor and F1 F0 ATP synthase and their prospects as molecular actuators in synthetic environments. Apart from the approaches based on biological or biomimetic engineering, synthetic or supramolecular chemistry has also been promising in developing actuators for molecular robots. In Sect. 5.5, prospects of water-soluble gels as actuators for any autonomous systems are discussed. Non-essentiality of an external control mechanism makes such gels ideal as the power source for the small flapping movement of molecular robots. Along with the natural molecules and hydrogels, inorganic layered crystals are also attracting attention as molecular actuators, which is described in Sect. 5.6. Despite the ongoing efforts, the synchronous and autonomous operation of various components of molecular robots has been a big challenge. An impending question is about the time when molecular robots will find real-life applications. By virtue of the recent multidisciplinary approaches, intelligence has been successfully introduced in molecular robots. However, the short lifetime of the components of molecular robots, particularly of the actuators, due to mechanical aging and thermal denaturation must be addressed. Improvement is also necessary to prevent degradation or functional inactivation of the molecular actuators. Introduction of autonomy in molecular components would enable molecular robots to autonomously perform a complex series of tasks like the natural machines. Further initiatives are required to address these existing challenges in order to ensure practical and sustainable applications of molecular robots in future. It is expected that, by overcoming these hurdles the much-desired maturity will be achieved in this new field which will permit real-life applications of molecular robots.

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5.1 Construction of Swarm-Type Molecular Robots Driven by Biomolecular Motors Arif Md. Rashedul Kabir and Akira Kakugo A. Md. R. Kabir · A. Kakugo Faculty of Science, Hokkaido University, Sapporo, Japan e-mail: [email protected] A. Kakugo Graduate School of Chemical Sciences and Engineering, Hokkaido University, Sapporo, Japan

5.1.1 Introduction Nature provides elegant examples of molecular machines that work in living organisms with extreme sophistication, high energy efficiency, and broad functional diversity [1]. In recent years, there has been a surge in interest in the fabrication of molecular machines using synthetic molecules or reconstructed biomolecular machineries [2]. With the rapid development of artificial intelligence (AI), the ‘technological singularity’, where AI surpasses human intelligence, has become a hot topic [3]. AI is the subject of research in the field of information science, whereas molecular machines belong to the field of applied science, such as chemistry and engineering. Controlling molecular machines with AI requires a novel approach that must connect both the fields. Amid such a background, a new academic field namely ‘molecular robotics’, has been created with an aim to fabricate molecular robots from molecularsized parts, following the methodologies involved in conventional robotics [4]. By taking the advantages of the ongoing developments and using the natural molecular components as building blocks, our research group at Hokkaido University is aimed at fabricating ‘swarm robots’, which are one of the most popular research subjects in molecular robotics. In the following sections, we would like to introduce our efforts till date.

5.1.2 What is a Swarm? In nature, complex and highly organized structures are created through self-assembly of constituent components [5, 6]. Local interactions among the components play an important role in their self-organization [7]. One of the striking examples of such selforganization is ‘swarms’ created by living organisms such as birds, fish, cells, and bacteria [8]. Even though, in the process of swarm formation, the organisms do not

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have any leader, they are able to read the information received from their surrounding through interactions with their neighbors and consequently change the shape and size of the swarm. The swarm formation by living beings can be ascribed to the following reasons [8]. By swarming, living organisms achieve ‘parallelism’ in the division of tasks, ‘robustness’ in reliable execution of tasks, and ‘flexibility’ in instantaneously responding to any changes in their environment. Since, ‘parallelism’, ‘robustness’ and ‘flexibility’ are guaranteed only at the expense of swarm formation, solitary individuals are unable to make use of these advantages. So far, swarming has been demonstrated in the field of conventional robotics [9], that deals with mechanical robots, with a view to employ a large number of robots for executing tasks in a concurrent manner. In the conventional swarm robotics, controlling the ‘size’ and ‘number’ of the individual robots in a swarm has remained a challenging task. The smaller the size and the larger the number of individuals that make up a swarm robot, the more flexible and scalable the swarm is. So far, the size of a mechanical robot has been reduced to the centimeter scale, and the number of such robots in a swarm was increased up to 1024 units [10]. Decreasing the size and increasing the number of individual robots has been highly desired in swarm robotics, which has been addressed through the development of ‘molecular robots’.

5.1.3 Fabrication of Molecular Robots What is a molecular robot? A molecular robot is defined as a nano- or micrometer-sized system equipped with the three elements necessary for a robot: (i) actuator (power generation unit), (ii) processor (information processing unit), and (iii) sensor (detection unit) [11]. Recent advances in chemistry (e.g., polymer chemistry, organic chemistry, supramolecular chemistry), biotechnology (e.g., genetic engineering, protein engineering), and nanotechnology (e.g., molecular assembly, DNA nanotechnology) have led to the creation of many functional molecular elements required for fabricating molecular robots. By integrating these molecular elements into a microscopic system in a bottom-up manner we have developed a ‘swarming molecular robot’ [12]. Among the many functional molecules, we have selected (i) a “biomolecular motor” as the actuator, (ii) a “DNA computational element” as the processor, and (iii) a “photosensitive molecule” as the sensor for the swarming molecular robot (Fig. 5.1). Features of the three elements of a molecular robot Biomolecular motors, the power source of molecular robots, are one of the smallest molecular machines working in living organisms and are reconstructed by following biotechnological methodologies. Biomolecular motors can convert chemical energy into mechanical work with remarkably high efficiency and specific power compared to man-made machineries [13]. DNA is a storage medium for genetic information with a high level of molecular recognition based on base sequence information.

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Fig. 5.1 Preparation of molecular robots from microtubules and swarming of the robots. a Schematic diagram shows microtubules conjugated with single-strand DNA with complementary sequence. b Microtubules shown in red and green color and gliding on a kinesin-coated substrate form swarm through self-organization due to hybridization of complementary DNA sequences. c Azobenzene, a photo-responsive molecule, is inserted in the DNA to facilitate photo-regulation of hybridization of the complementary sequences, which in turn offers a means to regulate the swarming of microtubules using light. Reproduced with permission from [12]. Scale bar: 20 μm

Nowadays, the chemical synthesis of DNA has become possible which permits using DNA for various applications [14]. These DNA-based computational elements function as processors of molecular robots. Photosensitive molecules can be used as a photo-switch to control functions of the DNA computational elements using light [15]. We decided to incorporate azobenzene derivatives into the DNA computational elements as the photosensitive molecule. This would facilitate switching the computational element to the ‘off’ state under ultraviolet light irradiation and ‘on’ state under visible light irradiation. Therefore, the photosensitive molecules are a potential candidate as visual sensors for molecular robots. Integration of sensor, processor, and actuator The biomolecular motor kinesin and its associated filamentous protein microtubule have been used in fabricating molecular robots. Kinesin is a linear motor that can produce force by consuming energy obtained from hydrolysis of ATP and move along microtubules. The basic unit of the molecular robot is obtained by conjugating microtubules with DNA strands through the copper ion free click reaction (Fig. 5.2). Furthermore, photoresponsiveness was incorporated into the robots by introducing an azobenzene derivative into the DNA. The kinetic properties of the integrated molecular robots were evaluated on a kinesin-coated substrate. DNA-conjugated microtubules exhibited translational motion on a kinesin-coated substrate in the presence of ATP. The basic unit of the molecular robot, i.e., the microtubules could retain ~ 85% of their kinetic properties (~600 nm/sec) [12].

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Fig. 5.2 Preparation of DNA-conjugated microtubules. Molecular robots i.e., the DNAconjugated microtubules were prepared by conjugating single-strand DNA to microtubules through copper-free click reaction (azide alkyne cycloaddition reaction). Azide functional groups were conjugated to the microtubules before the click reaction

5.1.4 Demonstration of Flocking by Molecular Robots Swarming of molecular robots mediated by DNA As described in the introduction, local interaction among the individuals plays an important role in swarm formation. The swarming of molecular robots has been realized by utilizing the molecular recognition ability of the DNA conjugated to microtubules to control their local interactions. By using DNA as an input signal, we have succeeded in creating swarms of a large number of molecular robots that glided on a kinesin-coated substrate [12]. The input DNA strands are designed to mediate attractive interaction among the motile molecular robots, i.e., microtubules. In a swarm, all the robots glided in the same direction which is governed by the polarity of the microtubules. The dissociation of the swarms can be also performed using another input DNA signal. The DNA signal undergoes a strand displacement reaction with the DNA strand that causes the attractive interaction between molecular robots, and eventually results in the dissociation of the swarms into solitary molecular robots. Logical operation by molecular robots Utilizing the ability of DNA to perform a logical operation, different mathematical operations such as YES, AND, OR gate etc. were demonstrated by the molecular robots [12]. In those operations swarming of molecular robots was the output that was regulated by suitable DNA signals as inputs. ‘YES’ logic gate was realized by using an input DNA signal, the presence of which facilitated the swarming of microtubules that were already equipped with DNA signals complementary to the input DNA signal. ‘AND’ logic gate was demonstrated by designing two different input DNA signals, which were partially complementary to the two DNA signals carried by two groups of molecular robots. Swarming of the molecular robots was

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observed as the output only when both the input DNA signals were present. ‘OR’ logic gate was operated by simultaneous operation of two swarming groups. In each group, two types of molecular robots were equipped with two different DNA signals. The two types of robots exhibited swarming independently when another input DNA signal partially complementary to the DNA carried by the robots was available. Both the swarm groups were operated in a concerted fashion when both the input DNA signals were available in the same swarm system [12]. Controlling the morphology of swarms The morphology of the swarms of molecular robots can be varied not only by tuning the local interactions among the robots using DNA, but also by tuning the length and stiffness of the microtubules that form the framework of the basic units. For example, the molecular robots fabricated from microtubules with bending stiffness of 60 × 10–24 Nm2 form bundle-shaped swarms and exhibit translational motion when the swarms were propelled by kinesins. On the other hand, when the stiffness of microtubules is reduced, the robots form ring-shaped swarms which exhibited rotational motion [12]. The stiffness of the microtubules was controlled by changing the polymerization condition of the tubulins. Such morphological changes in swarms were found to be related to the path-persistent-length of the molecular robots (Fig. 5.3).

Fig. 5.3 Regulating the morphology of swarms and dissociation of swarms. a Morphology of the swarms of molecular robots can be tuned by changing the mechanical properties, e.g., rigidity of the individual robots (microtubules). Relatively rigid microtubules form bundles whereas flexible microtubules form ring-shaped swarms. Bundle and ring-shaped swarms exhibit translational and rotations motions respectively. Scale bar: 20 μm. Reproduced with permission from [12]

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Variations in the morphology of the swarm robots will widen their applications in nanotechnology. Orthogonal swarming of molecular robots The highly specific molecular recognition capability of DNA allows to transmit the input signals to specific target molecules. By taking this advantage of DNA, it is possible to control the swarm formation by molecular robots independently even in an ensemble of robots. For example, we can design two sets of DNA input signals: one for rigid molecular robots and the other one for flexible robots. The required information for directing the robots to swarm is encoded in these two sets of DNA. The first set of DNA information will allow the swarming of the rigid robots in the form of bundles which exhibit translational motion. The second set of DNA is designed for less rigid molecular robots which will allow swarming of the robots in the form of ring-shaped assembly and result in their rotational motion. The two sets of DNAs can be designed to eliminate any possible mutual interaction which permits the formation of only translating swarms or only rotating swarms, or both translating and rotating swarms simultaneously and independently without any interaction between the bundles or ring-shaped swarms. Photo-regulated ON–OFF switching of swarming By incorporating a photosensitive molecule (azobenzene) into the DNA computation element, the swarming of the molecular robots or dissociation of the swarms into a solitary state can be controlled by irradiating the robots in a non-invasive manner with light. When irradiated with ultraviolet light (λ = 365 nm), the DNA computation element is in the OFF state due to trans-to-cis isomerization of azobenzene and the swarming of the robots is restricted. When irradiated with visible light (λ = 480 nm), azobenzene undergoes cis-to-trans isomerization and consequently, the DNA computation element is turned on and swarming of the robots is allowed. In addition to swarm formation and dissociation under suitable photo-irradiation conditions, it is possible to control the swarming behavior of robots such as translation and rotation by adjusting the physical properties of the molecular robot as described above (Fig. 5.4).

5.1.5 Conclusion By integrating biomolecular motors as actuators, DNA as computational elements, and photosensitive molecules as sensors, we have been dedicated to find solutions to the problems related to the ‘size’ and ‘number’ of individual units in the development of swarm robots. So far, we have succeeded in scaling down the size of robots from centimeter to nanometer scale and increasing the number from thousands to millions. Molecular robots capable of processing, retaining, and transmitting such information are expected to have a variety of applications [16–20]. For example, micro-sized ‘artificial muscles’, ‘imaging elements’ that can freely draw images by transforming

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Fig. 5.4 Photo-regulation of the swarming of molecular robots prepared from microtubules. The scheme shows cis/trans isomerization of azobenzene under ultraviolet (UV) and visible light. The cis form of azobenzene prevents hybridization of complementary DNA sequences and swarming of microtubules, whereas the trans-form permits hybridization of DNA and microtubule swarming. Fluorescence microscopy images show light-induced reversible regulation of swarming of rigid microtubules. Scale bar: 20 μm. Reproduced with permission from [12]

the swarm of molecular robots in response to chemical or physical stimuli, ‘genetic diagnosis kits’ that can visually display detected genetic information by drawing images by molecular robots, and ‘micromachines’ that can assemble nano-parts by using molecular robots. In addition, there are many other potential applications, such as ‘microreactors’ for assembling nano-parts and chemical plants. In this article, we have introduced a system that is able to select and integrate a certain combination of functional molecules from a number of choices. In the future, we expect to see the development of molecular robots with more complex functions in a completely new framework.

5.2 Peptide Actuator Kazunori Matsuura K. Matsuura Tottori University, Tottori, Japan e-mail: [email protected] The formation and dissociation of self-assembling peptide nanofibers can be dynamically controlled by a β-sheet structure responding to stimuli such as light, pH, and redox. Among these, light can induce structural changes in peptides in a relatively short time and can control the self-assembly timing of peptide nanofibers.

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Fig. 5.5 a Photo-induced peptide nanofiber growth system. b Formation of peptide nanofibers by photo-irradiation on a dT20 -immobilized glass substrate

We have developed a photoresponsive peptide-DNA-conjugate and succeeded in the spatiotemporal control of a photo-induced peptide nanofiber formation [21]. The photoresponsive peptide-DNA-conjugate 1 consists of a β-sheet-forming peptide (FKFEFKFE) and a single-stranded DNA (dA20 ), which are linked by a photo-cleavable amino acid (Fig. 5.5a). The DNA moiety not only suppresses the peptide self-assembly through electrostatic repulsion but also plays the role of addressing the peptide self-assembly position using DNA hybridization. Although conjugate 1 did not form nanofibers in an aqueous solution, the conjugate was cleaved by light irradiation at 365 nm and the released FKFEFKFE peptide self-assembled to form nanofibers. When conjugate 1 was hybridized on a glass substrate on which the complementary dT20 was immobilized, micrometer-sized fibril structures were formed by photo-irradiation (Fig. 5.5b). In contrast, conjugate 1 hardly formed fibril structures on a dA20 -immobilized glass substrate even after photo-irradiation. Then, we developed conjugate 2, which is photo-cleaved much faster than conjugate 1, and found that phase-separated giant liposomes asymmetrically modified with conjugate 2 were dramatically enhanced during the translational motion by photoirradiation (Fig. 5.6) [22]. The translational motion acceleration of the conjugate 2-modified giant liposomes using photo-irradiation was about six times larger than that of unmodified giant liposomes. A possible driving force for the propulsion of the conjugate 2-modified giant liposomes is the Marangoni effect caused by the surface tension gradient between the nanofiber-forming side and the other side of a liposome. Such translational motion of a liposome due to nanofiber growth is a phenomenon similar to the translational motion of Listeria and Shigella propelled by the actin nanofiber (actin comet tail) formation. Recently, we succeeded in constructing DNA microspheres modified with the photoresponsive peptide-DNA-conjugate, which shows a negative phototaxis (directional movement away from the light source) [23]. These findings serve as a design guideline for photo-controlled actuators for molecular robots.

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Fig. 5.6 Propulsion of giant liposomes driven by photo-induced peptide nanofiber growth

5.3 Photo-Regulation of Actin-Encapsulating Cell-Sized Giant Liposomes Kingo Takiguchi K. Takiguchi Nagoya University, Nagoya, Japan e-mail: [email protected] In order to understand the generating mechanism for various and complex movements exhibited by living cells, a bottom-up constructive approach that mimics and reproduces these movements is indispensable. As a first step, many attempts have been made to construct artificial cell models that deform and move using cell-sized liposomes with reconstructed cytoskeletons inside [24]. As a result, the number of reports of the successful reproduction of cell-like movement is increasing [25, 26]. Most of these reports used microtubules (MTs) that are stabilized with an inhibitor of depolymerization and kinesin, which is a molecular motor easily prepared. However, not only MT but also the actin cytoskeleton plays an important role in the morphogenesis and movement of cells. In addition, the cytoskeleton generates force not only by cooperating with its associating molecular motors but also by polymerizing/depolymerizing itself to cause elongation/shortening. Therefore, the utilization of actin or that of a cytoskeletal system without stabilization, i.e., one which is capable of polymerization/depolymerization, are also promising trials. An important issue to be solved in the trial will be the observation of repetitive movements similar to those shown by living cells [27], since so far almost all of the successful cases were irreversible reactions that could perform a series of movements only once or a movement that continued the same actions for long hours [25, 26]. Here, we report a repetitive deformation realized in liposomes encapsulating high concentrations of the actin filament (F-actin). In recent years, it has become possible

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Fig. 5.7 Repetitive deformation of spindle-shaped liposome (phase contrast images and the schematic diagram). ◼

to prepare liposomes at high frequency and reproducibility by transferring waterin-oil droplets containing the target substance through the oil–water interface [27, 28], enabling us to encapsulate actin into the liposomes even at a high concentration (100 μM or more) comparable to that in the cytoplasm. At high concentrations of more than 50 μM, F-actins aligned due to nematic liquid crystal formation, resulting in the deformation of liposomes into a spindle shape (Fig. 5.7, the leftmost phase contrast image in the upper row) [29]. A crucial point of this liposome deformation is that neither a system for continuously supplying ATP to drive the molecular motor nor an additional factor for regulating the arrangement of the cytoskeleton is required. It has been known that fluorescently labeled F-actin severs when irradiated with strong excitation light [29]. To investigate the effect of changing the length of the encapsulated F-actins on the liposome morphology, the deformed liposome was strongly irradiated with the excitation light of a fluorescence microscope. As a result, the central part of the spindle shape changed to a more spherical shape (Fig. 5.7, the second phase contrast image from the left in the upper row, also see Fig. 5.8), and the membrane protrusions extended from both its ends (Fig. 5.8, yellow arrows) [29]. After the irradiation was stopped, the liposome returned to the spindle shape while the protrusions were shortened (Fig. 5.7, the third phase contrast image from the left in the upper row), attributed to the re-elongation of the F-actin by spontaneous annealing (Fig. 5.7, lower right). This deformation could be repeated by turning on and off the light irradiation (Fig. 5.7). It should be noted that, regarding the protrusion, if its middle part occasionally adhered to the substrate during shortening, it did not shorten toward the liposome body, but rather pulled the body (Fig. 5.8), indicating that the force generated was sufficient to move a cell-sized object. These results suggest that the utilization of filamentous polymers with variable lengths, in addition to the DNA-origami technique and/or utilization of molecular motors, will be a promising approach as a drive unit to be mounted on molecular robots.

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Fig. 5.8 The body part of spindle-shaped liposome that moves by being pulled by shortening of the protrusion. Phase contrast (0 and 23–243 s) and fluorescence images (2–21 and 269 s), and schematic diagram of the movement (upper right) are shown. The “x” in the schematic diagram indicates the estimated attachment point between the protrusion and the substrate surface. ◼

5.4 Rotary Molecular Motors Shoichi Toyabe S. Toyabe Tohoku University, Sendai, Japan e-mail: [email protected] The fact that molecules are rotating in biological cells is one of the most astonishing facts in biology. These rotary molecular motors include the bacterial flagellar motor (BFM) and the F1 and FO of ATP synthase. The BFM is an electric motor with a dimension of around 50 nm. It rotates using the flow of ions such as protons and sodium ions through the motor. The ion flow is driven by the electrochemical potential difference across the cell membrane, where the BFM is embedded. The rotation rate depends on the species and reaches 1,700 Hz for some bacteria [30]. As well as such high-speed rotation, the rotational direction can be inverted by the biochemical “program” inside the cell. Furthermore, the motor implements load-dependent automatic transmission [31]. The motor has multiple stator units, which bind to and dissociate from the rotor dynamically. In a high-viscosity environment, more stator units bind to the motor and generate higher torque. In a low-viscosity environment, stators dissociate, probably, reducing futile ion flow. The rotation mechanism of the BFM remains to be elucidated due to its very complicated and huge system. Recent structural studies implied that the stator

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itself is a rotational motor [32]. It was proposed that the stator rotation drives the rotor rotation via gear-type coupling. The F1 (or F1 -ATPase) is the smallest rotary molecular motor found in nature with a dimension smaller than 10 nm (Fig. 5.9c). The F1 uses ATP as the fuel and rotates its central γ-shaft by using the free-energy difference of the ATP hydrolysis to ADP and phosphate (Pi ). The γ-shaft rotates 120 deg per ATP consumption. The rotation mechanism of F1 has been almost elucidated [33]. The β-subunit of the stator has a hinge-like structure and bends when it binds ATP, and the three βs adopt different chemical states (ATP-bound, ADP-phosphate bound, and empty). An ATP binding to the empty β triggers a 120° shift in the chemical state, which also rotates the bending state of β by 120°, thus drives the rotation of the γ-shaft by 120°. The F1 motor rotates in the cells of virtually all living forms on earth. A question that naturally arises is why they need to rotate in the cells. Actually, F1 does not exist alone but forms a complex with another electric motor, the Fo , in the cell (Fig. 5.9d). The Fo is embedded in the membrane and rotates with proton flow through it. Since

Fig. 5.9 Bacterial flagellar motor (a, b) and F1 -motor (c, d). a Bacterial flagellar motor. b The stator units bind to and dissociate from the rotor dynamically depending on the load and ion-flow driving force. c Isolated F1 -motor (α3 β3 γ) in the side view (left) and top view (right). d ATP synthase. Panels a and b were reproduced from Ref. [34] without change

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the rotor of Fo is connected to the γ shaft of F1 , the γ shaft is rotated forcedly. However, the rotation direction is opposite to that of the ATP hydrolysis by isolated F1 . Then, F1 synthesizes ATP from ADP and Pi instead of hydrolyzing ATP. Most of the ATP required for the biological activities is synthesized by this reversible mechanochemical energy conversion mechanism. Experiments have implied that this mechanism is highly efficient, and the internal dissipation due to friction is virtually negligible [35]. Macroscopic motors lose efficiency due to friction when they move fast. Frictional heat is wasted heat that is dissipated through microscopic degrees of freedom. Molecular motors, however, are themselves microscopic and may “see and control” the heat flow on the k B T energy scale to achieve high efficiency. The elaborate mechanisms of biological molecular machines are awe-inspiring. There seems to be much more to learn from molecular machines for the development of molecular robotics.

5.5 BZ Gel Actuators Yusuke Hara Y. Hara National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan e-mail: [email protected] Living organisms, including humans, directly and autonomously convert chemical energy obtained from the decomposition of foods into mechanical energy. Thus, organisms are extremely energy-efficient systems. BZ gel actuators, which are composed of water-soluble gels, are being investigated and developed with an aim of artificially creating autonomous systems [36–40] (Fig. 5.10). These water-soluble gels contain large volumes of water in their polymer networks, and the water content inside the gel changes in response to external stimuli or chemical reactions. The water content, which determines the gel volume, depends on the molecular design of the polymer main chain that composes the gel. By selecting the molecular design of the polymer main chain, the gels respond to desired external stimuli, i.e., changes in temperature, pH, light conditions, or electric field, etc., and chemical reactions that occur inside the gel. The molecular design of the BZ gel Fig. 5.10 BZ gel actuator

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actuator was determined to respond to the Belousov–Zhabotinsky (BZ) reaction that occur inside the gel. The BZ reaction, which is a self-oscillating chemical reaction, can simply occur by mixing an oxidizing agent such as sodium bromate, an organic acid such as malonic acid, and a strong acid, such as nitric acid or sulfuric acid, with a metal catalyst, such as tris(2,2’-bipyridyl)ruthenium. When the BZ reaction occurs inside the BZ gel actuators, the valence of the Ru catalyst covalently bonded to the polymer main chain changes periodically, as does the hydrophilicity of the gels at the same time. This occurs because the hydrophilicity of the polymer main chain that composes the gel depends on the Ru catalyst moiety. Therefore, the swelling– deswelling self-oscillation of the BZ gel actuators synchronizes with the period of the BZ reaction. The self-oscillating reaction can be confirmed by the periodic color change of the oxidized and reduced Ru catalyst moiety in the polymer main chain composing the BZ gel actuator. The BZ gel actuators do not require an external control device or power supply. The driving speed of the BZ gel actuator can be controlled by varying the reaction temperature and concentrations of the BZ substrates (malonic acid and sodium bromate). At present, the maximum confirmed driving speed in our study was approximately 2 Hz. In addition, because the BZ gel actuator has scale universality, similar to that of muscles, swelling–deswelling behavior can be observed even with gels of 1 mm3 or less. Our previous studies using AFM and QCM-D have confirmed the self-oscillating behavior of single BZ polymer chains [41, 42]. By using the scale universality of the BZ gel actuators, a small BZ gel pump for transporting liquid in the microchannels had been developed. The BZ gel pump for microchannels is expected to be applied to analytical systems that can be used anywhere. To synthesize small gel actuators without using templates, we are now developing the novel synthesis techniques using a laser beam (Fig. 5.11). The merit of novel synthetic techniques is that gel actuators can be directly synthesized inside microchannels without using templates. Moreover, since they do not require a power supply or external control devices, self-oscillating behaviors of small and light BZ gel actuators are expected to be used as power sources for small flapping robots like a butterfly (Fig. 5.11). Fig. 5.11 Novel synthesis technique using laser beam

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5.6 Inorganic Crystal Actuator Nobuyoshi Miyamoto N. Miyamoto Fukuoka Institute of Technology, 3-30-1, Wajirohigashi, Higashiku, Fukuoka, Japan e-mail: [email protected] Soft and wet materials such as motor proteins (tubulin/kinesin etc.) and stimuliresponsive hydrogels are usually considered as an actuator unit for molecular robots. However, optimization of response speed, torque, anisotropy and durability are still challenging issues. In this short column, an inorganic layered crystal is demonstrated as a new-type actuator unit, which would be a breakthrough for designing molecular robots in the future. Inorganic layered crystals such as mica and graphite are composed of stacked layers with a thickness of 1 nm. Various kinds of layered crystals have been synthesized and used to fabricate functional nanomaterials [43]. Recently, a layered iron titanate was found to show an interesting behavior [44]. When we put the plate-like crystals (Fig. 5.12a) in water and add dimethylaminoethanol (DMAE), the plate was one-dimensionally swollen and transformed into a long accordion-like shape in less than a second (Fig. 5.12a–d). Then, after adding hydrochloric acid, the accordion shrank back to the plate shape (Fig. 5.13e–g). Thus, this actuation is reversibly controllable. By X-ray scattering technique, the layer-layer distance was estimated as 1 nm in the plate crystal, while it increased to more than 90 nm after the addition of DMAE. Since the molecular size of DMAE is less than 1 nm, the large expansion of the layer-layer distance is explained by the accommodation of water molecules with DMAE. Usually, the large expansion of the layer-layer distance results in exfoliation and dispersion of the layered structure to single-layers nanosheets and they never go back to the plate crystal again. Thus, the present system is a very rare case. The fast, large, anisotropic shape change of the inorganic crystal would be suitable for application as actuator units.

212 Fig. 5.12 a SEM image of the layered iron titanate and schematic drawing of its swelling. The scale bar is 10 μm (Reprinted from Nature Commun. 2013 [44])

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Fig. 5.13 The optical microscopic observation of the swelling and deswelling of the layered ion titanate crystal. The scale bar is 100 μm (Reprinted from Nature Commun. 2013 [44])

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.

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Chapter 6

Molecular Material for Molecular Robots Akinori Kuzuya

Abstract Various molecular devices and materials that may extend the functionality of molecular robots and broaden the field of application such as DNA hydrogels utilizing G-quadruplexes, DNA hydrogels with branched DNA duplexes, DNA hydrogels that utilize physical entanglement and hybridization will be introduced. We also cover photoresponsive artificial DNA such as CNVK and CNVD, particularly, in addition to photo-isomerizing azobenzene residues, and their applications in gene regulation. Orthogonal artificial nucleic acids called XNA are also discussed. We also describe synthetic polymer material that is useful in DNA computing field including a comb-type cationic copolymer which accelerates strand exchange reactions. By fully utilizing such functional materials in combination, the construction of highly sophisticated molecular systems is strongly feasible.

In this chapter, various molecular devices and materials that may extend the functionality of molecular robots and broaden the field of application are introduced. Sections 6.1 and 6.2 are for intelligent hydrogel materials containing DNA. Liquidphase large-scale DNA synthesis using polyethylene glycol (PEG) substrates enables bulk-scale preparation of intelligent and biodegradable hydrogels (Sect. 6.1). An aqueous solution of DNA-PEG conjugates with quadruplex-forming short DNA segments rapidly turns into stable and stiff hydrogels when an appropriate alkaline metal ion is added. Hydrogels utilizing G-quadruplexes, particularly, may be useful for biomedical applications since the trigger of gelation is sodium ion, which is abundantly contained in various body related fluids. Section 6.2 explains more typical DNA hydrogels. Branched DNA duplexes such as three-way or four-way junctions give both chemical (covalently crosslinked) and physical (non-covalently crosslinked) hydrogels when assembled into a 3D DNA network. Combinations between synthetic polymers and DNA crosslinking points realize “intelligent” hydrogels. The section also covers quite a unique type of DNA hydrogels that utilize physical entanglement and hybridization. Very long single-stranded DNA produced by A. Kuzuya (B) Department of Chemistry and Materials Engineering, Kansai University, Osaka, Japan e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2022 S. Murata (ed.), Molecular Robotics, https://doi.org/10.1007/978-981-19-3987-7_6

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the rolling circle amplification (RCA) technique realizes such hybrid hydrogels as well as a system that shows metabolism-like dynamism in microfluidics. Sections 6.3 and 6.4, in contrast, are for photoresponsive artificial DNA. Efficient DNA/RNA photo-crosslinkers (CNV K and CNV D, particularly), in addition to photoisomerizing azobenzene residues, and their applications in gene regulation are first concisely explained in Sect. 6.3. Section 6.4 then focuses on the application of photoresponsive DNA in the DNA computing field. In Sect. 6.5, orthogonal artificial nucleic acids are discussed. Artificial nucleic acids, in which the main chain of natural DNA or RNA, particularly D-ribose or D-deoxyribose rings, are called XNA (for xeno-nucleic acid). Among these XNAs, PNAs (peptide nucleic acids), which are introduced amide bonds in the main chain, and TNAs (threoninol nucleic acids), which use a threoninol in place of a deoxyribose ring, exhibit orthogonality: they cannot form double strands with each other according to the chirality of the methyl group introduced in the main chain. This is equivalent to the relationship between natural DNA consisting of D-deoxyribose and its mirror image L-DNA consisting of L-deoxyribose. In the case of the same chirality, double-strand formation is possible between DNA, PNA, and TNA, and thus is expected to be utilized in various combinations. For example, if a seesaw gate, a very popular system used in DNA computing, is constructed using only D-TNA (which is orthogonal with natural DNA), the reaction is not inhibited even when an inhibitor made of DNA coexists. The use of universal SNA (serinol nucleic acid), which can bind to molecules of either chirality, is also briefly introduced in this section. Apart from the above sections dealing with DNA-related materials, Sect. 6.6 features synthetic polymer material that is useful in the DNA computing field. A comb-type cationic copolymer, poly(L-lysine)-graft-dextran (PLL-g-DEX), is found to accelerate strand exchange reaction between complementary oligonucleotides and acts like a molecular chaperone. It can enhance DNAzyme reaction by accelerating the turnover process. It is also able to boost toehold-mediated strand displacement reaction from the regime of minutes into seconds. By fully utilizing such functional materials in combination, the construction of highly sophisticated molecular systems is strongly feasible.

6.1 Large-Scale Synthesis of DNA and Its Application to Stimuli Responsive Gels Akinori Kuzuya A. Kuzuya Department of Chemistry and Materials Engineering, Kansai University, Osaka, Japan e-mail: [email protected]

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DNA hydrogels generally use complementary hybridization of DNA as a “physical” (i.e., reversible) crosslinking point to construct three-dimensional networks of the main components of the hydrogel (branched DNA duplexes or synthetic polymers). Most of these studies utilize conventional phosphoramidite solid-phase chemistry, which only produces µg- to mg-scale DNA, to synthesize DNA, and thus only miniature (mm- to cm-scale) gels are obtainable. The “liquid-phase large-scale DNA synthesis using PEG substrate” developed by Bonora et al. [1], on the other hand, can be used to prepare bulk-sized DNA hydrogels in combination with special higherorder structures other than DNA duplexes. A typical example is the “G-quadruplex gel” that utilizes a G-quartet structure consisting of four guanine base molecules (Fig. 6.1) [2]. While the original “liquid-phase large-scale DNA synthesis” uses monomethoxyprotected PEG (mPEG) as the substrate, unmodified PEG with OH groups at both ends is used for the preparation of DNA-PEG-DNA triblock macromonomer for Gquadruplex gels. For the DNA portions, only three or four deoxyguanosine residues each are required. In the presence of Na+ or K+ , four guanine bases can form a cyclic complex called G-quartet by Hoogsteen base-pairing. Especially in sequences with three or more consecutive guanine bases, the four strands form a very stable quadruplex. When Na+ (100–200 mM) or K+ (~50 mM) is added to an aqueous solution of dG4 -PEG-dG4 at a concentration of more than 5 wt%, accordingly, the solution instantly solidifies, and a very stiff hydrogel is obtained. The properties of the resulting hydrogels can be tuned by changing the structure of the PEG: the hydrogels obtained with four-arm branched PEG do not melt even when it is heated above 70 °C. This high stability is thought to be due to the fact that the G-quadruplex itself is more stable than DNA double strands, and also to the fact that the concentration of DNA segments in the macromonomer solution is between 10 to 30 mm, which is extremely high compared to those obtainable with chemically synthesized DNA. The most notable property of this material is that Na+ can be used as a trigger for gelation. Human body fluids usually contain Na+ , just as saline contains 140 mm Na+ . Not only phosphate-buffered saline (PBS), serum, artificial sweat, tear, saliva, or even cell-culture media can trigger gelation of hydrogels stable even above 37 °C. It is expected to be applied to the ultimate body-friendly implantable material consisting only of DNA and PEG. G-quadruplex gels prepared with cell-culture media are expected to be an efficient substrate for regenerative medicine [3]. Like alginate gels often introduced as artificial salmon eggs, gel beads can be easily prepared by dropping a polymer solution into a metal ion solution [4]. When such gel beads are added to an aqueous solution containing DNase, the beads disappear only when the enzyme is present, indicating that the G-quadruplex gel is biodegradable. When several nucleotides are additionally coupled after the consecutive dG residues, selective dissolution of the hydrogels triggered by toehold-mediated strand exchange in the presence of a fully matched complementary DNA strand. The process is completely sequence selective in that only one mismatch in the DNA strand results in failure of dissolution, showing the intelligence of G-quadruplex gels. It has also been confirmed that G-quadruplex gels have inherent self-healing properties based on the reversibility of the G-quadruplex. It has been found that meso-scale gels with

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Fig. 6.1 G-quadruplex gels prepared from dG4 -PEG-dG4 conjugates [2]

a particle size of 100 nm can be produced by adding Na+ to dilute polymer solutions, and microgel beads with a particle size of µm can be produced by using a microfluidic device. Both of them may be good candidates for future DDS carriers. In addition to G-quadruplexes, there is another DNA quadruplex that is often studied in the field of nucleic acid chemistry, i-motif. This is a special structure

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found in C-contiguous sequences, consisting of C–C+ unnatural base pairs between cytosine and protonated cytosine. As expected from the structure, the i-motif shows a sharp pH dependence. It is only formed in the weakly acidic pH range of 4–5, where exactly half the amount of cytosine can protonate. Such i-motif can also be applied to prepare pH-responsive hydrogel (i-motif gel) by combining it with the large-scale DNA synthesis using PEG (Fig. 6.2) [5]. For example, dC5 -PEG-dC5 , in which five dC residues are coupled using PEG as the substrate, can be synthesized in large quantities as in the case of dG4 -PEG-dG4 described above. If an aqueous solution of dC5 -PEG-dC5 around 10 wt% is prepared, this solution turns into gel only in the pH range of 3.5–5.5 at room temperature. The gelation is completely reversible, and by adding a branched structure to the PEG, properties such as mechanical strength and gelation pH range can be further enhanced. The pH range in which the i-motif gel is formed coincides with that of soft drinks and healthy human skin. Applications of i-motif gel in related industrial fields are being explored.

Fig. 6.2 The pH-responsive i-motif gel formed with dC5 -PEG-dC5 complex [5]

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Very recently, the one-pot system for liquid-phase oligonucleotide synthesis, which enables dramatic simplification of the procedure of the large-scale synthesis of DNA-PEG conjugates, has been developed [6]. The process was successfully applied to prepare 4-arm PEG-T20 and 4-arm PEG-A20 , which give reasonably stable hydrogels based on DNA duplex formation. DNA computing based on toehold-mediated strand exchange technique requires relatively long DNA strands. This simplified synthetic procedure together with DNA-PEG hydrogels would be catalysts to expand the abundant achievements in the field of DNA computing and DNA nanotechnology to the world of bulk materials.

6.2 DNA Hydrogel and Its Applications Shogo Hamada S. Hamada Department of Robotics, Tohoku University, Sendai, Japan e-mail: [email protected] DNA hydrogel is a material comprised of a crosslinked DNA network in solution. The material can be fabricated at ranges of scales from micro to macro. Notably, the material allows us to directly manipulate and interact at the bulk scale while retaining its biomolecular characteristics, providing unique applications in materials science, bioengineering, architecture, and robotics.

6.2.1 Design and Fabrication On a scale much longer than the persistence length,1 DNA can no longer be approximated as a “rigid rod” and thus regarded as a polymer chain. By following the general classification of hydrogels in polymer chemistry, DNA hydrogels can also be classified into two categories, chemical and physical gels, based on the driving forces of crosslinking. Chemical gels are defined by hydrogels with covalently bonded crosslinks. Typical DNA hydrogel achieves such network formation by a motif-based design. Branched DNA motifs, such as X- and Y-shaped motifs with sticky ends, are first annealed and then ligated to form a covalently-linked network (Fig. 6.3a) [7]. By incorporating photocrosslinking molecules to the sticky ends, repeated gelation and solation of DNA hydrogels triggered by photoirradiation have been achieved [8]. A recent example also demonstrated a new route to fabricate large-scale chemical DNA hydrogels directly from biomass DNA via aza-Michael addition-based crosslinking [9]. 1

Approximately 50 nm in case of ds-DNA.

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Fig. 6.3 DNA hydrogel. a Chemical gel. X-shaped motifs are hybridized and then ligated to form a network. b Physical gel. DNA synthesis by Φ29 DNA polymerase is initiated from a seed structure consisting of a circular template DNA and a short primer 1. Additional primers 2 and 3 are mixed during the reaction to further amplify the synthesis of DNA

On the other hand, non-covalently crosslinked hydrogels are categorized as physical gels. In the case of pristine DNA hydrogels, crosslinks of physical gels are formed by hydrogen bonding and physical entanglement of DNA. For example, physical gels are fabricated by using Y-shaped DNA motifs similar to the ones shown in Fig. 6.1a and linker DNA [10]. Instead of covalent bonding achieved by ligation, hybridization between sticky ends is utilized to form a network. Moreover, hybrid physical gels made of DNA and other types of polymers have been reported [11]. DNA is also utilized as an expandable crosslinker of polyacrylamide hydrogels, which allowed a sequence-directed shape-change via swelling [12]. Another strategy to create physical DNA hydrogels utilizes an enzymatic synthesis of DNA [13, 14]. The gelation occurs by a combination of physical entanglement and hybridization. One of the DNA polymerases, phi29 DNA polymerase, is especially known for its high processivity2 and strong strand displacement activity. Phi29 DNA polymerase synthesizes long single-stranded DNA with repeated sequences by using a circular template and a primer. This technique is known as Rolling Circle Amplification (RCA). An additional amplification process using secondary and tertiary primers (multi-primed chain amplification, or MCA) allowed DNA to form a hydrogel. By using this method, facile and cost-effective fabrication of DNA hydrogels with arbitrary repeated sequences is achieved.

6.2.2 Applications The uniqueness of DNA hydrogel is retaining its features as a biomolecule while forming bulk-scale network structures. One of the applications that can utilize such characteristics is cell-free protein synthesis [15]. DNA containing gene sequences, such as plasmids, were incorporated into hydrogels via ligation. When protein synthesis was performed from the hydrogel, target proteins were produced 300 times more efficiently than in free solution [16]. Several factors are considered contributing 2

Processivity is defined by an enzyme’s ability to continuously catalyze reactions before releasing its substrate. In the case of DNA polymerase, processivity is characterized by the number of nucleotides added to its product per association event of an enzyme with the template strand.

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to this enhancement, such as protection and an increased concentration of plasmids by incorporation into the network, and an increased transcription efficiency due to a lower diffusion coefficient. For instance, by using this protein-producing DNA hydrogel as a medium, a system that can realize one-to-one correspondence between genotype and phenotype was realized. Target proteins were synthesized and trapped inside a uniformly sized DNA microgel created in water-in-oil droplets [17]. Furthermore, envisioning the use of DNA hydrogels in further applications, for example, our team has reported the combination of protein-producing physical DNA hydrogel with ceramics [18, 19]. The material provided a spatial control of protein expression on the architectural components, allowing potential future use of the material in architecture and bioengineering applications. In addition, recent advancements in 3-D printing also allowed the formation of DNA hydrogels into designated shapes in situ [20]. A combination of Y-shaped motifs and light-activated DNA linkers achieved photolithographic shape control of DNA hydrogels [21]. Finally, DNA hydrogels could be used as a key material for future molecular robots. As a first step towards this goal, our team has successfully created dynamic DNA-based materials and constructed machines powered by artificial metabolism [22]. RCA-based physical hydrogel fabrication method was utilized as a basis of the design. By combining DNA synthesis/digestion and dissipative assembly process in a microfluidic device, the system achieved regeneration of meso-scale DNA materials in a spatiotemporal manner, mimicking the concept of metabolism in a simplified fashion. A new class of machines that can locomote and can even perform racing against each other was implemented by the material. DNA hydrogel has both remarkable characteristics as the macroscale structures and the nanoscale biofunctionality with sequence-based design. As shown in the examples mentioned above, utilizing and linking both characteristics could lead to further applications. In the future, DNA hydrogels may play a role as an interface across scales that can connect nanoscale molecular interactions and our living scales in artificial systems, especially in the slime-like molecular robots with life-like capabilities.

6.3 DNA/RNA Photo-Cross-Linker Kenzo Fujimoto K. Fujimoto School of Advanced Science and Technology, Japan Advanced Institute of Science and Technology, Ishikawa, Japan e-mail: [email protected] DNA is composed of four bases, adenine (A), thymine (T), guanine (G), and cytosine (C), and forms a double-stranded structure via Watson–Crick basepairing [23]. Oligodeoxynucleotide (ODN) sequence design enables the construction of nanoscale-controlled DNA structures [24] and molecular calculations [25].

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Currently, DNA strands of up to 100 bases can be chemically synthesized, and arbitrary sequences can be easily obtained. Research into functional artificial nucleic acids with functions that do not exist in nature is actively being conducted. A number of artificial nucleic acids have been reported to add new functions to DNA and are useful for applications aimed at manipulation, detection, and control of nucleic acids. In particular, photoresponsive artificial nucleic acids whose function can be controlled via photoirradiation allow temporospatial manipulation of their function through control of the timing and position of photoirradiation. Therefore, it is possible to impart optical response capability to a molecular robot and express it at the desired time and location. In this chapter, we will introduce some of the photoresponsive artificial nucleic acids reported to date and describe the method currently being developed by Fujimoto Laboratory for their regulated activation. Psoralen is a well-studied photoresponsive molecule that can be photo-crosslinked with a pyrimidine base via [2 + 2] photocyclization by photoirradiation (Fig. 6.4) [26]. With the development of DNA synthesis technology, it has become possible to introduce psoralen derivatives at arbitrary positions in a DNA strand, and their potential for various applications has been considered. Since the photo-crosslinking ability of DNA or RNA can be controlled via photostimulation, it is also used for functionalization as an antisense nucleic acid [27] and to add stability to DNA nanostructures [28]. Azobenzene displays the unique ability to change from trans to cis-form upon photoirradiation at a wavelength of 300–400 nm, and from cis to trans form by photoirradiation at wavelengths of 400 nm and longer (Fig. 6.5) [29]. The trans form has a planar structure, and it is possible to form double-stranded DNA even when azobenzene is inserted into the DNA. By contrast, the cis-form of azobenzene has a bulky structure, and DNA containing cis-form azobenzene cannot form double helix due to steric clashes [30]. It is possible to utilize this difference in properties to control reversible double-strand formation

Fig. 6.4 Photo-cross-linking of psoralen and thymidines

Fig. 6.5 Photoisomerization of azobenzene

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using multiple photoirradiation. This technology enables photochemical control of molecular motors [31] and DNA nanostructures [32] and is an indispensable tool for the construction of molecular robots. We have previously reported a photoresponsive artificial nucleic acid, 3cyanovinylcarbazole (CNV K), that can photo-cross-link with a pyrimidine base in complementary DNA or RNA via photoirradiation for a few seconds (Fig. 6.6) [32]. Photo-cross-linking between CNV K and thymidine proceeded to approximately 90% completion upon irradiation at 366 nm for 1 s. As it is possible to induce photosplitting by photoirradiation at 312 nm, the extent of photo-cross-linking can be finely controlled by choice of wavelength and time of photoirradiation. CNV K nucleotides display higher photoreactivity than conventional moieties such as psoralen and coumarin, and other features peculiar to CNV K continue to be discovered. 5-Carboxyvinyldeoxyuridine (CV U) was also developed in our laboratory and can photo-cross-link with adjacent pyrimidines instead of linking DNA duplexes (Fig. 6.7) [34]. The same function performed by the enzyme ligase can be stimulated by photoirradiation for approximately 1 min. Various other photoresponsive artificial nucleic acids have been reported to date, including the photoresponsive artificial nucleic acids stilbazole [35] and anthracene [36]. Using such photoresponsive artificial nucleic acids, our laboratory has developed methods for photochemical manipulation of nucleic acids, particularly ultrafast photo-cross-linking (UFC), using CNV K. Conventionally, DNA is ligated, cleaved, and amplified using different enzymes. However, the use of enzymes necessitates optimization of conditions and enzyme deactivation. Thus, their use has been limited

Fig. 6.6 Photo-cross-linking of CNV K (a) Structure of CNV K (b) Scheme of reversible DNA photocross-linking

Fig. 6.7 Photochemical ligation of CV U (a) Structure of CV U (b) Scheme of reversible photochemical ligation

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to extremely optimized conditions (including the use of enzyme-specific buffers). By contrast, nucleic acid manipulation using photoresponsive artificial nucleic acids has the following advantages: (1) it can be used under a wide range of conditions, (2) high operability (spatiotemporal control, irradiation energy/wavelength), and (3) it can be combined with enzymatic methods. These are considered useful tools for improving the operability of molecular robots. Photoresponsive artificial nucleic acids were designed and synthesized based on knowledge of organic chemistry. Reactivity varies greatly depending on DNA structure and variations in its substituents. Therefore, depending on the design, it is possible to produce a variety of photoresponsive artificial nucleic acids with fine-tuned functions and reactivities. Five types of photoresponsive artificial nucleic acids with similar structures are shown in Fig. 6.8. Their reaction rates differ, with a nearly tenfold difference in the first reaction between the fastest (CNV D) and slowest (CNV Gs) nucleic acids [37]. CNV D has a 3-cyanovinylcarbazole skeleton similar to CNV K, but the 2-deoxyribose moiety of CNV K is changed to D-threoninol, which has higher steric freedom than ribose, resulting in accelerated photo-cross-linking. The photo-cross-linking rate can be slowed down by using CNV L with its isomer L-threoninol. In addition, CNV Gr and CNV Gs, with shorter carbon chains, exhibited significantly slower cross-linking rates. It is very difficult to control the reaction rate when a conventional enzyme is used. However, photoresponsive artificial nucleic acids enable precise control at the molecular level and are extremely important for optimizing the functions of molecular robots. An example of an application that can be achieved by precise control at the molecular level is related to the photochemical regulation of DNA strand displacement. The construction of a biomolecule (DNA)-based information processing mechanism is crucial for molecular robots. Various enzyme-free DNA logic circuits have been reported. These include AND gates [38]. Amplifier circuits [39], and Seesaw gates [40]. The construction of these circuits features DNA strand displacement. This displacement comprises a multi-step equilibrium reaction termed branch migration and access to Toehold. Branch migration is the bottleneck to the reaction rate (Fig. 6.9a). The reverse reaction can be inhibited and branch migration accelerated using photo-cross-linking. We have been successful in accelerating the DNA strand exchange reaction by 21 times (Fig. 6.9b) [41]. The thermodynamic parameters of

Fig. 6.8 Photo-cross-linker libraries with different photoreactivity

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Fig. 6.9 Photochemical regulation of DNA strand displacement (a) DNA strand displacement; (b) Acceleration of DNA strand displacement using UFC; (c) Photochemical regulation dependent on photoirradiation energy; (d) Precise regulation of reaction rate by combining photo-cross-linker

this DNA strand displacement were investigated and simulated. When photo-crosslinking was used, the acceleration effect could be predicted based on the photo-crosslinking rate of the photocrosslinker (Fig. 6.9c). The simulation revealed that the DNA strand displacement rate can be controlled depending on the photo-cross-linking rate. Therefore, various DNA strand displacement rates can be realized by changing the photoirradiation energy and photo-cross-linker (Fig. 6.9d). It is possible to induce photochemical deamination as one of the applications of UFC. Typically, hundreds of years are required to convert cytosine to uracil. However, as shown in Fig. 6.7a, the reaction is induced by heating at 90 °C for 3 h using ultrafast photo-cross-linking [42]. Base conversion is achieved in the same way as genome editing technology. This method has attracted attention in recent years, and can be harnessed as a molecular robot to perform gene therapy. However, heating at 90 °C cannot be used in vivo. Studies including ours have explored rational in vivo applications. We succeeded in deamination at 37 °C by optimizing the surrounding base of the cytosine target [43]. The deamination reaction was accelerated 14 times when inosine was used as the counter base of cytosine [44], and a photo-cross-linker having a hydrophilic substituent was used as the photo-cross-linker [45]. By optimizing the chemical structure of the surrounding structure, this reaction succeeded in deamination at 37 °C. This could be useful for therapeutic applications of molecular robots (Fig. 6.10).

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Fig. 6.10 Photochemical C to U editing using UFC (a) Scheme of photochemical C to U editing (b) Chemical structure of Inosine and OHV K (c) Time course of C to U editing

UFC can be used intracellularly because of its high photoreactivity. Since it can form a very stable double-strand through covalent bonds with target DNA or RNA, it can be applied to antisense methods that inhibit translation by photocrosslinking mRNA. An antisense nucleic acid containing CNVK was introduced into GFP-HeLa cells by the lipofection method. Then, after photoirradiation for 10 s, the amount of GFP fluorescence was quantified by a confocal microscope. As a result, it was confirmed that GFP fluorescence was greatly reduced by photoirradiation [46]. It is considered that the formation of covalent bonds greatly inhibits translation. In addition, when CNVD, which has a higher photoresponsiveness than psoralen and CNVK, which have been conventionally used as photocrosslinking devices, was used and their antisense effects were verified, CNVD had a gene expression inhibitory effect of about 90%. It was. In addition, although the advantage of using light is high operability, it is possible to control gene expression in cells at the timing of photoirradiation. As shown in Fig. 6.11, the amount of GFP mRNA decreased immediately after photoirradiation, and we succeeded in controlling gene expression temporally at the timing of photoirradiation.

Fig. 6.11 Photochemical regulation of antisense effect using UFC (a) Photochemical regulation of antisense effect; (b) Amount of GFP mRNA with photoirradiation

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With the progress in DNA synthesis technology, it has become possible to easily obtain DNA from any sequence. Furthermore, research and development of functional artificial nucleic acids have progressed and various functions of molecular robots have been realized. It is anticipated that the light-based control of molecular robots will be realized. This will be an important technology supporting the evolution of molecular robots.

6.4 DNA Computing Using Photoresponsive Artificial Nucleic Acid Kenzo Fujimoto K. Fujimoto School of Advanced Science and Technology, Japan Advanced Institute of Science and Technology, Ishikawa, Japan e-mail: [email protected] DNA is considered as a candidate as a molecule that transmits information between devices in a molecular robot. Adleman et al. focused on the characteristics of DNA as information molecule, [47] and devised a calculation method using DNA ligation and amplification (Fig. 6.12a). On the other hand, a logic circuit using a DNA strand

Fig. 6.12 DNA computing using photoresponsive artificial nucleic acid. a DNA computing b DNA strand displacement c Acceleration of DNA strand displacement using ultrafast photo-cross-linking d Photochemical regulation of DNA strand displacement

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displacement as a non-enzymatic logic circuit [38] has also been reported. In addition, Winfree’s group implemented a logic circuit called the Seesaw gate in which multiple logic gates were cascaded [40]. These non-enzymatic logic circuits exchange information via DNA strand displacement. Using the single-stranded part(toehold) protruding from the double-stranded DNA as a foothold, another single-stranded DNA hybridizes, undergoes branch migration, and finally forms a stable doublestrand, and the output strand is released (Fig. 6.12b). However, as the scale of circuits increases, it is necessary to shorten the calculation time and synchronize the inputs. We succeeded in light-driven binary calculation using photochemical ligation and succeeded in constructing a full-adder circuit toward the realization of DNA calculation using photoresponsive artificial nucleic acid [48]. It has also been reported that the DNA strand exchange reaction can be accelerated about 21-fold when 3-cyanovinylcarbazole(CNV K), which is a photo-cross-linker, is incorporated (Fig. 6.12c) [41]. We have also found that the DNA strand displacement rate can be controlled by changing the photoirradiation energy. Furthermore, by using the photoresponsive artificial nucleic acids we have developed, such as CNV D and CNV L, it is possible to realize DNA strand displacement with various reaction rates by photoirradiation even within the same system (Fig. 6.12d), and cascade them. It is thought that the calculation time can be shortened and the synchronization of each input can be realized by the conversion (Fig. 6.13). Now that about 30 years have passed since molecular calculation using DNA was proposed, various applications are expected as an information processing mechanism for molecular robots. Since the photoresponsive artificial nucleic acid can control the speed of the DNA strand displacement, which is the basic reaction of DNA calculation, it enables new calculation models and controls that have never existed before. It has a circuit inside the molecular robot and outputs according to the input. Furthermore, the behavior differs depending on the presence or absence of photoirradiation. Based on this idea, DNA calculation using photoresponsive artificial nucleic acids may be widely used as a new technology for the construction of moving molecular robots. Fig. 6.13 Future of DNA computing

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6.5 Orthogonality of Nucleic Acids Keiji Murayama and Hiroyuki Asanuma K. Murayama · H. Asanuma Nagoya University, Nagoya, Japan e-mail: [email protected] H. Asanuma e-mail: [email protected]

6.5.1 Orthogonality of DNA In mathematics, orthogonality is defined as two vectors that cross perpendicularly to give a scalar product of zero. Derived from this definition, a relationship between two elements that have no interference with each other is referred to as the orthogonality regardless of field. For example, orthogonality in organic chemistry is high specificity of the substrate, such as when different protecting groups on the same functional group can be deprotected under different conditions [49]. In the field of nucleic acid chemistry, the sequence specificity of duplex formation is orthogonal. DNA and RNA oligonucleotides form the most stable duplexes with complementary strands, whereas mismatched sequences form less stable duplexes and scrambled sequences do not hybridize with each other. The orthogonality based on sequence complementarity makes DNA computing and logic gates possible. Molecular beacons are molecular machines that can detect target DNA and RNA in a sequence-specific manner via the orthogonality of the duplex formation. Sufficient orthogonality is available if the length of the strands are less than 8–10 nucleotides under physiological salt conditions, because a single base mismatch severely destabilizes a duplex of this length. However, short oligonucleotides would not form duplexes of sufficient stability and depending on the application, longer strands may be necessary to ensure sequence specificity. Moreover, the design of complicated and stable circuits requires long DNA strands, which can result in loss of orthogonality due to unintended crosstalk between strands of incomplete complementarity. In addition, the design of circuits composed of only DNA is restricted as there are only four natural bases. Thus, further evolution of DNA computing requires artificial nucleic acids that can expand the orthogonality.

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6.5.2 Expansion of Orthogonality by Artificial Nucleic Acids Recently, progress in organic synthesis has enabled the preparation of various artificial nucleic acids. Most artificial nucleic acids were designed to facilitate durability against enzymatic degradation, and are only partially modified relative to natural nucleic acids. In seeking to determine why nature chose the ribofuranosyl to carry the genetic code rather than some other structure, chemists have synthesized various unnatural scaffolds (Fig. 6.14). These artificial nucleic acids with modification on the D-ribose scaffold have been referred to as xeno-nucleic acids or XNAs. XNAs can be classified based on orthogonality as shown in Fig. 6.15. The XNAs in group A have chiral scaffolds that induce right-handed helicity. These XNAs can form duplexes with natural DNA and RNA. In other words, the orthogonality within group A is available only due to sequence specificity. XNAs in group B are constructed from building blocks that are enantiomers of the building blocks used to synthesize group A XNAs; these strands form left-handed helices. The orthogonality

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Fig. 6.15 Classification of XNA by orthogonality. Group A: XNAs that form duplexes with natural DNA (D-DNA). Group B: XNAs orthogonal with group A XNAs. Group O: XNAs that form duplexes with both group A and group B XNAs

within group B also results from sequence complementarity. XNAs in group A cannot hybridize with XNAs in group B even though they have complementary sequences due to the structural difference between right-handed and left-handed helices. This means that orthogonality can be derived not only from sequence specificity but also from helicity. Circuits that rely on orthogonality between groups A and B cannot be designed if these XNAs are completely independent of each other. To realize information transfer between groups A and B, XNAs composed of the achiral scaffold in group O can be used. Group O XNAs have achiral scaffolds. As a result, these XNAs can form duplexes with both right-handed group A and left-handed group B XNAs in a sequence-specific manner, because the achiral scaffold can conform to either helicity. Thus, even though group A and group B XNAs are orthogonal, they can communicate with each other through the mediation of group O. For example, a signal amplification circuit composed of only XNAs from group B cannot be activated by DNA, which is in group A. By using the interface composed of oligomer in group O, however, sequence information present in a DNA strand can be converted to an XNA in group B, activating the group B circuit. There are certain situations where a robust circuit cannot be designed from only the XNA of a single group. For example, contaminating DNA and RNA in a biopsy can cause incorrect activation of a signal circuit when the circuit is composed of group A

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XNA. A robust circuit that cannot be incorrectly activated by contaminating DNA or RNA is achievable using XNA in group B. Detection of the target DNA, orthogonal to this circuit, is possible via the interface that can convert the target DNA to an input of the group B circuit. Thus, the expansion of orthogonality by using different groups of XNA allows a function separation: The interface recognizes the target, and orthogonal XNA performs the signal amplification. In this manuscript, we focus on a seesaw gate, a signal amplification circuit that can be used to detect DNA or RNA in a sequence-specific manner, first reported by Winfree et al. [61].

6.5.3 Orthogonality Between DNA and D-aTNA DNA and D-aTNA, from group A and orthogonal group B, respectively, were used as components of a seesaw gate circuit. In this circuit, the termini of Output and Gate were labeled with a fluorophore and a quencher, respectively. Fuel has the same sequence as Output without fluorophore (Fig. 6.16, upper). At the initial state, Output and Gate form the Output/Gate duplex, and Fuel is a single strand (Fig. 6.16, lower, state a). At this stage, the strand exchange between Fuel and Output is impossible due to the absence of a toehold region. Upon addition of Input, which is complementary to the overhang region of the Gate, a toehold exchange reaction occurs that results

Fig. 6.16 Operation of a seesaw gate. Upper: Sequences used in the operation of a seesaw gate. Lower: Schematic diagram of steps involved in the operation of a simplified seesaw gate

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in the formation of the Input/Gate duplex and the dissociation of Output from Gate, which produces a fluorescence signal (Fig. 6.16, lower, states b and c). The generated Input/Gate duplex can interact with Fuel which is in excess amount in this system. The toehold exchange with Fuel regenerates the single-stranded Input and produces Gate/Fuel duplex as waste (Fig. 6.16, lower, states d and e). Continuing the cycle, the regenerated Input again causes a toehold exchange reaction with the Output/Gate duplex (Fig. 6.16, lower, states e and b). These reactions continuously generate a single-stranded Output strand, resulting in a fluorescent signal, until the amounts of Output/Gate duplex and the waste are similar. Thus, the seesaw gate composed of only DNA can detect target DNA or target RNA (i.e., the Input) detected as an increase in fluorescence intensity. We evaluated the reaction of the seesaw gate composed of only D-aTNA by measuring fluorescence intensity change [62]. The D-aTNA seesaw gate was activated at 50 °C (Fig. 6.17a), whereas the DNA seesaw gate was activated at 25 °C. The increase in optimal temperature of the D-aTNA circuit was due to the extremely high stability of the D-aTNA homo-duplex compared to the DNA duplex of the same sequence. Only a fully complementary Input could activate the D-aTNA seesaw gate; neither an Input strand containing a mismatch nor a scrambled sequence caused signal amplification (Fig. 6.17b). This confirmed signal amplification of the D-aTNA circuit resulted from orthogonality based on the sequence. The orthogonality between the DNA seesaw gate and the D-aTNA seesaw gate was examined by the addition of Input strands of the other scaffold into each system [62]. A DNA Input did not activate D-aTNA seesaw gate (Fig. 6.18a) and a D-aTNA

Fig. 6.17 Influence of temperature and Input sequence on D-aTNA seesaw gate. a Effect of temperature on the signal generation from D-aTNA seesaw gate. b Effect of sequence of D-aTNA Input on signal. The circuit was operated at 50 °C. Partially modified and reprinted from ChemistrySelect 2017 [62]

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Fig. 6.18 Orthogonality of DNA and D-aTNA seesaw gates. Operations of (a) D-aTNA and (b) DNA circuits in response to D-aTNA (red) or DNA (black) Input. D-aTNA and DNA circuits were operated at 50 °C and 25 °C, respectively. Partially modified and reprinted from ChemistrySelect 2017 [62]

Input did not activate the DNA circuit (Fig. 6.18b). Thus, the D-aTNA circuit and the DNA circuit are completely orthogonal: Only a fully matched Input composed of the same scaffold as the circuit resulted in activation. The effects of contaminating DNA and RNA on the signal from a seesaw gate circuit were examined by the addition of DNA with partial complementarity to Gate as a model of a contaminant (Fig. 6.19a) [62]. The DNA circuit was not activated in the presence of the DNA contaminant (Fig. 6.19b), because Input hybridized with the contaminant rather than Gate (Fig. 6.19a). Thus, if a DNA circuit were used to detect a nucleic acid sequence in a patient biopsy, the circuit operation could be impaired by contaminating DNA or RNA. In contrast, the D-aTNA circuit, with a scaffold orthogonal to that of the contaminating DNA, worked correctly despite the presence of the contaminant (Fig. 6.19c). This demonstrated that a robust detection system can be constructed using a D-aTNA circuit that is not affected by contaminating DNA and RNA. The D-aTNA circuit cannot, however, detect an Input with a DNA scaffold.

6.5.4 SNA Interface Converts RNA Signal into D-aTNA Signal To activate a D-aTNA circuit with natural DNA and RNA, the sequence information of the DNA or RNA must be converted to D-aTNA. We reasoned that an XNA from group O that can cross-pair with DNA or RNA and with D-aTNA would function as an interface between these two orthogonal XNAs. As a target RNA, we choose miR21, a cancer-associated microRNA. As an interface, we used SNA (Fig. 6.15). The SNA

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Fig. 6.19 Effect of contaminating DNA on activation of circuits. a Scheme of deactivation by binding of a DNA contaminant to Input. b and c Responses of (b) DNA and (c) D-aTNA circuits in the presence of DNA contaminant. D-aTNA and DNA circuits were operated at 50 °C and 25 °C, respectively. Partially modified and reprinted from ChemistrySelect 2017 [62]

synthesized is fully complementary to the target RNA and to the region of the Input D-aTNA containing the overhang required for the circuit reaction (Fig. 6.20a). At the initial state, the D-aTNA seesaw gate is not activated by Input D-aTNA, due to duplex formation with the Interface SNA. In this situation, the toehold region of the Input D-aTNA is completely hybridized to the Interface SNA, so the circuit is not activated. Upon addition of target RNA, fluorescent amplification of D-aTNA seesaw gate occurs through a release of Input D-aTNA from the Interface SNA due to a toehold-mediated strand exchange reaction that generates a duplex between the Interface SNA and the target RNA. The SNA/RNA duplex was designed to be longer than SNA/D-aTNA duplex, because SNA has higher affinity for D-aTNA than for RNA. The addition of the target miR21 resulted in fluorescent signal that increased over time, whereas no reaction was observed in the absence of the target (Fig. 6.20b). The circuit was also not activated without Interface SNA, demonstrating the interface function of the SNA [62]. In conclusion, a combination of orthogonal XNA and interface XNA enabled the development of a novel molecular circuit. This system can detect a small amount of RNA such as a microRNA expressed in cells and present in biopsy tissue. As detection of natural nucleic acid by a D-aTNA requires an interface molecule, the

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Fig. 6.20 RNA-mediated activation of D-aTNA circuit via an SNA interface. a Schematic of use of SNA as an interface to convert to enable a D-aTNA circuit to detect RNA and the sequences used. The SNA prevents activation by D-aTNA input in the absence of RNA. Addition of RNA allows the release of D-aTNA Input to activate the circuit. b Signal generation of D-aTNA circuit with SNA interface in the presence (red) and absence (black) of target RNA. D-aTNA circuit was operated at 40 °C

presence of contaminating nucleic acid will not activate the circuit. A variety of amplification circuits composed of nucleic acids have been reported [63]. As with the example of the seesaw gate described here, we can design highly sensitive and robust detection systems for trace amounts of target RNA using signal amplification circuits composed of orthogonal XNAs of group B and interface XNAs of group O.

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6.6 Regulation of DNA Reaction by Cationic Comb-Type Copolymer as an Artificial Nucleic Acid Chaperone Naohiko Shimada and Atsushi Maruyama N. Shimada · A. Maruyama Tokyo Institute of Technology, Nagatsuta 226-8501, Yokohama, Japan e-mail: [email protected] A. Maruyama e-mail: [email protected]

6.6.1 Nucleic Acid Chaperone Activity of Cationic Comb-Type Copolymer Accurate hybridization or folding of nucleic acids in vitro is conducted by an annealing process. Instead of the annealing, nucleic acid chaperone proteins mediate the folding of nucleic acids into a thermodynamically stable structure in vivo. We have reported that cationic comb-type copolymers such as poly(L-lysine) grafted with dextran(PLL-g-Dex, Fig. 6.21) showed nucleic acid chaperone activities. In this section, we introduce the effect of the copolymer as an artificial nucleic acid chaperone on the DNA reaction and also introduce applications using the copolymer in DNA-based nanotechnologies. The copolymers thermodynamically stabilize DNA double-strands [64] and triple strands [65] under physiological conditions. The copolymers also accelerate DNA hybridization by two orders [66]. These effects are due to the release of counterions around DNA by binding of the copolymer increases the entropy in the system resulting in stabilization and acceleration of DNA hybrids. A strand displacement reaction is an exchange reaction between a double-stranded (ds) DNA and its fully complementary single-stranded (ss)DNA. The reaction is initiated by partial hybridization of the ssDNA to the ds-DNA which partially melts Fig. 6.21 Structural formula of poly(L-lysine)-graft-dextran (PLL-g-Dex)

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Fig. 6.22 a Schematic illustration of DNA strand displacement reaction. b Acceleration of strand displacement reaction by the addition of PLL-g-Dex(CCC). Reproduced with permission from ref [67]. Copyright 2002 American Chemical Society

at the terminal end, followed by branch migration to replace with a new strand (Fig. 6.22a). However, the reaction is extremely slow owing to the formation of thermodynamically unfavorable three-stranded intermediate as shown in Fig. 6.22b. The copolymer showing a stabilization effect on DNA hybrids allowed us to stimulate the strand displacement reaction because the copolymers promote the formation of the three-stranded intermediate [67]. The copolymers showed a better effect in strand displacement acceleration than a naturally occurring chaperone protein, NCp7 which accelerates the strand displacement [68]. Guanine-rich DNA oligonucleotides form a mixture of hetero four-stranded complex, quadruplex, containing metastable conformation because of their conformational polymorphism. Although reassembling to a thermodynamically stable quadruplex is very slow, the copolymers quickly reassembled hetero quadruplexes into a thermodynamically stable quadruplex [69]. This is because the nucleic acid chaperone activity of the copolymer significantly increases both the association rate and the dissociation rate of the quadruplex. Thus, cationic comb-type copolymers promoting reaction to form thermodynamically stable structures behave as an artificial nucleic acid chaperone.

6.6.2 Enhancement of DNA Enzyme Activity by Cationic Comb-Type Copolymers DNAzymes are enzymes composed of DNA. Some DNAzymes cleave RNA as a substrate in the presence of metal ions. The DNAzymes consists of a catalytic core region flanked by two substrate binding arms. The substrate binds to the arms to be cleaved. The cleaved substrate then is released to load a fresh substrate for turnover. Compared with protein enzymes, DNAzymes have the advantage of being easier

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Fig. 6.23 Schematic illustration of DNAzyme reaction (a) and temperature dependence of DNAzyme multiple-turnover reactions at 5 mM Mn2+ with or without PLL-g-Dex(B-E). Samples containing 200 nM substrate and 6.7 nM DNAzyme were incubated in the absence or presence of PLL-g-Dex at (b) 25 °C, (c) 50 °C, and (d) 60 °C. (E) Temperature dependence of rate constants, k obs , estimated in 5 mM Mg2+ (square) and in Mn2+ (circle) in the absence (dotted lines) and presence (solidlines) of the copolymer. Reproduced with permission from ref [70]. Copyright 2015 The Royal Society of Chemistry

to synthesize and showing activity under a wider range of temperatures. Unfortunately, DNAzyme reaction is much slower than protein enzymes. One of the ways to improve the slow reaction rate is an increase in the turnover rate. We hypothesized that the copolymer having chaperone activity could enhance the DNAzyme activity owing to an increase in the turnover rate. As expected, the DNAzyme reaction was enhanced at various temperatures by the addition of copolymer (Fig. 6.23) [70]. At 50 °C, the optimal temperature for the DNAzyme in the absence of the copolymer, the copolymer increased the DNAzyme k cat /K M by 50-fold. Multi-component nucleic acid enzymes (NMAzymes) are composed of two fragments, which are split catalytic core of DNAzyme, conjugated with the specific sequence for binding to a target nucleic acid. The binding of the target activates NMAzyme to cleave the substrate. Because MNAzyme cleaves multiple substrates and amplifies the signal corresponding to target, MNAzyme system has utilized as biosensors or components of DNA nanomachines. As same as a problem for DNAzyme, MNAzyme reaction has a bottleneck in the turnover process. The addition of copolymer successfully improved MNAzyme reactivity, resulting in target DNA detection at a picomolar concentration which is 100-time lower detection concentration than the detection concentration without copolymer [71, 72]. The copolymer chaperoning DNA-based sensors have the potential for use as highly sensitive assays for nucleic acid.

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6.6.3 Boosting of DNA Logic Gate by Cationic Comb-Type Copolymers DNA computation is performed by cascades of sequence specific reactions including toehold-mediated strand displacement. However, a long computation time is required to output the answer because of the slow reaction of toehold-mediated strand displacement. Even in the presence of Mg2+ , which accelerates the reaction, the computing often takes several hours. Boosting-molecules, which accelerate the reaction faster is necessary before the computer will be useful. The effect of the copolymer as a candidate of the boosting-molecule on toehold-mediated strand displacement reaction rate was evaluated by using ds-DNA having five base toehold and its complementary ssDNA [73]. In the presence of MgCl2 but in the absence of the copolymer, it took 34 min for 50% of the displacement reaction to proceed (DR50 ). In the presence of the copolymer, the displacement took place quickly with a DR50 of only 30 s, indicating that the copolymer accelerated toehold strand displacement better than Mg2+ . The copolymer was added to the DNA seesaw gate [74] designed by coupling multiple strand displacement reactions to examine whether the operation of DNA gates was accelerated by the copolymer (Fig. 6.24). For AND gate, in the absence of the copolymer, the output signal increased significantly only when both inputs were added (1^1). However, it required several hundred minutes of operation time as previously reported. On the other hand, the operation was completed within a few minutes in the presence of the copolymer while maintaining input selectivity. The result indicated that the response of the gate was considerably improved by the copolymers. Furthermore, the copolymer permitted the operation of the logic gate even in the presence of DNase that degrades DNA. This allows us to operate the DNA gate in biological environments containing DNase. Thus, chaperoning of the cationic comb-type copolymer will expand the utility of the DNA logic gate.

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Fig. 6.24 a Schematic illustration of DNA gate based on the seesaw gate. b Output signal changes with the time of AND gate in the absence (left panel) or the presence of PLL-g-Dex (right panel). Reproduced with permission from ref [73]. Copyright 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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Chapter 7

Medical Application of Molecular Robots Taro Toyota

Abstract Molecular robots have a high affinity with the pharmaceutical and medical fields. It will contribute to the sophistication of drug delivery systems, for which much research is already underway based on polymer and colloid chemistry. In addition, molecular robots also play a major role in the concept of system nucleic acid medicine, which innovatively develops nucleic acid medicine of the practical stage in recent years. In both cases, an artificial molecular system introduced into the body makes a diagnosis (molecular computing) using multiple inputs, and in situ produces compounds that can be used as a drug. Moreover, the control technology of stem cells such as iPS cells by molecular robots will enable the production of artificial organs in the future by the controlled differentiation induction with artificial organelles composed of RNA–protein complexes. A more advanced application is the molecular robot that can perform sample returns in vivo, the so-called molecular Hayabusa.

This chapter gives a glimpse of the future that molecular robotics is aiming for. In what fields and how molecular robotics is applied? Whether the relationship with the nearby field of synthetic biology is complementary or competitive? By reading this chapter, the readers will comprehend that molecular robotics is a versatile technology that has the potential to become the next generation of industrial foundations. The major difference between synthetic biology technology, including cell engineering, and molecular robotics is that molecular robotics is based on pure chemistry and produces molecular devices composed of DNA, RNA, peptides, etc., all of which have known well without any black box. It also differs from cell engineering technology in synthetic biology, which is based on living organisms (cells), in that molecular robots do not proliferate unless they are intentionally programmed to do so. Cell engineering technology in synthetic biology is superior for the power play such as mass production of useful molecules (Fujiwara introduces the latest research examples in this chapter). On the other hand, delicate and effective molecules even in T. Toyota (B) The University of Tokyo, Komaba, Japan e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2022 S. Murata (ed.), Molecular Robotics, https://doi.org/10.1007/978-981-19-3987-7_7

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a very small amount are highly expected to be produced in situ and molecular robots can likely meet such demand. Therefore, in the viewpoint of molecular systems engineering, molecular robotics is about to be rapidly developed in an application where life and artificial objects are fused. Since molecular robots are made of biomolecular materials such as DNA, RNA, peptides, and lipids, they have a high affinity with the pharmaceutical and medical fields. Molecular robots will contribute to the sophistication of drug delivery systems, for which much research is already underway based on polymer and colloid chemistry. This is discussed in detail by Toyota and Zhang. In addition, as Fujimoto introduces his latest research, molecular robots also play a major role in the concept of system nucleic acid medicine, which innovatively develops nucleic acid medicine of the practical stage in recent years. In both cases, instead of targeting a single gene or biomarker, an artificial molecular system introduced into the body makes a diagnosis (molecular computing) using multiple inputs, and in situ produces compounds that can be used as a drug. Moreover, the control technology of stem cells such as iPS cells by molecular robots will enable the production of artificial organs in the future by the controlled differentiation induction with artificial organelles composed of RNA–protein complexes. Ohno, Komatsu, and Saito describe the fundamental technologies and application examples. A more advanced application is the molecular robot that can perform sample returns in vivo, the so-called molecular Hayabusa (Hayabusa is a spacecraft developed for sample returns from extraterrestrial asteroids by Japan Aerospace Exploration Agency). Murata proposes the concept of the molecular Hayabusa with the detailed necessary technology. Molecular Hayabusa is a project that needs to develop a series of processes based on artificial cells. They mount peptide-based devices for collecting molecular samples (such as miRNA and exosomes) on the surface of artificial cells, and the trace amount of molecular samples brought back is analyzed by the nanopore system. If a molecular robot can directly bring back molecular samples from inside the body, it will be possible to approach the inner part of the ovary and pancreas, which are previously difficult to perform with biopsy. This means a new medical technology for intractable diseases that are difficult to detect. By reading this chapter, which introduces concrete ideas and cutting-edge researches for such application fields, readers will be able to imagine the appearance of molecular robots in the near future.

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7.1 Cell-Fate Control by RNA/RNP Hirohisa Ohno, Shodai Komatsu and Hirohide Saito H. Ohno · S. Komatsu · H. Saito Kyoto University, Kyoto, Japan e-mail: [email protected] S. Komatsu e-mail: [email protected] H. Saito e-mail: [email protected] In this section, we describe the regulation of cell functions by artificial RNA/RNP (RNA–protein complex) systems, focusing on our research. The technologies presented here, RNA nanotechnology platforms, biological modular design, and synthetic biological circuits, are aimed at controlling cells and serving as components for molecular robots.

7.1.1 Synthetic RNA Switches for Gene Regulation Cellular function and cell fate are regulated by gene expressions (the transcription of a gene into mRNA and its subsequent translation into protein). Conversely, the artificial control of gene expressions enables exogenous control of a cell. Accordingly, synthetic biologists have developed various artificial gene regulation systems. However, most are based on DNA and transcription factors and regulate transcription. Similarly, complex genetic circuits have, in general, been constructed using multiple transcriptional modules. In contrast, while the translation of mRNA also regulates gene expressions, it is less exploited for artificial control. An example of translational regulation is riboswitches [1]. Riboswitches are complexes of folded RNA domains that control gene expressions by altering their structure in response to specific metabolite binding. Another example is microRNAs (miRNAs) [2]. miRNAs interact with complementary sequences of the target mRNAs to induce mRNA degradation and translational repression. These translational regulation systems provide an additional layer of gene expression control to transcriptional modules, allowing for more precise control. Indeed, we have used such translational regulation systems to develop several artificial RNA-based systems that control gene expressions in response to target molecules in the cell. The protein-responsive mRNA OFF switch is synthetic RNA whose translation can be repressed by a specific protein (Fig. 7.1a) [3]. There are an estimated 20,000 to 25,000 protein-coding genes in the human genome. Gene expression patterns are affected by various factors, including the cell type, cell cycle and the environment.

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Fig. 7.1 Translational control by switch mRNA. a Protein-responsive “OFF” switch. b Proteinresponsive “ON” switch. c MicroRNA-responsive “OFF switch”

Therefore, by designing mRNA whose translation is regulated by a target protein, we can modulate the cellular condition. As a proof of concept, we used the kink-turn (k-turn) RNA motif, which is recognized by L7Ae, an archaeal ribosomal protein [4]. When the k-turn motif is inserted into the target mRNA upstream of the open reading frame, interaction with L7Ae prevents the ribosome machinery from initiating translation (Fig. 7.1a). Indeed, the translation of the L7Ae-responsive mRNA OFF switch was modulated by the L7Ae protein level in human cells. We have

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confirmed other RNA motifs recognized by specific proteins can also be used in this protein-responsive switch design [5]. Similarly, we have developed a protein-responsive mRNA ON switch whose translation can be initiated by a specific protein (Fig. 7.1b) [6]. In this system, short hairpin RNAs (shRNAs) are used for translational regulation. To construct the ON system, we designed a synthetic shRNA containing a protein-binding motif in its stem and loop regions, where Dicer processes the shRNA to its mature form for the cleavage and degradation of the target mRNA. Binding by the protein to the designed shRNA interferes with Dicer-catalyzed processing, preventing the mRNA degradation. Synthetic shRNAs retain RNA interference activity in the absence of the protein in human cells, but in the protein’s presence, target mRNA is degraded and translation is repressed. This ON switch design has been used to control human cell fate by modulating the translation of apoptosis regulatory proteins [6]. As with OFF switches, other RNA motifs can be applied to protein-responsive ON switches [7]. We have also developed an mRNA switch that responds to miRNAs. miRNAs are small non-coding RNA of approximately 20 nucleotides long. They repress protein synthesis by binding to mRNA with a complementary sequence to inhibit translation. The human genome encodes more than 2,500 mature microRNAs, but their expression patterns vary depending on the cell type and cell state [8]. This fact suggests that miRNA can be used to distinguish cell types. We confirmed this conjecture by showing synthetic mRNA with an miRNA target site on its untranslated region was degraded by the corresponding miRNA (miRNA-responsive OFF switch; Fig. 7.1c). miRNA-responsive ON switches, which consist of an miRNA-responsive OFF switch and a protein-responsive OFF switch, have also been designed. Finally, we have developed various synthetic gene circuits by combining previous translational switches that respond to specific proteins and miRNAs. A combined gene circuit that is comprised of miRNA-responsive switches was able to selectively induce apoptosis in cancer cells. We also developed a multilayer gene circuit whose translation was regulated by three different switches and a circuit in which two switches cross-repress each other, resulting in an RNA-based toggle switch [9].

7.1.2 Artificial Molecular Scaffolds for Spatio-temporal Regulation of Biomolecules There are numerous strategies to regulate cellular functions, including the modulation of gene expressions and post-translational modifications. One commonly used mode of regulation is to control protein localization. The spatial and temporal organization of molecules within a cell increases the efficiency of interactions between individual partner molecules and facilitates enzymatic reactions. Molecular scaffolds play an important role in assembling relevant molecular components [10, 11]. Therefore, their artificial design would enable the control of molecular localizations, adding a

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new layer for the precise control of cellular functions. Based on this idea, attempts have been made to construct artificial molecular scaffolds using RNA and RNPs. The first example of artificial molecular scaffolds made of RNA was demonstrated in E. coli [12]. Artificial RNA modules were constructed using an RNA motif that binds two bacteriophage proteins, MS2CP and PP7CP (Fig. 7.2a), thus assembling the modules into 1D and 2D scaffolds. MS2CP and PP7CP were fused to ferredoxin and hydrogenase, which catalyze the reduction of protons to hydrogen through electron transfer. The addition of RNA scaffold resulted in protein-RNA assembly in vivo and a 4.0-fold increase in hydrogen production compared with no scaffold. Furthermore, the extended assemblies with the 1D and 2D scaffolds resulted in a 11- and 48-fold increase in hydrogen production, respectively. RNA scaffolds have also been used to facilitate pentadecane and succinate synthesis by assembling more than two enzymes [13]. These works demonstrated that the intracellular scaffolding of RNA can enhance multiple enzymatic reactions. Our group has used RNA scaffolds in mammalian cells for similar purposes (Fig. 7.2b) [14]. One example is a series of triangular RNA scaffolds containing three box C/D RNA motifs and three L7Ae proteins. We fused L7Ae to Galectin-1, a cell-surface receptor, to induce apoptosis via dimerization on the cell surface [15]. The triangular RNA scaffold induced the assembly of galectin-1 on the cell surface and apoptosis. However, extending the length of the three sides of the triangular RNA scaffold reduced apoptosis, indicating that galectin-1 assembly was controlled by the size of the RNA scaffold. Our group has also developed RNA scaffolds that function not on the surface but inside cells [16]. For example, we designed RNA scaffolds to assemble Caspase-8, which induces apoptosis via oligomerization. We constructed an mRNA that encodes L7Ae-fused Caspase-8 and RNA scaffolds that contain multiple boxC/D motifs for L7Ae binding. L7Ae-fused Caspase-8 assembled in human cells and induced apoptosis in the presence of the RNA scaffolds. We also fused Caspase-8 to Lin28 protein, a stem cell marker, instead of L7Ae. This system induced apoptosis in human cells unless they expressed Lin28 even in the presence of the RNA scaffold, indicating that endogenous Lin28 inhibited the assembly of Lin28-fused Caspase-8 on the RNA scaffold by competitive binding (Fig. 7.2c). These studies show that artificial RNA/RNP scaffolds can regulate the localization and assembly of a variety of molecules, both on the cell surface and inside cells, to control cellular function and cell fate.

7.1.3 CRISPR-Cas System and Genetic Manipulation The CRISPR-Cas system is a prokaryotic immune system that confers resistance to foreign genetic elements such as phages. Many bacteria rely on this system to store memory of the DNA sequence derived from invading viruses and use them to protect cells from reinfection. The CRISPR-Cas system consists of two key molecules: a Cas nuclease and a guide RNA (gRNA) (Fig. 7.3a). gRNA specifically binds to the target sequence in foreign genomic DNA and directs Cas nuclease to a target site

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Fig. 7.2 A molecular scaffold made of RNA. a By accumulating multiple enzymes on RNA (i) or in a two-dimensional structure (ii), improvement of enzyme reaction efficiency in Escherichia coli could be realized. b Control of the distance between receptors on the cell membrane surface using a triangular RNA structure. c Scaffold RNA that detects endogenous proteins and controls the generation of cell death signals

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Fig. 7.3 Applications of CRISPR/Cas system. a By using the CRISPR/Cas system consisting of a Cas protein having DNA-cleaving activity and a guide RNA responsible for recognizing a target sequence, any gene on the genome can be knocked out or a gene can be introduced. By fusing various effector proteins to Cas protein mutants that have lost DNA-cleaving activity, it can also be used for b labeling arbitrary regions on the genome, c controlling gene expression, d epigenetic control such as DNA and histone modification, and e base editing

for cleavage, resulting in a double-strand break. Because RNA-guided Cas nuclease can function in other species, the CRISPR-Cas system has been used for many applications, including gene knockout, transgene knock-in and the correction of genetic defects. The CRISPR-Cas system also provides high target specificity and ease of use compared with other existing gene-editing technologies and is currently regarded as the most reliable tool for genome editing and engineering [17, 18]. Introducing point mutations into the catalytic domain eliminates the nuclease activity of Cas protein but does not impact its binding to its target DNA. This nuclease dead Cas (dCas) protein has been used as a programmable DNA targeting protein. The dynamic and multiplexed imaging of genomic loci in living cells has been achieved by fusing or recruiting fluorescent proteins to either dCas protein or gRNA (Fig. 7.3b) [19, 20]. Similar approaches have been applied to various gene modifications, including transcriptional activation and repression using transcription factors (Fig. 7.3c) [21–23], epigenetic editing using histone-modifying enzymes (Fig. 7.3d) [24–28] and nucleotide substitutions using base editing enzymes (Fig. 7.3e) [29–31]. Some CRISPR-Cas systems are also capable of RNA targeting [32]. and thus RNA editing [33]. Overall, CRISPR-Cas systems are used as both DNA and RNA editing platforms and enable a wide range of cell control [34]. However, the possibility of off-target effects by the CRISPR-Cas system remains a concern. Gene-targeting by the CRISPR-Cas system mainly depends on gRNA specificity, and sequence mismatches between the target DNA and gRNA can result in

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off-target effects. The target and cleavage efficiencies are also affected by chromatin accessibility or epigenetic factors that vary across cell types. To overcome these issues, several approaches have been applied. Engineering Cas nuclease can improve the target specificity and reduce off-target effects [35–37]. The use of chemically modified gRNAs can also enhance genome editing efficiency [38]. The delivery of recombinant Cas nuclease and synthesized gRNA into human cells, including hardto-transfect cells and early embryos, enables efficient and precise genome editing compared with plasmid transfection [39, 40]. Our group has applied the CRISPR-Cas system to miRNA-responsive switches to regulate Cas nuclease activity by miRNA [41]. Using this system, we demonstrated that cell type specific genome editing can be achieved in heterogeneous cell populations.

7.1.4 RNA/RNP for iPS Cell Research and Application Induced pluripotent stem (iPS) cells are generated from adult somatic cells by forcing the expression of reprogramming factors, with the Yamanaka factors, Oct3/4, Sox2, Klf4, and c-Myc, being the most common. Unlike other pluripotent stem cells, iPS cells do not need embryo tissue for their generation. However, like all pluripotent stem cells, iPS cells have great promise for regenerative medicine, disease research and drug discovery [42–44]. One of the challenges for their application, however, is the purity of the differentiated cell populations. No differentiation process is 100% efficient and leaves contaminating undifferentiated iPS cells and partially differentiated cells. These contaminating cells may cause tumors following transplantation and can also negatively affect drug screenings. Current cell purification is based on cell surface markers, and differentiated cells are isolated using fluorescence-activated cell sorting (FACS). However, this purification method requires expensive systems, such as antibodies and equipment (cell sorters). Furthermore, commercially available antibodies do not cover all cell surface antigens. A simpler, more cost effective and reliable method for cell identification and purification is desired. Our group has used miRNA-responsive mRNA switches to purify several cell types, including cardiomyocytes, differentiated from human iPS cells (Fig. 7.4) [45]. Cardiovascular disease is one of the most common causes of death worldwide, and the transplantation of cardiomyocytes derived from iPS cells is a promising therapy. However, cardiomyocytes have no specific cell surface markers that facilitate their identification and purification by FACS. On the other hand, internal markers, namely miRNAs, do exist. Accordingly, miRNA-responsive switches that respond to these miRNAs were used to efficiently purify cardiomyocytes at >95% purity (Fig. 7.4a). Furthermore, an miRNA-responsive switch that translates Bim, a pro-apoptotic protein, enabled the autonomous purification of cardiomyocytes by inducing apoptosis only in non-target cells. Using this same strategy, the same report showed

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Fig. 7.4 Cell-type-specific selection by switch mRNA. a By using switch mRNA that responds to microRNA specifically expressed in cardiomyocytes, it was possible to specifically detect cardiomyocytes or specifically remove only undifferentiated cells. In addition, b by using switch mRNA that responds to microRNA specifically expressed in undifferentiated iPS cells, differentiated nerve cells can be detected, and only undifferentiated cells can be specifically eliminated

that miRNA-responsive switches efficiently purify endothelial cells, hepatocytes and insulin-producing cells differentiated from iPS cells. Later, miRNA-responsive switches were used to detect and eliminate undifferentiated iPS cells from dopaminergic neuron populations (Fig. 7.4b) [46]. miRNA302 is highly and specifically expressed in human pluripotent stem cells but not in dopaminergic neurons. Thus, an miRNA-302-responsive switch identified dopaminergic neurons in a heterogeneous population by expressing a fluorescent protein. Moreover, the switch showed higher sensitivity than immunofluorescence staining. Additionally, an miRNA-302-responsive switch that encodes puromycin resistance gene was expressed only in iPS cells to efficiently eliminate residual iPS cells through puromycin selection, resulting in the autonomous purification of neuronal cells.

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We have applied miRNA-responsive mRNA switches to viral RNA-based reprogramming for iPS cell generation [47]. Another miRNA-302-responsive switch encoding Yamanaka factors was introduced into human embryonic fibroblasts through virus infection. The fully reprogrammed iPS cells expressed miRNA-302, thus repressing the transgene expression and suppressing virus replication. This system also allowed us to visualize changes in the miRNA expression pattern in living cells during the differentiation process. Protein-responsive switches and RNP molecular scaffolds have also been applied to iPS cell research [48, 49]. We developed Lin28-responsive switches and Lin28driven RNA nanostructured devices to purify differentiated cells and induce the selective apoptosis of undifferentiated cells [5, 16]. Thus, RNA/RNP systems provide a promising tool for clinical stem cell research.

7.1.5 Perspective and Challenges As we explained above, artificial RNA/RNP systems have been used to regulate a variety of cell types. However, many issues remain before realizing precise and diverse cell regulation. In order to expand the range of applications of protein-responsive mRNA switches, it is necessary to identify marker proteins that define more cell types and cell states and to develop switches that can detect these target proteins with high sensitivity and specificity. Further, when constructing complex genetic circuits, as described in Sect. 7.1.1, it is necessary to use an orthogonal RNA/RNP pair that does not interfere with other protein and RNA functions. The development of such proteinresponsive mRNA switches requires the design of RNA motifs that specifically bind the target proteins. However, RNP interactions vary between RNA motifs, and there is no universal design rule to optimize the interaction. To overcome this problem, the establishment of robust design rules and methodologies is necessary. In the case of miRNA-responsive mRNA switches, the lack of miRNA expression profiles and databases for cell types and cellular events has limited their application. In order to accurately identify diverse cell types, multiple miRNAs, rather than only one miRNA, should be used. Our group is currently developing a miRNAresponsive genetic circuit that responds to two miRNAs separately and expresses the reporter proteins as programmed outputs [50]. In the future, we will develop a genetic circuit that can perform more complex computational processing in response to multiple miRNAs. Ultimately, we aim to establish a system that can accurately detect differences in intracellular conditions. A variety of proteins and RNAs act as molecular scaffolds to regulate cellular functions [51, 52]. However, there are few studies in which artificial molecular scaffolds are used in cells due to the difficulty of designing functional scaffolds. Our RNA/RNP-based molecular scaffolds consist of small components and are easy to change in shape and size [14]. Their components can also be supplied by transcription in cells, making them easier to use than DNA-based scaffolds. In the future, more

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functional RNA/RNP-based molecular scaffolds will be developed by expanding the available RNP motifs and increasing the size and complexity of the structures. Cell control has been achieved mainly by controlling protein expression through transcriptional regulation, but we have succeeded in developing technologies to control the translation and spatial organization of molecules within a cell. We have also succeeded in developing tools to control other stages of gene expression, such as mRNA splicing patterns [53]. These technologies are expected to make it possible to control a variety of gene expression processes and provide powerful tools to a wide range of research fields. In conclusion, various RNA/RNP-based technologies have been developed and enabled us to control cellular function and cell fate. They can also provide versatile modules for the design and construction of molecular robots.

7.2 Giant Vesicles Actively Involved in Biological Systems Taro Toyota and Yiting Zhang T. Toyota · Y. Zhang The University of Tokyo, Komaba, Japan e-mail: [email protected] Y. Zhang e-mail: [email protected] Artificial cells, which are cell-sized closed capsules capable of exchanging compounds and energy with the environment, have been attracting attention for therapeutic applications such as artificial hematocytes, as well as a basic model of living cells. A giant vesicle (GV), which is a closed bilayer membrane consisting of amphiphilic molecules, is one of the artificial cell models similar to the cell membrane. GV can afford not only compartmentalization owing to semipermeable membranes but also can express functional membrane proteins and exhibit morphological changes due to their low bending modulus. This section introduces recent designs and applications of GVs chemically linked to biological systems from the viewpoint of molecular robotics.

7.2.1 Artificial Cells and Their Practical Applications Artificial cells are regarded as capsule-like structures of cellular size, ranging from sub-micrometer to centimeter, that can exchange molecules, ions, and heat with the environment through their membranes [54, 55]. Chang published a remarkable paper in 1964 explaining the basic properties of enzymes encapsulated in a semipermeable polymer capsule [56]. This is regarded as the pioneering study pertaining to artificial

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Fig. 7.5 Schematic illustration of artificial cell provided from Chang [56]

cells that can take a substrate as an input and release a product as an output via an enzymatic reaction (Fig. 7.5). In other words, a primitive form of a molecular robot, an automatic molecular device combining sensor, processer, and actuator, is embodied in such polymeric capsules. Chang’s research group has aimed to construct artificial hematocytes where oxygen and carbon dioxide can be accumulated and released in response to the change in the concentration of each gas based on the circumstances in the body [57]. In recent years, the encapsulation of living cells, instead of enzymes, inside artificial cells has enabled the formation of organ-like structures and has been applied in therapeutic transplant treatments for diabetic patients [58]. The preparation methods for polymeric artificial cells are known as microencapsulation techniques not only in the pharmaceutical field but also in other industrial fields [59]; microcapsules containing insecticides [60], fragrance [61], and fertilizers [62] have already been commercially available as “practical” artificial cells. A tubular artificial cell encapsulating a sex steroid pheromone, etonogestrel, has been developed and clinically used as a birth control implant for women [63]. At the beginning of the development of these artificial cells, which are introduced into living bodies or used in the natural environment, polymer materials that have little interaction with biological systems and are chemically and physically stable have been derived from industries. For example, the membrane of the artificial erythrocytes reported for the first time from Chang’s group was composed of Nylon 6,10 [56] and the birth control implant is made of an ethylene vinyl acetate membrane [63]. Polyethylene oxide, polyvinyl alcohol, silicone, and Teflon have also been used. However, the difficulty of decomposing these polymeric materials for a long time becomes a problem sometimes. For example, the potential risk of cardiovascular disorders such as blood clots was reported when these durable polymers were used as a stent in the living body [64]. In addition, the emergent problem of microplastics in the natural environment has been highlighted [65]. Therefore, in recent years,

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functional organic molecules and polymer materials, that are not only biocompatible and biodegradable but also actively interact with biological systems, have been developed successively with the aim of solving these problems and increasing the usefulness of such materials. One of these newly developed materials is an amphiphilic molecule. Amphiphilic molecules can dissolve in both water and oil, and when their concentration exceeds a certain level (known as critical aggregation concentration or CAC), some of them form micelles, which are spherical aggregates, and others form vesicles, which are closed bilayer membranes. Vesicle-based artificial cells can be prepared by the selfassembly of amphiphilic molecules in water without the assistance of specific microcapsule encapsulation techniques. While micelles are rapidly decomposed upon dilution and the proportion of amphiphiles dissociating from micelles into monomers is high, the proportion of amphiphiles dissociating into monomers from vesicles is quite small (i.e., the CAC for vesicles is much lower than that for micelles), and thus vesicles are decomposed slowly upon dilution [66]. Both micelles and vesicles have been utilized as carriers to deliver drugs by introducing them into the circulatory system of the human body. In particular, the design of such drug delivery carriers using polymers originated from the pioneering concept proposed by Ringsdorf [67]. Kataoka’s group is currently leading the development of drug delivery carriers based on polymeric micelles [68]. Phospholipid vesicles, which are also known as liposomes, have been utilized as drug delivery carriers; some of these carriers have been clinically used, since phospholipids are cell membrane components and can be degraded in the body. In Japan, four types of liposomal drugs have been approved and released for use in clinical practice: AmBisome® (liposomal formulation of antifungal drugs) [69], DOXIL® [70] and Onivyde® [71] (liposomal formulation of anticancer drugs), and Visudyne® [72] (photodynamic therapy agent for suppression of the elongation of new blood vessels in the retina under laser irradiation). For example, DOXIL is a liposomal formulation that uses a mixture of natural phospholipids and synthesized one, which bears a polyethylene oxide chain at the polar head, and carries the anticancer drug, doxorubicin. It is passively trapped in the vascular system with a specific width or gap inside the tumor after being introduced into the patient’s body; the liposomal membrane molecules are then dissociated, thus resulting in the release of the drug. The mRNA vaccine, which has been rapidly developed since 2020 against the COVID-19 pandemic and has been inoculated globally, is also a sub-micrometer-sized capsule made of lipids including phospholipids (Fig. 7.6) [73, 74]. After intramuscular injection, the lipid capsule is taken up by cells via endocytosis, and proteins are produced by the translation of mRNA released into the cytoplasm. The produced proteins are fragmented in the proteasome and the fragments are taken up by the endoplasmic reticulum and displayed on the cell surface by exocytosis, resulting in the activation of the body’s immune system. This strategy assisted by defense mechanisms at the molecular level in human bodies will prove to be a powerful contribution to future clinical therapies.

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Fig. 7.6 Schematic illustration of the lipid-nanoparticle of mRNA vaccine and proposed mechanism for stimulation of immune system. Among cells taking up the nanoparticles, dendric cells translate proteins including spike proteins of SARS-CoV-2 and then displays those spike proteins and other proteins (MHC class II and CD80/CD86) for stimulation of the immunological cells (B cells and T cells)

7.2.2 Giant Vesicles A vesicle is a closed bilayer membrane of amphiphilic molecules with hydrophobic parts facing each other. Based on previous studies [75], the thickness of the membrane has been observed to be approximately 4 nm. Nanometer-sized vesicles are characterized as either small vesicles (less than approximately 100 nm) or large vesicles (approximately 100 nm to 1 μm). Vesicles with a diameter of 1 μm or more are called giant vesicles (GVs). GVs are of the same size as that of bacteria and cells and can be individually observed in real time under an optical microscope. Small and large vesicles can be relatively effortlessly homogenized in size, shape, and internal structure; hence, they are widely used as mentioned above. On the other hand, since GVs are considered difficult to handle quantitatively with good reproducibility, research on GVs has not been prevalent over the past 50 years. GVs prepared using conventional methods exhibit significant variations in their size, shape, and internal structure. In recent years, GV preparation methods have been greatly improved, and active research pertaining to the applications of GVs is being conducted [76]. GVs include giant unilamellar vesicles (GUVs) consisting of a single bilayer membrane and giant multilamellar vesicles (GMVs) that are enclosed by several overlapping bilayer membranes. GMVs have fewer inner aqueous regions than GUVs of the same diameter, making it difficult to encapsulate water-soluble substances and water-dispersed particles with a high volume ratio. The multiple nested membrane

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structure of GMVs causes a fault in the control of the membrane dynamics of sensing and releasing. Therefore, the preparation methods for GUVs encapsulating biological macromolecules and sub-micrometer-sized particles have primarily been developed [77]. For example, Hayashi’s group has developed GV-based cell/tissue markers using the GUV preparation process. In recent years, with a declining birthrate and aging population, minimally invasive treatments such as endoscopic surgery are in high demand for improving patients’ quality of life. Palpation of the objects is no longer available under these techniques, and surgeons need to locate target lesions inside the organs with the aid of only video and/or images of the objects. This complicates the surgical procedure. For example, laparoscopic surgeries in patients with early gastric cancer, occasionally require additional assistance from a gastroendoscope to localize the lesion during surgery [78, 79]. Hayashi’s group proposed the concept of a cell/tissue marker, based on the GV constructs in which multiple contrast agents can be encapsulated [80, 81]. When GV aggregates containing a near-infrared (NIR) fluorescent dye were administered preoperatively around the lesion of interest, they allowed direct localization of the target under NIR fluorescence laparoscopy. The group also demonstrated that preoperative administration of the same GV aggregates containing an X-ray contrast medium as well as NIR fluorescence dye also enables the positioning of undetectable lesions with X-ray computed tomography exams, aiding in preoperative simulations of surgeries [82]. Such multimodality of GUVs is deemed useful for further medical applications.

7.2.3 Self-contained Chemical Sensor GUVs have drawn considerable attention as new sensing devices. It provides a micrometer-sized reaction field that can possess membrane proteins anchored in the phospholipid bilayer membrane, which act as effective and specialized molecular sensors. Yanagisawa’s group reported that KcsA, a potassium ion channel, can change its function in inward and outward directions when incorporated into GUVs [83]. By using patch-clamp techniques, Shoji’s group demonstrated that the combination of synthesized antibody-tagged phospholipids and membrane-penetrating peptides enhances ion current induction by GUVs upon exposure to antigens [84]. Matsuura’s and Yokobayashi’s groups collaboratively constructed a GUV sensor for histamine, a membrane-permeant molecule, by encapsulating a cell-free protein synthesis system that contains RNA that responds to histamine as a riboswitch [85]. Ueda’s group constructed an immunodetecting GUV by inserting a chimera of the membranespanning receptor with an enzyme [86]. GUVs that can bind to phages via a transmembrane protein, Fhu A, and absorb their DNA was constructed by Dezi et al. [87]. Hamada et al. synthesized an insect pheromone receptor, which is a complex of two membrane proteins, BmOR1 and BmOrco, in GUVs and demonstrated that this receptor functions in GUVs using the patch-clamp technique [88]. As shown

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Fig. 7.7 Schematic illustration of a GUV system responsive to insect pheromone [82]

in Fig. 7.7, at a folding potential of −70 mV, inward currents of several picoamperes were recorded while the pheromone was added to the GUVs. Since the current was within the range of the amplitude for the single-channel conductance of insect pheromone receptors found in the living cells [89], it was indicated that BmOR1 and BmOrco folded properly and formed heteromeric complexes on the vesicular membrane. Not only functional membrane proteins (called ion channels) but also synthesized peptides and artificial molecules spanning the vesicular membrane have been developed as supramolecular transducers that convert the chemical signals between the inner and outer regions of GUVs [90, 91]. GUVs that actively interact with living cells through contact or fusion has also been developed. Kaneda et al. reconstituted connexin, a transmembrane protein that forms gap junctions between cells, into a GUV membrane by gene expression outside of the GUVs [92]. Through the gap junction of the cell, the molecule encapsulated in the GUV was transported into the cell and a reporter protein was produced in the cell. Nomura’s group provided another methodology for changing the cytoplasm of living cells. They fused GUVs into living cells by applying voltage [93]. These techniques for cell-GUV communication are expected to be applied as a new tool for cell therapy. Our lives are inseparable from light. The emergence of cyanobacteria boosted the oxygen concentration on the earth to just under 20% by the invention and evolution of photosynthesis which converts sunlight into chemical energy. To construct a minimum biological system for photosynthesis, Kuruma’s group has developed GUVs encapsulating ATP synthase, an enzyme for ATP production, and bacteriorhodopsin, a transmembrane protein that couples light energy sensing with intramembrane ion transmission [94]. ATP synthase and bacteriorhodopsin were buried into large vesicles, and these large vesicles were encapsulated in GUVs with mRNA of these proteins and cell-free protein synthesis reagents. They found that de

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novo photosynthesized bacteriorhodopsin and the parts of ATP synthase integrated into the large vesicles and enhanced the ATP photosynthetic activity through positive feedback of the products. This achievement not only demonstrated the construction of a self-contained light sensor system based on GUVs but it was also the first breakthrough for energetically independent functional GUVs using light.

7.2.4 Low-Bending-Modulus Compartment That Changes Its Morphology Since amphiphilic molecules have a small molecular weight and they self-assemble into a vesicular membrane by means of non-covalent bonds, the elastic modulus of the vesicular membrane is significantly smaller than that of a polymer membrane. Therefore, when cytoskeletal proteins such as actin fibers and microtubules are included in GUVs, GUV deformation occurs only by the force generated by the proteins, affording a GUV-based actuator. Cortese et al. first reported that GVs deformed by encapsulating actin and tubulin and reconstructing actin fibers and microtubules inside [95]. Takiguchi’s group found that, when actin was encapsulated in GUVs at a higher concentration than that found in living cells, it resulted in a nematic phase where actin fibers were aligned inside the GUVs [96]. These GUVs were also polarized according to the response of the environment to maintain the dynamic state of polymerization and depolymerization of the actin fibers. Dogic’s group enclosed microtubules and kinesins (a motor protein moving on microtubules) in GUVs together with a high concentration of polyethylene oxide [97]. The microtubules and kinesins were localized closer to the surface of the inner leaflet of GUVs due to the depletion effect of polyethylene glycol, thus resulting in a high orientation field (called a dynamic nematic phase) inside the GUVs. They observed that at the defect point of the nematic phase, the balance of the sliding motion of microtubules and kinesins was lost and GUVs formed protrusions. Loiseau et al. demonstrated GUV blebbing (foam-like morphological changes) when a part of the reconstituted actomyosin (complex of actin and myosin, which is a motor protein that binds to actin) was bound to the inner leaflet of GUV [98]. Sato et al. developed photoresponsive GUVs showing small blebbing, which was induced only during light irradiation [99]. They conjugated a part of kinesin to the inner leaflet of GUVs containing photoresponsive DNA and encapsulated the kinesin-DNA complex and microtubules in the GUV. These results are expected to contribute to the elucidation of mechanisms underlying cell polarity and motility.

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7.2.5 Self-propulsion GVs can move unidirectionally in water with the assistance of proteins. Van Oudenaarden’s group reported the self-propulsion of GVs equipped with actin comets, which consisted of actin and actin binding proteins, on the surface of GV membranes [100]. Takeuchi’s group installed flagella on the GV membrane, conferring GVs with the ability to migrate [101]. Kojima et al. further established a GUV-E.coli docking system for GUV transportation by linking the surface of GUV to that of E.coli with a membrane-tagged antibody [102]. Dogic’s group also showed that GUVs created by the abovementioned system [97] moved around on the substrate by changing the center of gravity of the GUV owing to the protrusions. There are some reports of GUV self-propulsion driven by a mechanism other than motor proteins. The vesicular membrane is a semipermeable membrane that can permeate water and other molecules. Ban’s group demonstrated that this allows the GUVs containing dextran to self-propel in an aqueous solution of polyethylene oxide [103]. They discussed the driving force of the self-propelled GUVs with respect to the transient interfacial energy produced by the permeation of polyethylene oxide and its mixing with dextran. Joseph et al. enclosed glucose oxidase and catalase into a vesicle composed of two amphiphilic block copolymers to create a self-propelled micro-object against a glucose concentration gradient [104]. The driving force for this micro-object was thought to be unidirectional phoresis caused by the heterogeneous concentration field (called self-diffusiophoresis [105]) formed by glucose consumption around the vesicle. The research group aimed to develop a new drug delivery carrier that reached the blood–brain barrier (highly selective semipermeable boundary composed of endothelial cells located between the brain nervous system and circulating blood) which has been difficult to achieve for conventional drug delivery carriers. By binding peptides that serve as substrates for the receptors of the blood–brain barrier onto the micro-object, they established an innovative “magic bullet” (this term is a concept developed by P. Ehrlich in the 1900s to refer to drugs, i.e., antibiotics, that are harmless to the human body but effective against microbes [106]) that reached the blood–brain barrier in rats in response to a glucose concentration gradient in the blood.

7.2.6 Cascade Reaction Systems for Information Conversion The closed space inside a GUV has been explored for signal processing of the chemical reaction networks. Peng et al. proposed a DNA-based nanopore gate and a signaling network that switches the gate in the presence of ATP [107]. Danelon’ group constructed a DNA amplification reaction using a feedforward circuit with DNA polymerase encoded from the DNA sequence [108]. GUVs that produce a quorum-sensing chemical signal perceived by bacteria were constructed by Stano’s

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Fig. 7.8 Schematic illustration of a GUV system which responds to glucose and releases insulin [101]. Insulin secretion is driven by membrane fusion of large vesicles containing insulin through the complexation of two membrane-anchored peptides (peptide K and E) which are tagged the large vesicles and the inner leaflet of GUVs respectively

group [109]. One of the highly important medical studies pertaining to the applicability of GUVs capable of signal processing was carried out by Gu’s group, who demonstrated the construction of artificial pancreatic β-cells that release insulin in response to glucose (Fig. 7.8) [110]. In their study, the GUVs encapsulated polyethylene oxide-coated large vesicles containing insulin. Because the inner leaflet of the GV and the outer leaflet of the large vesicles were modified with membrane fusion peptides, the polyethylene oxide chain coating the large vesicles prevented membrane fusion unless the chain was decomposed upon exposure to a low pH. In response to glucose stimulation, insulin was released from the inside of the GUV during the sequential process of pH decrease by glucose oxidase and catalase and the subsequent membrane fusion of the large vesicles driven by decomposition of the coating polymer. The pH in the GUVs was repeatedly recovered by gramicidin A embedded in the GUV membrane. When a hydrogel package including these GUVs was transplanted into mice with high blood glucose levels, the blood glucose levels decreased and returned to normal levels. Recently, Luo et al. demonstrated another medical applicability of GVs comprising a plasma membrane and modified by functional DNA for targeting tumor cells [111]. These GVs contained anticancer drugs and near-infrared phototherapy reagents. After administering the GVs into a tumor in mice and irradiating the tumor with near-infrared light, the weight of the tumor efficiently decreased. In addition to polymeric artificial cells, encapsulation of living cells inside GUVs enables the formation of organ-like structures. Elani et al. encapsulated living cells or bacteria inside GUVs to mimic the roles of cell organelles, and successfully built chemical sensors in which the cells were incorporated into one module of the chemical reaction network [112, 113]. Morita et al. demonstrated a GUV-based

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microbial incubator and examined how bacteria repeatedly divide and proliferate inside GUVs [114]. In addition, Dittrich’s group reported a new system in which the preparation channel and the observation chamber for GUVs containing bacteria were integrated into a microfluidic device [115].

7.2.7 Summary: Toward GUV-Based Molecular Robotics Among artificial cells, GUVs are a promising supramolecular platform for the construction of highly selective sensors, information convertors with chemical reaction networks, and flexible actuators. We have reviewed here that each functionalized compartment based on GUVs can work with biological systems including proteins, cells, bacteria, and even the entire body. When these GUVs are developed as building blocks for constructing a scalable system, a new molecular science pertaining to hybrid systems that utilize functionalized GUVs and living cells for practical molecular robotics is expected to get established.

7.3 Towards Artificially Controllable Nucleic Acid Drugs Kenzo Fujimoto K. Fujimoto Japan Advanced Institute of Science and Technology, Ishikawa, Japan e-mail: [email protected] One of the goals of molecular robots is in vivo medical applications. Molecular robots that can think and act independently will be able to sense information in the body, process it, output diagnostic results, and directly connect to treatment. This column describes nucleic acid drugs, which are candidates for drugs encapsulated in molecular robots, and the potential for pharmaceutical applications enabled by molecular robots. In recent years, research on nucleic acid medicine has been actively carried out using oligonucleotides as direct drugs. Antisense [116] and antigene [117] methods exert their medicinal effects by binding to target nucleic acids and inhibiting transcription or translation (Fig. 7.9a). In addition, the high sequence specificity of nucleic acids makes it possible to develop drugs with fewer side effects. The medical application aimed at molecular robotics is one of the important outputs of nucleic acid medicine since it is considered to be a useful therapy for genetic diseases. In nucleic acid drugs, the stronger the binding force to the target nucleic acid, the more effective the inhibition of transcription or translation. Ultra-Fast photo-Crosslinking (UFC), developed in the Fujimoto laboratory, is capable of photo-crosslinking with target nucleic acids in a few seconds of photo-irradiation, thus enabling highly efficient gene expression inhibition (Fig. 7.9b, c). We introduced antisense nucleic acid including photoresponsive artificial nucleic acid into GFP-HeLa cells

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Fig. 7.9 a Nucleic acid drug. b DNA/RNA photo-cross-linker. c Antisense method using RNA photo-cross-linking. d Inhibition effect of photo-induced antisense method. e Photo-induced double duplex invasion DNA

by lipofection method and irradiated them with light [118]. As a result, the gene expression of the GFP gene was successfully suppressed by nearly 90% after 10 s of light irradiation (Fig. 7.9d). The timing of light irradiation is also an advantage of the photo-manipulation method because the reaction can be controlled, and the gene expression can be suppressed by the timing of light irradiation. In addition, this photomanipulation technique can be applied to the antigene method by cross-linking to double-stranded DNA. Currently, we have successfully developed a photo-induced double duplex invasion (pDDI) technique for double-stranded DNA by using 5cyanouridine as well as 3-cyanovinylcarbazole, which is a light-responsive artificial nucleic acid [119]. Therefore, the photo-cross-linking technology is expected to be applied to the antigene method in the future (Fig. 7.9e). We believe that one of the goals of molecular robotics is a molecular doctor who can perform the entire process of detection, diagnosis, and treatment. Until now, diseases have been diagnosed and treated (drugs administered) by doctors. However, this is because molecules are not equipped with the ability to think independently. Molecular robots, on the other hand, are able to think independently. Nucleic acid medicine therapy using molecular robots is expected to develop as a new therapeutic method that is not available in conventional antibody drugs or small molecule drugs (Fig. 7.10).

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Fig. 7.10 Gene therapy using molecular robots

7.4 Dream of Molecular Hayabusa Satoshi Murata S. Murata Department of Robotics, Tohoku University, Sendai, Japan e-mail: [email protected] As an application of molecular robotics in the near future, sample-and-return exploration of living organisms by molecular robots (Molecular Hayabusa) is being considered, just as the Hayabusa satellite1 brought back samples from asteroid Itokawa to Earth. Molecular robots are expected to be able to enter deep into living organisms and bring back molecular samples to check health conditions and diagnose diseases that cannot be detected using ordinary diagnostic techniques. The specific procedure for “molecular Hayabusa” is as follows: A solution containing a large number of microbe-sized molecular robots is injected into a blood vessel, and the robots are carried by the bloodstream to every corner of the body. Each molecular robot collects information regarding the presence or absence of a particular molecule in the bloodstream. When a particular molecule binds to a receptor on the surface of the robot, it is transmitted inside the robot, and the molecular computer inside the robot records the presence, absence, and concentration of the molecule. Additionally, if the robot determines that the surrounding environment meets certain conditions, it can collect molecular samples from that location. For example, if the concentration of a particular cancer marker molecule is high, the molecular robot strips the molecule from the nearby cell surface and takes it inside. Molecular robots do not have the ability to swim on their own; however, because they are scattered throughout the body, a small number of them can be retrieved 1

Hayabusa (“Peregrine falcon”) was a robotic spacecraft developed by the Japan Aerospace Exploration Agency (JAXA) to return a sample of material from a small near-Earth asteroid named 25,143 Itokawa to Earth for further analysis. It landed on the asteroid, collected samples and returned to Earth in 2010.

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Fig. 7.11 Concept of molecular Hayabusa

by the collection dock (blood collection patch). Vehicles that are not recovered are naturally broken down by enzymes in the blood. When a small amount of sample is brought back by the molecular robots to the collection dock and concentrated and passed through a nanopore, the weak current pattern will provide information on the sample molecules. By analyzing this information using bioinformatics, a precise diagnosis is thus possible. The realization of molecular Hayabusa will require the development of various technologies such as receptors to capture specific molecules, molecular devices to acquire molecular samples, reaction circuits to store molecular inputs, analysis technology for trace molecules, and advanced bioinformatics (Fig. 7.11).

7.5 Engineered Cell Kei Fujiwara K. Fujiwara Keio University, Yokohama, Japan e-mail: [email protected]

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Fig. 7.12 An illustration of engineered cells

Molecular robotics is a discipline belonging to chemistry and engineering that designs molecules and treats them as robots. Biology also has a research field in which cells are remodeled as robots to express desired functions. These kinds of fields are called synthetic biology or cell engineering. As introduced in another chapter, synthetic biology includes studies in the chemical synthesis of living cell-like materials by combining elements of living cells. This chapter focus on the mainstream of synthetic biology that uses living cells as a platform for engineering. It should be noted that there is no barrier between this kind of synthetic biology and cell engineering. Although these two fields are sometimes distinguished from the degrees of modification, it is almost meaningless. In this chapter, these two fields are integrated, and the obtained product is called Engineered Cell, and the concept is compared with molecular robotics (Fig. 7.12).

7.5.1 Engineered Cells The definition of engineered cells is simple: the cells that have a different function from the original cells due to artificial manipulation. In many cases, the manipulations result from genetic modification, such as gene transfer or disruption. Whereas molecular robotics uses chemical molecules as a base of the construction, engineered cells are built up from living organisms. Modification of the parts of living cells to create cells with robot-like is the basis of engineered cells. If you ask what engineered cells can do, the answer is that they can do a lot. A very wide variety of engineered cells have been constructed, from plant cells that are rich in nutrients that are difficult to synthesize in nature, to microorganisms that synthesize precursors for stimulants, and even microorganisms that process perceived ambient information and execute internal functions like a microcomputer. Engineered cells are materials based on living cells and are created by gene manipulation (genetic modification or genome synthesis). Many engineered cells have been developed and they work similar to molecular robots.

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In order to understand engineered cells, it is necessary to understand the term “gene expression”. Organisms maintain homeostasis and achieve self-reproduction by converting information from genes encoded in genomic DNA into proteins. This process is called gene expression. There are many types of proteins, but three are important in understanding the “grammar” of engineered cells. The first is the receptor, which receives environmental signals and transmits them to the inside of the cell. Since cells have no eyes, molecular recognition by receptors plays a role in the eyes. As an aside, the fact that humans perceive light with their eyes also comes from the function of receptors. The second is transcriptional factors, which mainly control the ON and OFF of gene expression. Although in some cases it is used to regulate the intermediate levels between ON and OFF, ON and OFF are sufficient for understanding in the context of molecular robotics. Transcriptional factors control ON and OFF by receiving a signal from a receptor, or they control ON and OFF by sensing a compound in a cell. The third is an enzyme, which converts a compound into a catalyst. There are too many types of enzymes to list, but components like pheromones, flavor, nutrition and pharmaceutical chemicals that animals, plants, and microorganisms synthesize in nature are all synthesized by the action of enzymes in cells. What is important is that all three of these elements are proteins that have been converted from genes. As an example, consider creating cells that emit the flavor of jasmine. This can be done by using the genes in jasmine for the enzyme that synthesizes cis-Jasmone and methyl jasmonate, which are the flavor components of jasmine. By expressing these genes in the target cells, the cells will be able to emit the flagrance of jasmine flavor. Consider an application of this method to synthesize jasmine-flavor cells. A gene encoding a photoreceptor and a transcriptional factor that receives the signal of the photoreceptor and turns on the expression of the gene only when it is not exposed to light are introduced into the target cell. If the gene that turns ON at this time is a gene that synthesizes the components of jasmine flavor, cells that emit jasmine flavor only at night. Because this engineered cell has the ability to operate according to the situation, it is possible to regard these engineered cells as a kind of molecular robot. Engineered cells developed so far are classified into 5 categories; (i) chemical plants, (ii) functional materials with sensing ability, (iii) biosensors, (iv) microcomputer-like objects, and (v) cells created by biological interests or chemical interests, including remodeling of basic components of cells and attempting to understand the origin of life.

7.5.2 Engineered Cell as a Chemical Plant The most popular use of engineered cells is to treat them as a compound production factory by utilizing the fermentation ability of cells. Microbes synthesize alcohols like wine and beer and foods like cheese and yogurt from glucose or milk. This process is called fermentation. The fermentation is carried out by the enzymes within the cells. As mentioned above, an enzyme is a protein synthesized based

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on genetic information. Hence, the introduction of a gene for producing wine into cells that cannot produce wine confers them to the ability of wine fermentation. For example, these kinds of modifications are applied to create novel rice. Natural rice does not contain β-carotene, which is a precursor of vitamin A, but rice cells can synthesize β-carotene if genes for synthesizing vitamin A were introduced. In fact, such rice is sold as Golden rice. Synthesizing various industrial products and pharmaceutical ingredients. Engineered cells created by similar strategies can produce butanol and isopropanol which are usually obtained from fossil fuels, opioids which are anesthetic components taken from plants, and artemisinin which is a therapeutic agent for malaria, and synthesize insulin which is a critical diabetes medicine. To create the fermentative engineered cells, the information needed is how the chemical reaction is catalyzed by enzymes and the gene sequences of the enzymes. At present, it is possible to synthesize any size of DNA, and therefore, “genes” can be prepared once the genetic information of target enzymes is known. With over 10,000 types of enzyme-catalyzed reactions, the varieties of fermentation by engineered cells will be further expanded. In many cases, production levels by the fermentative engineered cells do not reach the levels sufficient for industrial usage, and it is not necessarily possible to synthesize compounds without another organic chemical reaction. However, they are suitable for the synthesis of rare compounds and the provision of intermediate materials for organic compounds. Future studies will expand the possibility of fermentative engineered cells.

7.5.3 Engineered Cell as a Functional Material with Sensing Ability Microbes are everywhere around us, and most of them are harmless. Some microorganisms cause food spoilage, but some can play a role in environmental purification. For example, when heavy oil spills from a tanker, some special bacteria that accidentally arrive decompose them and grow around it, and as a result, the heavy oil is removed from the environment. A plastic, polyethylene terephthalate (PET), was thought to be unable to be decomposed naturally, but bacteria that can decompose PET were also found around the PET disposal site, and studies demonstrated that an enzyme (PETase) plays the role. Cellulose, which is a component of paper, is indigestible by humans, but some microorganisms can decompose it into glucose as their nutrient. However, heavy oil, PET, and paper are needed for our daily life, so they should be decomposed only when they are wasted. Therefore, there is a need for cells that sense the environment and function only when desired. Creating something like this is one area of Engineered cells. There is another interesting example. Since concrete gradually deteriorates in the environment and loses its strength, its maintenance is a very important issue. In this regard, attempts are being made to repair cracked concrete using engineered cells. Some bacteria synthesize calcium carbonate as their shell and also have the

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ability to form a stable structure called a spore. Spore is an inactive state of the cells, but germinates when exposed to water and becomes an active state that can synthesize the original calcium carbonate. When the spore cells are sprinkled on cracked concrete, they penetrate the crack and become a material that fills the gap with calcium carbonate. If this spore was mixed with concrete in advance, it will automatically repair when the crack occurs. This repair system does not necessarily use engineered cells because natural cells have this ability, but it is a good example of using cells as a molecular robot. Engineered cells as medical robots are also being considered. For example, an engineered cell called CAR-T is a modification of our immune cells that is programmed to sense and specifically kill cancer cells. There are various other examples, though they are not mentioned here. All of them can be considered molecular robots, because they express the desired function by processing information obtained by the signals or changes in the environment and the target.

7.5.4 Engineered Cell as a Biosensor It is not necessarily safe to drink water in the environment. Even if the water looks clean at first glance, if there are poisons such as arsenic dissolved in it, long-term ingestion will be harmful to your health. If you are just going on a short trip, you can just bring your own food and drink and come back. However, if you are going to stay for a long time and live there, the situation is different. There are many things to be concerned about, such as whether the water contains arsenite and whether there are any pathogens. The availability of drinking water is a very important issue in many countries. At the very least, one should be able to immediately and easily determine the presence of any known harmful substances. Furthermore, it is desirable to be able to test in places where there is no electricity. The sensing and processing capabilities of cells can be used for such applications. There are sensor proteins for a wide variety of compounds, including heavy metals and environmental pollutants, not limited to arsenite. Although receptors may play a role, transcriptional regulators that are activated by toxins are often used. For example, a transcription factor called ArsR turns on the expression of a specific gene in the presence of arsenite, and if the gene expressed is visible, such as a fluorescent protein like GFP, it is possible to determine if the water contains arsenite. In fact, engineered cells with gene circuits that enable such arsenic detection have been developed and put to practical use.

7.5.5 Engineered Cells Behave as a Microcomputer The calculation and operation of computers are based on logical bits using the states of 0 and 1. Since turning off and turning on the expression of genes in a cell also

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corresponds to 0 and 1, the more gene expression control is increased, the more logical operations, or computing, can be performed. Of course, cells are capable of sensing enzymatic reactions and receptors that produce larger changes than the logical operations produced by 0 and 1. However, sometimes it is necessary to change the output by making conditional decisions like a microcomputer. For example, we can imagine a function in which bacteria exist as engineered cells in the intestines, releasing drugs when the gastrointestinal tract is irritated, and stopping the synthesis of drugs upon recovery. In this case, if there is a way to replace the phenomenon of gastrointestinal upset with a signal molecule, it is easy to create engineered bacterial cells that can sense the signal and release the drug. However, if it is necessary to make a decision based on a number of signals, or if judgment based on a number of conditional branches is required, it is necessary to engineer the cells to synthesize a number of drugs within a single cell. In such cases, the computing processes inside cells are essential. In the 1990s, the idea of implementing microcomputer-like computing circuits into cells seemed like a dream. However, recent studies have made it possible to assemble genetic circuits like electronic circuits, as in the construction of genetic circuits that oscillate like a clock, and genetic circuits that can transition between two states as a toggle switch. Furthermore, researchers have become clear on how to create gene circuits that do not interfere with each other, and finally, a tool called Cello has been created to automatically design gene circuits that realize desired logic gates. At this point, we are still in the stage of improving the parts and making them controllable. In the future, as the tools become more accurate and practical, the number of combinations of parts for decisions and functions will increase enormously, and one day, an engineered cell may be announced as a molecular robot with judging functions like computers.

7.5.6 Engineered Cell to Understand Life and Its Origin The most remarkable development in the field of engineered cells in the 2010s is the synthesis of genome-size DNA. The ability to synthesize the genome of an organism has led to the creation of engineered cells for deeper intellectual curiosity and practical needs. For example, the codon table of Escherichia coli was rewritten by the genome synthesis. Information about genes was translated by converting three-letter DNA sequences (codon) to the corresponding amino acids. Since 64 (=43) codons were linked to 20 amino acids, there is redundancy. In the engineered cells, several redundant codons were unliked to amino acids to be linked to other non-natural amino acids if needed. Examples of this kind of cell engineering are not limited to the modification of the codon table. In genomes of living organisms, genes have been scattered during the process of evolution, and their positions seem to be settled through a stochastic process. Research using genome synthesis conducts to confirm the extent to which this randomness can be reduced by genome synthesis. Cell engineering has also

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shown that cells can possess two types of ribosomes, which convert RNA into protein using different initiation signals. In addition, studies have shown that engineered cells have the capacity to store information, not genes, similar to USB memory or HDD. The engineered cell, driven by this interest, is also expected to be used to solve practical problems, such as the development of plants that are immune to viral infections, low-cost information distribution media, and as a driving force for constructing cells with a large circuit of metabolism for fermentation.

7.5.7 Summary and Futures in Engineered Cells The development of engineered cells progresses day by day (For a more detailed overview of the field, see Ref. [120]). As with the increase of research fields that molecular robotics can be applied, the scope of research applied by engineered cells is also increasing year by year. As mentioned at the beginning, they have the same goal, only the difference is whether they use molecules or are cell-based. Since both are hot research, the information in this chapter may be out of date in 10 years. The future in which engineered cells acquire abilities that were unexpected at the time of writing this chapter and behave like a sophisticated robot will come true.

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Chapter 8

Social Acceptance of Molecular Robots Akihiko Konagaya

Abstract Molecular robotics is an emerging technology that has the potential to significantly impact society. To promote beneficial research on molecular robotics, careful consideration must be made from the viewpoint of ethical, legal, and social implications (ELSI). This chapter presents an overview and background of ELSIs in molecular robotics, with particular focus on discussions and practices concerning ELSIs in ongoing research. Lessons learned from previous cases provide direction for considering the ELSIs of molecular robotics, and examining such guidance highlights a starting point for discussing the broad impacts of molecular robotics that need to be communicated to society in the future. To that purpose, this chapter focuses on cases of genetically modified organisms (GMO) and synthetic biology as essential references for examining the ELSIs of molecular robotics from the perspective of responsible research and innovation (RRI).

Molecular robotics is an emerging technology that has the potential to significantly impact society. To promote beneficial research on molecular robotics, careful consideration must be made from the viewpoint of ethical, legal, and social implications (ELSI). To that end, a molecular robotics ELSI project has been conducted since 2016 in joint research between molecular robotics researchers and researchers in the social sciences and humanities supported by the Human–Information Technology Ecosystem (HITE) at RISTEX within Japan’s JST. This chapter presents an overview and background of ELSIs in molecular robotics, with particular focus on discussions and practices concerning ELSIs in ongoing research. Lessons learned from previous cases provide direction for considering the ELSIs of molecular robotics, and examining such guidance highlights a starting point for discussing the broad impacts of molecular robotics that need to be communicated to society in the future. To that purpose, this chapter focuses on cases of genetically

A. Konagaya (B) Keisen University, Tama, Japan e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2022 S. Murata (ed.), Molecular Robotics, https://doi.org/10.1007/978-981-19-3987-7_8

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modified organisms (GMO) and synthetic biology as essential references for examining the ELSIs of molecular robotics from the perspective of responsible research and innovation (RRI).

8.1 Ethics in Molecular Robotics: Issues and Needs Akihiko Konagaya

8.1.1 Background Molecular robots are artifacts made of biological molecules and materials and that have the functions of sensors, processors, and actuators—that is, the three functions well-known as the basic components of robots. Unlike electromechanical robots, molecular robots have better synergy with human bodies because they are made of biomolecules and biomaterials such as liposomes, proteins, and nucleic acids [1]. However, administering molecular robots into the human body requires biomedical guidelines that consider safety, effectiveness, and quality to be in place before clinical trials and research are initiated. It is also important to gain social consensus regarding the administration of molecular robots in the human body [2]. To understand and accommodate the ELSIs of molecular robotics, a molecular robotics ethics project [3, 4] was launched as part of HITE within JST from 2017 to 2020. Focusing on ELSIs, RRI, and the technology assessment (TA) of molecular robotics, the project was promoted by molecular robotics researchers in collaboration with researchers in the social sciences and humanities [5–8]. During the project period, eight workshops, three international symposia, and four online workshops were held. Throughout the symposia and workshops, the project promoted understanding of ELSI in molecular robotics, molecular robotics TA, and the formulation of guidelines as well as ELSI practices for molecular robotics in the Biomolecular Design Competition Student Contest JAPAN (BIOMOD JAPAN), as detailed in the following sections.

8.1.2 Ethical, Legal, and Social Issues (ELSI) in Molecular Robotics ELSI is a technical term proposed in the context of the human genome sequencing project in 1990 to study various topics, including the sanctity of life, the deterrence of discrimination, information protection, and the right to know, that emerged with A. Konagaya Keisen University, Tama, Japan e-mail: [email protected]

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the identification of human genome sequences [9]. Since then, the term “ELSI” has been adopted in many large, advanced research and development projects, especially in the life and medical sciences. To study the ELSIs of molecular robotics, the case of synthetic biology represents one of the most important references. The “Environmental Risk Assessment and Management Forum Report” published by the ministry of economy, trade, and industry of Japan (METI) in 2013 summarizes the social implications, legal issues, and future ethical issues to be discussed concerning synthetic biology. Although molecular robotics is similar to synthetic biology—both aim to develop artificial cells by using DNA sequences to control biomolecular systems—they fundamentally differ in the sense that the final products of molecular robotics and synthetic biology are categorized into bioengineered products and living things, respectively. Therefore, it is important to discuss the ELSIs of molecular robotics from the viewpoint of artifacts, even if molecular robots behave similar to living things. The difference between bioengineered products and living things may encourage different interpretations of the Cartagena Protocol (“Act on the Conservation and Sustainable Use of Biological Diversity through Regulations on the Use of Living Modified Organisms”). Because the objective of the Cartagena Protocol is the regulation of living modified organisms, it may not be applicable to molecular robots, which raises further questions concerning the boundaries of living modified organisms, for the Protocol also regulates viruses and phases as living modified organisms. From the viewpoint of biosafety, molecular robots also have unique ELSIs regarding their environmental survivability, because the emergence of unforeseen traits could pose risks to existing ecosystems. As for social implications, molecular robotics have the same potential risk of dual-use similar to synthetic biology, for both can be used to improve the quality of life or bring about the opposite.

8.1.3 Technology Assessment of Molecular Robotics TA is an activity undertaken to support decision-making by analyzing social implications and problems during the early development of technology. TA is useful for evaluating advanced innovative technologies that are difficult to compare with conventional research and development systems, innovation systems, and legal systems [10]. In the past two decades, the TA of social aspects of emerging science and technology have been discussed along with the concepts of real-time TA (RTTA) and RRI. On the one hand, RTTA aims to extract early warnings and lessons from previous cases and give feedback on natural scientific discovery, engineering innovation, and related scientific policies during the early development of technology—that is, not retrospectively but in real time [11]. On the other, RRI aims to guide innovations for socially desirable technologies by discussing positive and negative impacts as well as normative issues in the early development of technology in consideration of the redesign of enhanced innovation ecosystems [12]. The RTTA and RRI activities undertaken in the project on ELSIs in molecular robotics included the following.

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The real-time TA of molecular robotics was conducted during the collaboration of two groups in JST’s HITE project: the “Co-Creation and Communication for RealTime Technology Assessment (CoRTTA) on Information Technology and Molecular Robotics” project (Ryuuma Shineha, Osaka University) and the “Co-Creation of Molecular Robotics ELSI and Real-Time Technology Assessment Research” project (Akihiko Konagaya, Keisen University). The CoRRTA group developed the subject of discussion co-creation platform (NutShell) to promote research on real-time TA. NutShell is designed to gather feedback from policymakers and various stakeholders, including citizens, by means of disclosing information online. NutShell is expected to issue feedback for research and development by extracting opinions from the general public outside the research community. As an RRI activity, the “Molecular Robotics ELSI/RRI Insight” workshop was held at TITECH’s Tamachi Campus in February 2018. During the workshop, 38 people including molecular robotics researchers, ethics researchers, and journalists discussed the desirable and undesirable futures of molecular robots. The participants were divided into four groups to discuss the following opposing axes: (1) from the near future to distant future, (2) from the individual to the social, (3) from individual decision-making to social decision-making, and (4) from not yet done to already done activities, from will-be-realized to will-not-be-realized activities, and from profitable to welfare activities. Insights from various axes forecast the desirable future of the “rapid progress of interdisciplinary research” and “medical applications” and the undesirable future of the “delay of research,” “military use,” and “uncontrollable use.” More information is available on Molecular Robot Ethics website [2].

8.1.4 Formulating Guidelines for Molecular Robotics The Molecular Robotics ELSI and Real-Time Technology Assessment Research cocreation group has formulated ethical guidelines for research on molecular robotics. The group adopted the same strategy used by legal systems for formulating guidelines, principles, basic research guidelines, and application guidelines (e.g., pharmaceutical and agricultural guidelines). At the annual Symposium on Molecular Robotics in 2017, Naoto Kawahara (Kyushu University) presented a draft proposal of principles for molecular robotics that were later approved after a year of discussion with up-and-coming researchers in molecular robotics [2]. Ethical Principles of Molecular Robotics (ver. 1.0) Preamble: Making much progress in creativity and ingenuity of technology, a new device or system appears continuously. But there are concerns that ethical scope of molecular robotics ranges widely. In Japan, there has been promoted research and development of molecular robotics, taking advantage of an important elemental technology concerning sense, motion, and intelligence. It is feasible that more complicated

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configurations of molecular robotics will be applied to informatics, engineering, chemistry, and biology in near future. This could be also applied to medicine. So, it is an issue of capital importance to establish an ethical framework with a new view of material, information, and life according to such a progress of technological development. In this context, we formulate the following ethical principles. We also request any person who engages in molecular robotics to comply with the principles. Ethical Principles: 1.

Comprehensive assessment of risk and benefit

2.

Any person who engages in molecular robotics shall make a comprehensive assessment of burdens to be caused on human and environment as well as predicted risks and benefits. Then they shall also take measures to minimize those burdens and risks. Consideration for safety and environment

3.

Any person who engages in molecular robotics shall take containment and safety measures for artificially modified organisms which may affect environment. This includes ethical responsibility and consideration for future generations. Paying attention to security and dual-use issues

4.

Any person who engages in molecular robotics shall investigate security measures in consideration of physical, personnel, transport, material, and information aspects. They shall also pay attention to dual-use issues. Ensuring accountability and transparency Any person who engages in molecular robotics shall ensure accountability and transparency for the public good, making progress of the research and development rooted in social justice.

The above principles were translated from the Japanese version into English, on the July 28, 2018.

8.1.5 Molecular Robotics ELSI Practices in BIOMOD Japan For young researchers and students to practice adherence to the ELSI guidelines set for molecular robotics, the organizers of BIOMOD Japan have required student teams to address their works possible ELSIs in their project-related wikis [13]. The use of ELSI-oriented wiki pages originated from the International Genetically Engineered Machine (iGEM) student contest in synthetic biology [14]. It should be noted that such ELSI practices took more than three years to gain attraction in the iGEM community, which suggests the importance of keeping ELSI practices as a part of BIOMOD in order to make consideration of ELSIs commonplace in the molecular robotics community.

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8.1.6 Summary This chapter has described the current status of ethics and ethical issues in molecular robotics including ELSIs, TA, guideline formulation, and BIOMOD’s ELSI-oriented practices. Molecular robotics is considered to be an emerging technology that will greatly impact society. Although it may be too early to discuss ELSIs when we look at the current status of research on molecular robotics, ethics in molecular robotics is a social experiment working toward a bright future in the early phase of research and development.

8.2 Ethical, Legal, and Social Issues (ELSI) in Molecular Robotics: An Introduction for Further Discussion Ryuma Shineha Research and development in molecular robotics will bring many scientific insights and broad impacts to society. At the same time, experts are expected to consider various ethical, legal, and social issues (ELSIs) and how to communicate molecular robotics research to society. Understanding the lessons from previous cases is essential for examining ELSIs related to molecular robotics.1

8.2.1 Lessons from Genetically Modified Organism (GMO) Controversies Previous controversies involving genetically modified organisms (GMO) provide several lessons to consider when it comes to the ELSIs of emerging science and technology. Particularly, I would like to focus on lessons from public dialogue in the UK. In the UK, the collapse of trust in expertise became a significant issue concerning the relationship between science and society after the Bovine Spongiform Encephalopathy (BSE) affairs in the 1990s. The “GM Nation?” public debate was conducted as a nation-wide public dialogue on GMOs under the pressure to recover trust in expert authorities. Several reflective lessons on ELSIs and science communication were gained through “GM Nation?” The significant implications are summarized below [15–20]:

R. Shineha Osaka University, Osaka, Japan e-mail: [email protected] 1 For example, a technology assessment on the social aspects of biotechnology was conducted by the research group SOKENDAI in 2014 [29].

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• People’s diverse concerns: we need to look at concerns that are not limited to scientific and technical aspects such as food and environmental safety, but also include social and political issues. • Understanding of risk and benefit: as we learn about the benefits, we also become more concerned about the risks (especially risks that require long-term observation). • Backlash against easy commercialization: more testing, a solid regulatory framework, and presentation of benefits to society at large (not just producers) are required. • Distrust of governments and multinational corporations: people were concerned that “GM Nation?” was just for alibi-making, that the results would be ignored, and that the interests of multinational corporations would be prioritized. Although participants acknowledged the benefits of GM crops, their doubts about multinational corporations were not dispelled. • Further information and pilot studies: there were requests for more information from reliable sources and further tests. • Special attention to the situation in developing countries: there was recognition that GM crops can contribute to developing countries through increased food production and other effects. At the same time, however, people saw the importance of promoting fair trade, better food distribution systems, improved incomes, and increased status of the countries concerned. • Welcoming and valuing debate: participation in dialogue and discussion was welcomed, and opportunities were created to express one’s own views and to listen to and discuss the views of others, including experts. In addition to these implications, one of the significant lessons in “GM Nation?” is the importance of continuous dissemination and communication from an early stage. In other words, the sharing of information and discussion even before GMOs became controversial was considered a prerequisite for building a better social agenda around the technology in question.2 How has the social discussion around GMOs developed in Japan? GMOs rapidly became a topic of discussion in political and social contexts in Japan with the development of GM foods and the establishment of safety evaluation systems from the mid-1990s. It was in this context that negative reactions to the use of GM foods appeared, and people’s sense of avoidance toward GMOs remains strong [21]. In response to this situation, the Hokkaido local government issued the “Hokkaido Ordinance on the Prevention of Hybridization Due to the Cultivation of Genetically Modified Crops” on March 31, 2005, due to concerns about harmful rumors concerning GMO. This ordinance set much stricter standards for field trials of GM crops than the national standards. Scientists voiced concerns that this ordinance may hinder their research. One of the most significant moves on the part of scientists in response to the Hokkaido ordinance was a proposal issued in January 2005 by a joint 2

A consensus conference led by the Hokkaido government was held as an example of a dialogue on GMOs in Japan. In these discussions, many issues similar to those of “GM Nation?” were raised [30].

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statement of six academic societies in plant science, called “Seeking a system to promote appropriate acceptance of genetically modified plants in society.”3 Unfortunately, however, the joint statement was released just before the enforcement of the Hokkaido ordinance (and, of course, discussions on the ordinance had started earlier yet). At the time the statement was released, peak interest in mass media had already passed.4 In addition, the tone of media coverage concerning GMOs was positive until the mid-1990s with expectations for medical and industrial applications, and then became negative [22].5 In this case, it can be said that the GMO controversy is an example of researchers’ failure to participate in the process of agenda building.

8.2.2 Synthetic Biology Case Studies To gain insight into ELSIs concerning molecular robotics, previous discussions in the field of synthetic biology should not be overlooked. Those in synthetic biology learned from the bitter experiences of the GMO controversy, paying more attention to ELSIs and policy from the very beginning of the formation of the field. Thus, active discussions have taken place in both domestic and international research communities. For example, a session on ELSIs was held at iGEM, an international student competition in synthetic biology, and other active discussions have been organized. Also, in Japan, presentations and discussions on ELSIs have been held in the Japanese Society for Cell Synthesis Research from the beginning. In this context, major ELSIs and science policy issues related to synthetic biology have been raised based on the implications of the GMO controversy. In this section, we summarize the discussion points in a table in order to weigh the implications of GMOs and synthetic biology as we consider future discussions on molecular robotics (see Table 8.1) [15–20, 23–25].6

3

This is a joint statement by six academic societies: the Japanese Society of Plant Physiologists, the Japanese Society of Agricultural Chemistry, the Japanese Society of Breeding Science, the Japanese Society for Plant Cell and Molecular Biology, the Horticultural Society of Japan, and the Japanese Society for Plant Chemical Regulation: https://jspp.org/16appeal/teigen2005.html. (Access to this site is broken as of May 2018. However, you can read it by entering the URL into the Wayback Machine of the Internet Archive.) In addition, before this joint proposal by the six societies, there had been efforts such as a statement by the Japanese Society for Plant Arrangement and speech activities on blogs by individual researchers. However, such systematic social communication as the six societies’ joint proposal was exceptional. 4 The peak of media coverage was around 2000, when news focused on the anxiety and risks surrounding food use [22]. 5 Looking beyond Japan, the debate over GMOs has often been discussed in a positive tone in the context of medical and industrial applications, whereas in food use it has been discussed in a negative tone, especially in Europe [31–34]. 6 It should be noted that the discussion around synthetic biology is often conducted with GMOs as a reference point, and that there are many common issues between the two fields.

Risk of environmental releases under uncontrolled conditions Security issues, bioterrorism How to think about artificial life and biological organisms How to understand the potential impacts A cautious or judgmental attitude Conflict with the precautionary principle Limitations of technology assessment

A wide range of concerns and interests, including not only scientific and technical aspects such as food and environmental safety, but also social and political issues The need for testing and research is recognized

As people learn about the benefits, they also become more concerned about the risks (especially those risks that require long-term observation)

Backlash against easy commercialization Request of appropriate regulatory framework Presentation of benefits not only to producers but also to society at large

Interest in developing countries

People’s concerns and interests

Understanding risk–benefit

Commercialization

Fair distribution of wealth (social/distributive justice)

Intellectual property Various debates over biological patents right

Mainly from the 2000s

Mainly from the 1990s

How can we present the benefits to a wide range of people?

How do we understand the medium- to long-term effects on health and the environment? How do we establish an assessment system?

What are the ELSI and political issues concerning Molbot? How to consider dual use issues

Implications for molecular robotics ELSI

Treatment of intellectual property rights Open source innovation

(continued)

Necessity of discussion on intellectual property rights and standardization

Issues on technology trade How do we gain a broader perspective on Infrastructure and environment for research the North–South issue of advanced science and technology?

–

Synthetic biology

Genetically modified organisms (GMO)

Table 8.1 To consider the ELSI on molecular robotics: discussion points in previous emerging science and technology

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ELSI sessions at the international student competition (iGEM) ELSI discussions in the Japanese Society for Cell Synthesis Research –

Actions of academic Concern that research will be limited communities Researchers’ failure to participate in the process of building a social agenda

Media attentions

Initially, there were many positive articles with strong expectations for medical and commercial applications, but then negative articles in food use became apparent

Awareness of the keywords is increasing Lack of understanding about the future scenarios of synthetic biology and its utilization

Governance and regulation The treatment of risk management in policies Responsibility for management of technology and transparency of information disclosure

Mainly from the 2000s

Mainly from the 1990s

Distrust of authority Isn’t ELSI just an alibi-making and its arguments will be ignored? Fairness and transparency Request of more information from reliable sources

Synthetic biology

Genetically modified organisms (GMO)

Communication and Do citizens feel welcome to reflexivity - participating in dialogues and discussions, - expressing their own opinions, - opportunities to hear and discuss the opinions of others, including experts

Building a trust with society

Table 8.1 (continued)

What kinds of themes are discussed similar to? Early understanding and warning, considearing overseas situations

How do we demonstrate the autonomy of Molbot researchers Active ELSI discussions and communicate them to the society

Ensuring a public engagement scheme for various stakeholders How do we conduct co-creation of future scenario?

Consideration of measures to ensure fairness and transparency How to ensure accessibility of a wide range of information?

Implications for molecular robotics ELSI

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Public attitudes toward synthetic biology have also been investigated. For example, a survey in the United States published by Hart Research Associates found that awareness of the key term “synthetic biology” has been increasing over the years. Simultaneously, it showed that people took a “wait and see” or precautional attitude to the relationship between risk and benefit of synthetic biology [24, 26].

8.2.3 Issues in Communication with Society: A Case Study of Regenerative Medicine What are the points that need to be considered when it comes to communication between science and society? As part of the Risk Communication Model Formation Project, funded by the Ministry of Education, Culture, Sports, Science, and Technology (MEXT), a survey was conducted by the Japanese Society for Regenerative Medicine (JSRM) involving 1,115 members of the JSRM and 2,160 respondents from the general public. Based on the results, differences in communication concerns between the general public and experts on regenerative medicine were examined. The results of the survey point to gaps between what the general public “wants to know” and what JSRM members “want to tell.” JSRM members tend to emphasize the scientific validity and mechanisms of scientific research and medical applications. On the other hand, the general public tends to be interested in matters that come about after the realization of the technology, such as risks, treatment costs, measures to deal with contingencies, and responsibility systems [27].7 In addition, the general public had a strong sense of avoidance toward the creation of human-animal chimeras, in contrast with their highly positive and supportive attitudes toward regenerative medicine research. In other words, the survey results suggested that even those who are positive about a new technology may feel avoidance depending on the content of the application. Therefore, for research involving the collection of biological materials, depending on the intended use, it is necessary to take sufficient care, for instance, in obtaining informed consent [28]. In summary, it is necessary to share the vision of how the new technology is to be implemented with the society as far upstream as possible in the research and development process and to conduct communication activities that keep in mind the difference in interest between experts and the general public.8 7

The “Compensation System for Clinical Research on Regenerative Medicine,” which the Japanese Society for Regenerative Medicine is conducting jointly with Mitsui Sumitomo Insurance Co., will go further than the usual insurance for clinical research to cover cases in which doctors and medical institutions are not legally liable for compensation, and will also cover compensation for patients, which is not provided for in the Law for Securing the Safety of Regenerative Medicine: http://www. mskhoken.com/dantai/jsrm/index.html (last accessed February 17, 2018). 8 When researchers participate in communication and information sharing, it is especially important to improve the environment in terms of time, place, and evaluation system, and to give consideration to the burden that researchers have and to provide institutional support [35]. In the case of the medical field, if some exaggeration is included in the press release issued by the researcher or research

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8.2.4 Responsible Research and Innovation We have reviewed the cases that seem relevant to ELSIs and communication with society in the field of molecular robotics. Based on these past findings, it is necessary to construct a system that will highlight the positive impact of molecular robotics and that takes into appropriate consideration various ELSIs. In the discussions of molecular robotics ethics that have already begun, questions have arisen such as “what kind of ELSIs should be considered in the field?” and “what are the issues regarding medical applications, industrialization, standardization, and quality assurance?” The accumulation of discussions with medical professionals, medical ethics experts, and governmental agencies has been recognized. In addition, the importance of communicating with society to build social agendas has been emphasized.9 Issues related to the dual-use problem and eugenics, as well as the examination of worst-case scenarios, have also been pointed out. Discussing the innovation ecosystem in a way that considers ELSIs and appropriate care for society from the upstream stage of research and development is called Responsible Research and Innovation (RRI).10 Currently, RRI has attracted attention in science and technology policy frameworks such as the European Commission’s Horizon 2020. How can RRI be realized in molecular robotics? What kinds of perspectives and education are necessary for RRI in molecular robotics? These questions should be discussed within the academic community through trial and error.11 Those trials in molecular robotics will become the model for RRI in other advanced fields. To return to the case of GMOs, the origin of regulations and guidelines for GMOs in various countries lies in the spontaneous discussion among scientists as represented by the Asilomar Conference. The importance of the Asilomar Conference’s approach, relying on early autonomous discussion and dissemination, has not changed in the field of molecular robotics.

institute, the media coverage will also include exaggeration to a large extent. On the other hand, if the press release is written in a restrained manner, the ratio of exaggeration in the media report is reduced to about one-fifth [36]. It is necessary to take this dynamic into account in communication activities. 9 The description is based on the discussion in the Joint Workshop on Molecular Robotics/JST Molecular Robot Ethics held on January 22, 2017 and February 11, 2017. 10 RRI has been described thus: “RRI stands for care for the future through the collective management of science and innovation in the present” [37]. 11 A situation in which researchers do not know much about or are indifferent to the policy foundations, institutional conditions, or socially relevant incidents surrounding their field requires monitoring and educational response within the academic community. For example, in the field of regenerative medicine, a total of 27.9% of researchers answered “I have heard of it, but I do not know the outline” or “I do not know it at all” when asked about the three regenerative medicine laws, which are the institutional basis for the field. In addition, 38.4% of respondents answered that they “did not know” about the stem cell administration case. Researchers have argued for the importance of countermeasures against this situation [27].

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