Peptide Nucleic Acids: Methods and Protocols (Methods in Molecular Biology, 2105) 107160242X, 9781071602423

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Methods in Molecular Biology 2105

Peter E. Nielsen Editor

Peptide Nucleic Acids Methods and Protocols Third Edition

METHODS

IN

MOLECULAR BIOLOGY

Series Editor John M. Walker School of Life and Medical Sciences University of Hertfordshire Hatfield, Hertfordshire, UK

For further volumes: http://www.springer.com/series/7651

For over 35 years, biological scientists have come to rely on the research protocols and methodologies in the critically acclaimed Methods in Molecular Biology series. The series was the first to introduce the step-by-step protocols approach that has become the standard in all biomedical protocol publishing. Each protocol is provided in readily-reproducible step-bystep fashion, opening with an introductory overview, a list of the materials and reagents needed to complete the experiment, and followed by a detailed procedure that is supported with a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice. These hallmark features were introduced by series editor Dr. John Walker and constitute the key ingredient in each and every volume of the Methods in Molecular Biology series. Tested and trusted, comprehensive and reliable, all protocols from the series are indexed in PubMed.

Peptide Nucleic Acids Methods and Protocols Third Edition

Edited by

Peter E. Nielsen Department of Cellular and Molecular Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark

Editor Peter E. Nielsen Department of Cellular and Molecular Medicine Faculty of Health and Medical Sciences University of Copenhagen Copenhagen, Denmark

ISSN 1064-3745 ISSN 1940-6029 (electronic) Methods in Molecular Biology ISBN 978-1-0716-0242-3 ISBN 978-1-0716-0243-0 (eBook) https://doi.org/10.1007/978-1-0716-0243-0 © Springer Science+Business Media, LLC, part of Springer Nature 2020 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, 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 Humana imprint is published by the registered company Springer Science+Business Media, LLC, part of Springer Nature. The registered company address is: 1 New York Plaza, New York, NY 10004, U.S.A.

Preface The PNA molecule has close to 30-year anniversary being publicly born in 1991. During this period, it has visited many areas of science, with the main attractions being organic chemistry, chemical biology, and drug discovery, in particular within hybridization probe applications and antisense/gene targeting drug discovery, and new sides, new chemistry, and new applications of this family of DNA mimics are still being uncovered. The intention of the present Methods in Molecular Biology edition of “Peptide Nucleic acids from Chemistry to Animals” was to turn a little more focus toward in vivo properties and behavior and applications of PNA while maintaining contributions on both chemistry and nucleic acid recognition. Thus, it is an addition to the previous edition rather than an update. I am deeply indebted and grateful to my colleagues, collaborators, and friends for once again without hesitation supporting this effort and contributing to this book with their expertise and innovative approaches. It is my hope that this shall inspire discovery and pursuit of new exciting avenues in the broad area of “artificial DNA.” Copenhagen, Denmark

Peter E. Nielsen

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Contents Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1 Fmoc-Based Assembly of PNA Oligomers: Manual and Microwave-Assisted Automated Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ashif Yasin Shaikh, Anna Mette Hansen, and Henrik Franzyk 2 A Robust Method for Preparing Optically Pure MiniPEG-Containing Gamma PNA Monomers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Wei-Che Hsieh and Danith H. Ly 3 Synthesis of Pyrrolidinyl PNA and Its Site-Specific Labeling at Internal Positions by Click Chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Boonsong Ditmangklo, Penthip Muangkaew, Kotchakorn Supabowornsathit, and Tirayut Vilaivan 4 Synthesis of PNA-Peptide Conjugates as Functional SNARE Protein Mimetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Barbara E. Hubrich, Patrick M. Menzel, Benedikt Kugler, and Ulf Diederichsen 5 Lipid-Modified Peptide Nucleic Acids: Synthesis and Application to Programmable Liposome Fusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Philipp M. G. Lo¨ffler, Alexander Rabe, and Stefan Vogel 6 Facile Preparation of PNA-Peptide Conjugates with a Polar Maleimide-Thioether Linkage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Anna Mette Hansen, Ashif Yasin Shaikh, and Henrik Franzyk 7 PNA-Encoded Synthesis (PES) and DNA Display of Small Molecule Libraries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Jacques Saarbach, Sofia Barluenga, and Nicolas Winssinger 8 Transcription Inhibition by PNA-Induced R-Loops . . . . . . . . . . . . . . . . . . . . . . . . . Boris P. Belotserkovskii, Sum-yan Ng, and Philip C. Hanawalt 9 Nucleobase-Modified Triplex-Forming Peptide Nucleic Acids for Sequence-Specific Recognition of Double-Stranded RNA. . . . . . . . . . . . . . . . . Nikita Brodyagin, Dziyana Hnedzko, James A. MacKay, and Eriks Rozners 10 In Vitro Cellular Delivery of Peptide Nucleic Acid (PNA) . . . . . . . . . . . . . . . . . . . Takehiko Shiraishi, Mahdi Ghavami, and Peter E. Nielsen 11 Reactive Quantum Dot-Based FRET Systems for Target-Catalyzed Detection of RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oleksandr Zavoiura, Ute Resch-Genger, and Oliver Seitz 12 Peptide Nucleic Acids for MicroRNA Targeting . . . . . . . . . . . . . . . . . . . . . . . . . . . . Roberto Gambari, Jessica Gasparello, Enrica Fabbri, Monica Borgatti, Anna Tamanini, and Alessia Finotti

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119 141

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Detection of Microorganisms by Fluorescence In Situ Hybridization Using Peptide Nucleic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ricardo Oliveira, Carina Almeida, and Nuno F. Azevedo PNA Antisense Targeting in Bacteria: Determination of Antibacterial Activity (MIC) of PNA-Peptide Conjugates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lise Goltermann and Peter E. Nielsen In Vivo Administration of Splice Switching PNAs Using the mdx Mouse as a Model System. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Camilla Brolin, Ernest Wee Kiat Lim, and Peter E. Nielsen Near-Infrared In Vivo Whole-Body Fluorescence Imaging of PNA. . . . . . . . . . . . Ernest Wee Kiat Lim, Camilla Brolin, and Peter E. Nielsen Poly(Lactic-co-Glycolic Acid) Nanoparticle Delivery of Peptide Nucleic Acids In Vivo. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Stanley N. Oyaghire, Elias Quijano, Alexandra S. Piotrowski-Daspit, W. Mark Saltzman, and Peter M. Glazer Preparation of Conjugates for Affibody-Based PNA-Mediated Pretargeting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mohamed Altai, Anzhelika Vorobyeva, Vladimir Tolmachev, Amelie Eriksson Karlstro¨m, and Kristina Westerlund

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Contributors CARINA ALMEIDA • LEPABE—Laboratory for Process Engineering, Environment, Biotechnology and Energy, Faculty of Engineering, University of Porto, Porto, Portugal; INIAV—National Institute for Agrarian and Veterinarian Research, Vairao, Portugal; CEB—Centre of Biological Engineering, University of Minho, Braga, Portugal MOHAMED ALTAI • Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden NUNO F. AZEVEDO • LEPABE—Laboratory for Process Engineering, Environment, Biotechnology and Energy, Faculty of Engineering, University of Porto, Porto, Portugal SOFIA BARLUENGA • Department of Organic Chemistry, NCCR Chemical Biology, University of Geneva, Geneva, Switzerland BORIS P. BELOTSERKOVSKII • Department of Biology, Stanford University, Stanford, CA, USA MONICA BORGATTI • Department of Life Sciences and Biotechnology, Ferrara University, Ferrara, Italy NIKITA BRODYAGIN • Department of Chemistry, Binghamton University, Binghamton, NY, USA CAMILLA BROLIN • Department of Cellular and Molecular Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark ULF DIEDERICHSEN • Institute for Organic and Biomolecular Chemistry, University of Go¨ttingen, Go¨ttingen, Germany BOONSONG DITMANGKLO • Organic Synthesis Research Unit, Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok, Thailand ENRICA FABBRI • Department of Life Sciences and Biotechnology, Ferrara University, Ferrara, Italy ALESSIA FINOTTI • Department of Life Sciences and Biotechnology, Ferrara University, Ferrara, Italy HENRIK FRANZYK • Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark ROBERTO GAMBARI • Department of Life Sciences and Biotechnology, Ferrara University, Ferrara, Italy JESSICA GASPARELLO • Department of Life Sciences and Biotechnology, Ferrara University, Ferrara, Italy MAHDI GHAVAMI • Department of Cellular and Molecular Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark PETER M. GLAZER • Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT, USA LISE GOLTERMANN • Department of Cellular and Molecular Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark PHILIP C. HANAWALT • Department of Biology, Stanford University, Stanford, CA, USA ANNA METTE HANSEN • Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark DZIYANA HNEDZKO • Department of Chemistry, Binghamton University, Binghamton, NY, USA

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WEI-CHE HSIEH • Institute for Biomolecular Design and Discovery (IBD), Carnegie Mellon University, Pittsburgh, PA, USA; Department of Chemistry, Carnegie Mellon University, Pittsburgh, PA, USA BARBARA E. HUBRICH • Institute for Organic and Biomolecular Chemistry, University of Go¨ttingen, Go¨ttingen, Germany AMELIE ERIKSSON KARLSTRO¨M • Department of Protein Science, School of Engineering Sciences in Chemistry, Biotechnology and Health, KTH Royal Institute of Technology, Stockholm, Sweden BENEDIKT KUGLER • Institute for Organic and Biomolecular Chemistry, University of Go¨ttingen, Go¨ttingen, Germany PHILIPP M. G. LO¨FFLER • Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Odense, Denmark ERNEST WEE KIAT LIM • Department of Cellular and Molecular Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark DANITH H. LY • Institute for Biomolecular Design and Discovery (IBD), Carnegie Mellon University, Pittsburgh, PA, USA; Department of Chemistry, Carnegie Mellon University, Pittsburgh, PA, USA JAMES A. MACKAY • Department of Chemistry and Biochemistry, Elizabethtown College, Elizabethtown, PA, USA PATRICK M. MENZEL • Institute for Organic and Biomolecular Chemistry, University of Go¨ttingen, Go¨ttingen, Germany PENTHIP MUANGKAEW • Organic Synthesis Research Unit, Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok, Thailand SUM-YAN NG • Department of Biology, Stanford University, Stanford, CA, USA PETER E. NIELSEN • Department of Cellular and Molecular Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark RICARDO OLIVEIRA • LEPABE—Laboratory for Process Engineering, Environment, Biotechnology and Energy, Faculty of Engineering, University of Porto, Porto, Portugal; INIAV—National Institute for Agrarian and Veterinarian Research, Vairao, Portugal STANLEY N. OYAGHIRE • Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT, USA ALEXANDRA S. PIOTROWSKI-DASPIT • Department of Biomedical Engineering, Yale University, New Haven, CT, USA ELIAS QUIJANO • Department of Genetics, Yale University School of Medicine, New Haven, CT, USA ALEXANDER RABE • Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Odense, Denmark UTE RESCH-GENGER • Division Biophotonics, Federal Institute for Materials Research and Testing (BAM), Berlin, Germany ERIKS ROZNERS • Department of Chemistry, Binghamton University, Binghamton, NY, USA JACQUES SAARBACH • Department of Organic Chemistry, NCCR Chemical Biology, University of Geneva, Geneva, Switzerland W. MARK SALTZMAN • Department of Biomedical Engineering, Yale University, New Haven, CT, USA; Department of Chemical & Environmental Engineering, Yale University, New Haven, CT, USA OLIVER SEITZ • Department of Chemistry, Humboldt University of Berlin, Berlin, Germany ASHIF YASIN SHAIKH • Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark

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TAKEHIKO SHIRAISHI • Department of Cellular and Molecular Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark KOTCHAKORN SUPABOWORNSATHIT • Organic Synthesis Research Unit, Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok, Thailand ANNA TAMANINI • Department of Molecular Pathology and Diagnostics, University Hospital of Verona, Verona, Italy VLADIMIR TOLMACHEV • Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden TIRAYUT VILAIVAN • Organic Synthesis Research Unit, Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok, Thailand STEFAN VOGEL • Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Odense, Denmark ANZHELIKA VOROBYEVA • Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden KRISTINA WESTERLUND • Department of Protein Science, School of Engineering Sciences in Chemistry, Biotechnology and Health, KTH Royal Institute of Technology, Stockholm, Sweden NICOLAS WINSSINGER • Department of Organic Chemistry, NCCR Chemical Biology, University of Geneva, Geneva, Switzerland OLEKSANDR ZAVOIURA • Division Biophotonics, Federal Institute for Materials Research and Testing (BAM), Berlin, Germany; Department of Chemistry, Humboldt University of Berlin, Berlin, Germany

Chapter 1 Fmoc-Based Assembly of PNA Oligomers: Manual and Microwave-Assisted Automated Synthesis Ashif Yasin Shaikh, Anna Mette Hansen, and Henrik Franzyk Abstract Exploration of PNA-peptide conjugates as potential antisense antibiotics necessitates a fast and efficient synthesis protocols for amounts that facilitate determination of structure-activity relationships and in vivo studies in animal infection models. Fmoc/Boc-protected PNA monomers are here used for assembly of oligomers by optimized protocols involving either a manual synthesis method at room temperature or automated microwave-assisted coupling of monomers on a peptide synthesizer. Key words Peptide nucleic acid, Solid-phase synthesis, Coupling conditions, Fmoc deprotection, Microwave heating, Antisense oligomers, P. aeruginosa

1

Introduction Since their introduction almost 20 years ago, peptide nucleic acids (PNAs) have been recognized as stable, nontoxic synthetic mimics of DNA and RNA [1], and thus they are still receiving considerable attention in many fields of science from pure chemistry, over (molecular) biology, and drug discovery to nanotechnology and prebiotic chemistry. In particular, PNA oligomers are applied as biological probes, nano-scaffold components, and as diagnostic tools for genetic diseases. Consequently, a very wide variety of PNA derivatives and conjugates have continuously been in demand, and hence a large number of synthesis protocols have been developed in the last two decades as summarized below. Originally, PNA oligomers were synthesized by solid-phase methods involving assembly of monomers with a tert-butyloxycarbonyl (Boc)/benzyloxycarbonyl (Z ¼ Cbz) protection scheme for the primary amine of the (2-aminoethyl)glycine backbone and nucleobase amino functionalities, respectively [2–4]. However, these protocols require the use of harsh conditions (typically HF

Peter E. Nielsen (ed.), Peptide Nucleic Acids: Methods and Protocols, Methods in Molecular Biology, vol. 2105, https://doi.org/10.1007/978-1-0716-0243-0_1, © Springer Science+Business Media, LLC, part of Springer Nature 2020

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or F3CSO3H) to release the final oligomer from the solid phase. Following the development of solid-phase synthesis (SPS) of peptides by assembly of fluoren-9-ylmethoxycarbonyl (Fmoc)protected amino acid building blocks, a related Fmoc-based SPS protocol for PNA oligomers was reported; this was based on monomers displaying an Fmoc/Z protection scheme [5]. In this seminal work, stable, activated pentafluorophenyl esters or the corresponding carboxylic acids in combination with 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU)/N-hydroxybenzotriazole (HOBt) were employed to promote coupling of monomers, while 30% piperidine in 20% dimethyl sulfoxide (DMSO) in N-methyl-2-pyrrolidone (NMP) was used for Fmoc removal. Also, leaving the N-terminal Fmoc protecting group intact prior to cleavage from the resin facilitated purification by high-performance liquid chromatography (HPLC) as impurities arising from deletion sequences then readily could be separated. A final Fmoc deprotection in aqueous medium followed by a second preparative HPLC purification provided oligomers of high purity [5]. Subsequently, a number of synthetic protocols involving backbone Fmocprotected building blocks, displaying a variety of nucleobase protecting groups, have been reported: Bhoc (¼ benzhydryloxycarbonyl) [6–13], Cl-Bhoc [12], F-Bhoc [12], 4-OMe-Z [12], (2-trimethylsilyl)ethoxycarbonyl (Teoc) [12], or 4-monomethoxytrityl (Mmt) [14]. So far, the most commonly used monomers for Fmoc-based oligomerization possess Bhoc as protecting group for the nucleobases, while different coupling conditions have been explored as briefly summarized below. Typically, 2-(1H-7-azabenzotriazole-1yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU) has been considered the most efficient coupling reagent (for activation of monomers) in the presence of excess diisopropylethylamine (DIPEA)/2,6-lutidine base mixture in N,Ndimethylformamide (DMF) and/or NMP with subsequent Fmoc removal by treatment with 20% piperidine in DMF [7, 10]. Also, the less reactive HBTU (under similar conditions) has successfully been employed together with a range of PNA building blocks in Fmoc-based oligomer assembly [9, 12]. Although it was reported that the use of benzotriazole-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) as coupling reagent in the oligomerization of Fmoc/Bhoc-protected monomers may give rise to modification of guanine side chains [13], subsequent protocols also employing PyBOP proved successful both in automated synthesis [8] and when using microwave (MW) heating to further expedite the coupling cycles

Fmoc Manual and Microwave-Assisted PNA Synthesis

3

[11]. Potential advantages of the use of PyBOP are that preactivation is not required and that formation of by-products displaying N-terminal guanidinylation may be avoided [13]. In addition, alternatives to Fmoc protection as temporary backbone amine protecting group have been proposed (with the nucleobase protecting groups also stated in brackets): 4-monomethoxytrityl (Mmt; anisoyl/4-tBu-benzoyl/isobutanoyl) [15], 4-monomethyltrityl (Mtt; Boc) [16], allyloxycarbonyl (Bhoc) [17], 1-(4,4,-dimethyl-2,6-dioxacyclohexylidene)ethyl (Mmt) [18, 19], p-nitro-Z (bis-Boc) [20], and benzothiazole-2-sulfonyl (Boc) [21]. Also in continuous automated Fmoc-based SPS of PNA-peptide conjugates, Fmoc/Bhoc-protected PNA monomers are the most commonly applied, since this allows for a convenient simultaneous acid-catalyzed removal of all protecting groups and cleavage from the resin [6, 8, 10–12, 22, 23]. To our knowledge, only Fmoc-based building blocks displaying the traditional Bhoc protection of the nucleobases are commercially available. Nevertheless, these Fmoc/Bhoc-protected monomers are expensive and possess reduced solubility in the solvents usually employed for SPS (i.e., DMF, NMP, or CH2Cl2) [23–26]. Thus, for automated synthesis, both guanine monomer and in particular the cytosine monomer typically need laborious preparation of reactant solutions involving prolonged agitation/sonication to avoid crystallization over time. Addition of DMSO may promote dissolution, but when using MW-assisted synthesis, different coupling conditions with other MW settings may be needed for the cytosine monomer due to the large difference in the degree of MW energy absorption between DMF/NMP and DMSO, thus complicating the methodology. In traditional Fmoc/tBu-based peptide synthesis, the Boc group is preferred for side-chain protection of amino and heteroaromatic functionalities (e.g., Lys and Trp) due to its compatibility with weakly acidic conditions employed for obtaining partially protected peptides. By contrast, it is somewhat surprising that the use of Boc as protection of nucleobases in PNA synthesis is limited to a few reports [12, 23–28]. Two different types of Fmoc-based building blocks have been developed, displaying either (1) bis-Boc protection of the nucleobases [23–25] or (2) mono-Boc protection of the nucleobases [26–28]. For the Fmoc/bis-Boc type of monomers, oligomerization was reported to be performed under conditions also feasible for assembly of Fmoc/Bhoc-protected PNA monomers [6, 23, 24], but no experimental details were included. Oligomerization of Fmoc/Boc-protected monomers (4 equiv) was

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carried out at room temperature (with coupling times of 30 min) by using HBTU (3.5 equiv) in the presence of DIPEA (4.0 equiv) and 2,6-lutidine (6.0 equiv) in NMP [12]. In order to obtain relatively large amounts of PNA-peptide conjugates as potential antisense antibiotics [29], we are currently exploring different synthesis methods for obtaining amounts that facilitate structure-activity studies and testing in animal infection models. Hence, MW-assisted automated assembly of PNA oligomers (on a 0.1 mmol scale) was considered a convenient methodology compatible with continuous peptide synthesis. Previously, Fmoc/Bhoc-protected monomers have been employed in protocols for MW-assisted SPS of PNA-peptide conjugates either via separate syntheses of peptides and PNA oligomers followed by conjugation [30] or by continuous synthesis of the entire PNA-peptide conjugate [11]; an alternative method for the latter involves an Fmoc/Dde protection scheme [19]. Recently, we reported improved syntheses of Fmoc/Boc-protected PNA monomers (originally reported by Sugiyama et al. [27]) and showed their utility in automated MW-assisted continuous synthesis of PNA-peptide conjugates [26]. Here, chain elongation of PNA oligomers was conveniently performed by using Fmoc/Bocprotected monomer building blocks (5 equiv) in combination with HBTU (5 equiv) as coupling reagent (in DMF) and DIPEA in NMP as activator base. Fmoc deprotection was performed with 20% piperidine in DMF at 45  C (1 min + 5 min), while monomer couplings were conducted at 45  C for 10 min [26]. When a manual synthesis of a PNA oligomer was carried out by using an adapted protocol with conventional heating to 45  C, the main fraction (after careful purification by preparative HPLC), containing the desired Fmoc-PNA oligomer, was consistently contaminated with one or more deletion oligomers missing only a single residue (as determined by MALDI-TOF and visible as shoulders on the peak in analytical HPLC; especially impurities with a missing T or C residue proved impossible to remove). Different approaches such as repeated couplings, when introducing the so-called difficult residues (i.e., T onto G or C onto A as previously reported [7]), did not lead to improved purity of the crude as judged by analytical HPLC and MALDI-TOF. In an earlier study, it was found that during Fmoc-based PNA oligomer SPS two side reactions may occur under highly basic conditions [5]: (a) migration of nucleobase-acetyl moiety to the N-terminal amine, which may lead to only partial coupling of the next monomer on the resulting sterically hindered secondary amine, or (b) deletion of the N-terminal residue by an intramolecular acyl migration to form a lactam (see Fig. 1).

Fmoc Manual and Microwave-Assisted PNA Synthesis

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Fig. 1 Side reactions that may occur under basic conditions [5]

As Fmoc deprotection with 20% piperidine under heating might induce such migrations, this step was performed at room temperature. Also, an excess of DIPEA was used during monomer coupling, and therefore it was attempted to utilize less equivalents of DIPEA and perform the couplings at room temperature as well in order to alleviate the issues with deletion sequences. Somewhat surprisingly, it was the latter modification that had the most pronounced effect, and in combination, these altered conditions led to increased efficiency as judged by analytical HPLC of test cleavage samples of Fmoc-protected partially assembled oligomers. Hence, a protocol in which all reactions are performed at room temperature appeared promising: 20% piperidine in DMF for Fmoc deprotection (2  10 min) and monomer coupling (5 equiv of HBTU for 1-h couplings), while capping was accomplished with Ac2O-DIPEA-NMP. After complete assembly of the desired 11-mer (H-CTCATACCTTGNH2), a MALDI-TOF spectrum of the crude product only displayed a single major peak without the presence of substantial impurities lacking a single monomer. To ensure that the desired crude purity is obtained, it is generally recommended to verify this for the PNA oligomer prior to removal of the N-terminal Fmoc group, since the presence of deletion sequences is more readily detected as individual peaks in analytical HPLC and FmocPNA oligomers appear to be more readily analyzed by MALDITOF as well. Ultimately, a preparative HPLC purification step at the stage of the Fmoc-PNA oligomer may be required for longer oligomers. When our recently reported protocol, developed for a 10-mer oligomer devoid of guanine (i.e., H-CTCATACTCT-NH2) [26], was employed for the above 11-mer (which is an antibacterial anti-acpP oligomer targeting P. aeruginosa), a careful MALDITOF analysis of the apparently pure product (as judged by analytical HPLC) revealed the presence of small amounts of several impurities corresponding to deletion sequences. Thus, the general

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conditions developed for manual synthesis were implemented in a modified automated synthesis of the desired guanine-containing 11-mer PNA. Assembly on a Biotage Syro Wave synthesizer enabled a reduction in monomer coupling time from 1 h to 20 min by applying MW heating to 45  C, which previously was found to be the limit for prolonged stability of the Fmoc/Bocprotected PNA monomers [26]. After screening of different Fmoc deprotection and monomer coupling conditions (such as temperature, coupling reagent, and time), we now report an optimized protocol in which all couplings are performed at 45  C (with MW heating for 20 min), while Fmoc deprotection is performed at room temperature (with 30% and 15% piperidine in DMF for 3 and 12 min, respectively). This protocol was also shown to be compatible with the commercially available Fmoc/Bhocprotected monomers. However, besides circumventing the solubility issues, another advantage of applying the Fmoc/Boc protection scheme is that Boc constitutes a “traceless protecting group” when employed in combination with a TFA-H2O cleavage mixture (when appropriate for PNA-peptide conjugates, triisopropylsilane may be included). Thus, when using Boc protection of the nucleobases, it is possible to avoid any sample cleanup prior to purification by HPLC.

2 2.1

Materials Chemicals

1. Fmoc/Boc or Fmoc/Bhoc PNA monomers (Boc, [12, 26]; Bhoc, ASM Research Chemicals GmbH or Link Technologies Ltd). 2. 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU; Iris Biotech GmbH). 3. Piperidine (Iris Biotech GmbH, peptide grade). 4. Dimethylformamide (DMF, VWR, peptide synthesis grade). 5. Dimethylformamide (Acros Organic, 99.8%, Extra Dry over Molecular Sieve, AcroSeal®). 6. N-Methyl-2-pyrrolidone (NMP, Iris Biotech GmbH). 7. Dichloromethane (CH2Cl2, VWR, HPLC grade). 8. Acetonitrile (MeCN; VWR, CHROMANORM® for LC-MS). 9. Acetic anhydride (Sigma-Aldrich). 10. Diisopropylethylamine (DIPEA, Iris Biotech GmbH). 11. Triisopropylsilane (TIS, Sigma-Aldrich). 12. Trifluoroacetic acid (TFA, Alfa Aesar, HPLC grade).

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7

13. α-Cyano-4-hydroxycinnamic acid (ACCA, Sigma-Aldrich). 14. Rink Amide ChemMatrix® resin (Matrix Innovation, with particle size of 100–200 mesh; see Note 1). 2.2 Solutions and Eluents

1. Fmoc deprotection solutions: 30%, 20% or 15% piperidine in DMF. 2. Capping solution: NMP-2,6-lutidine-Ac2O (89:6:5). 3. Cleavage cocktail solution: TFA-CH2Cl2 (9:1) or TFA-TISH2O (95:2.5:2.5). 4. MALDI-TOF-MS solution: 10 mg ACCA in 1 mL MeCNH2O-TFA (500:475:25). 5. HPLC eluent A: 0.1% trifluoroacetic acid (TFA) in H2OMeCN (95:5). 6. HPLC eluent B: 0.1% trifluoroacetic acid (TFA) in MeCNH2O (95:5).

2.3

Instruments

1. Activo-PLS synthesizer (www.activotec.com). 2. Biotage® Syro Wave™ (www.biotage.com). 3. PTFE (teflon) reactor (10 mL, www.activotec.com) for manual synthesis. 4. Preparative HPLC Phenomenex Luna C18(2) column (particle size: 5 μm; 250  21.2 mm). 5. Analytical UHPLC Phenomenex Luna C18(2) HTS column (particle size: 2.5 μm; 100  3.0 mm). 6. MALDI-TOF-MS (Bruker). 7. Sonicator (Thermo Fisher).

3

Methods Herein, synthesis of an 11-mer PNA antisense oligomer (H-CTC-ATA-CCT-TG-NH2) in 0.05 mmol scale is demonstrated as an example.

3.1

Preparations

1. Clean the reaction vessel and make sure it does not leak. 2. Weigh out the required quantity of Fmoc/Boc- or Fmoc/ Bhoc-protected monomer for each coupling in separate vials for manual synthesis (1 equiv for the loading step and 5 equiv for elongation steps) or in the reactant containers used for the automated synthesizer. 3. Weigh out the required quantity of coupling reagent (HBTU) for each coupling in separate vials for manual synthesis (1 equiv for the loading step and 5 equiv for elongation steps) or in the reactant containers used for the automated synthesizer.

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Ashif Yasin Shaikh et al.

3.2 Assembly of PNA Oligomers and Final Cleavage 3.2.1 Manual Synthesis of H-CTCATACCTTG-NH2

1. Weigh out 0.05 mmol of Rink amide ChemMatrix® resin (loading, 0.53 mmol/g; used directly without any downloading) in a vial, and transfer it into the teflon reaction vessel with DMF-CH2Cl2 (1:1). 2. Swell the resin in DMF (3  5 mL; each for 5 min, draining in between) and then drain. 3. The resin is treated with 20% piperidine in DMF (5 mL) to remove the Fmoc protecting group on the Rink amide linker. Deprotection is performed at room temperature (2  10 min, each with 5 mL) with a DMF (5 mL) wash in between. Following deprotection, the resin is washed with DMF (3  3 min, each with 5 mL). 4. Loading of the first PNA monomer (see Note 2): Dissolve the preweighed HBTU (1 equiv; relative to the resin loading) in DMF (1.5 mL), and add the solution to the monomer (1 equiv) followed by DIPEA (1 equiv), after which the mixture is left for activation for 10 min before being added to the Fmoc-deprotected resin. Loading is continued for 30 min followed by draining. 5. Upon loading of the C-terminal residue, the resin is washed with DMF (2  3 min, each with 3 mL), and then unreacted linker sites are capped with NMP-2,6-lutidine-Ac2O (89:6:5; 2  5 min, each with 5 mL). 6. Fmoc deprotection is performed by treatment with 20% piperidine in DMF (2  10 min, each with 5 mL) followed by washing with NMP (2  3 min, each with 5 mL) and DMF (1  3 min, 5 mL). 7. Coupling of PNA monomers: Dissolve the preweighed HBTU (5 equiv) in DMF (2 mL), and add the solution to the PNA monomer (5 equiv) followed by DIPEA (5 equiv), and then the mixture is left for activation for 10 min before being added to the Fmoc-deprotected resin. Coupling is continued for 1 h followed by draining. Capping (as in step 5) is optional but may for some sequences improve the purity of the crude PNA oligomer. 8. Repeat steps 6 and 7 till completion of the desired sequence. 9. At the end of the synthesis, Fmoc deprotection is performed as described in step 6. 10. After the final Fmoc deprotection, the resin is washed with DMF and CH2Cl2 (both 3  5 min, each with 5 mL). 11. The washed resin is treated with TFA-CH2Cl2 (9:1, 2  30 min each with 5 mL) after assembly of Fmoc/Bocprotected monomers or TFA-TIS-H2O (95:2.5:2.5) after

Fmoc Manual and Microwave-Assisted PNA Synthesis

9

assembly of Fmoc/Bhoc-protected monomers. The cleavage mixture is concentrated and the crude product is obtained, and it is stored at 20  C until purification. 3.2.2 MW-Assisted Solid-Phase Synthesis of H-CTCATACCTTG-NH2 Using Biotage® Syro Wave™ (at 45  C)

1. Weigh out 0.05 mmol (94 mg) ChemMatrix® resin (loading 0.53 mol/g; used directly without any downloading) into a sample vial from which it is transferred to the reaction vessel using the CH2Cl2-DMF solvent mixture. 2. Allow the resin to swell further in DMF for 5 min, and start the automated synthesis by using the assigned program on the Biotage Microwave synthesizer. 3. First Fmoc deprotection is carried out with an initial treatment with 30% piperidine in DMF (3 min at room temperature) followed by a second treatment with 15% piperidine in DMF (10 min at room temperature), and finally the resin is washed with DMF-CH2Cl2-DMF (2:1:2, 2.6 mL). 4. Next, monomer and coupling reagents are added in NMP: 0.25 mmol PNA monomer (Fmoc/Boc- or Fmoc/Bhocprotected), 0.24 mmol HBTU, and then 0.25 mmol DIPEA. Coupling is performed for 20 min under microwave heating to 45  C, followed by washing with DMF, CH2Cl2, and DMF (2.6 mL each). 5. Steps 3 and 4 are repeated until the desired monomer sequence is assembled. Depending on whether Fmoc is desired to remain on the PNA oligomer (e.g., for initial HPLC purification) or not, final deprotection is performed. 6. Test cleavage is performed for both Fmoc-CTCATACCTTGNH2 and H-CTCATACCTTG-NH2 by using TFA-CH2Cl2 (9:1, 200 μL, for 30 min). 7. The cleaved Fmoc-PNA sample is dissolved in MeCN-H2O (50:50, 0.1% TFA), and analytical HPLC is performed by using a gradient of 0–70% eluent B during 10 min (flow rate 0.5 mL/min), while the sample of H-CTCATACCTTG-NH2 is dissolved in MeCN-H2O (5:95, 0.1% TFA) and analyzed by using a gradient of 0–20% B during 10 min. HPLC analysis: >85% crude purity; MALDI-TOF spectrum: the desired mass peak is observed as the only major peak. 8. Final cleavage is performed with TFA-CH2Cl2 (9:1; 2  30 min, each 2 mL) after assembly of Fmoc/Boc-protected monomers or with TFA-TIS-H2O (95:2.5:2.5) after assembly of Fmoc/Bhoc-protected monomers. The cleavage solvents are evaporated, and the resulting crude product is treated with diethyl ether to obtain a white solid, which then is dissolved in 2 mL of MeCN-H2O (5:95, 0.1% TFA) and immediately subjected to purification by preparative HPLC.

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Ashif Yasin Shaikh et al.

3.3 Purification and Characterization

1. A sample of the crude material (~0.5 mg/mL) is prepared in HPLC buffer solution A, and an analytical HPLC is run to obtain the chromatogram of the crude (see Note 3). 2. MALDI-TOF-MS is used to verify that the correct mass is present as the main peak (see Note 3). 3. The preparative HPLC is equipped with a Phenomenex Luna C18(2) column (particle size, 5 μm; 250  21.2 mm) and detection is at 260 nm. From the analytical profile, the gradient for purification is chosen. 4. The crude material is dissolved in HPLC eluent A (~100 mg/ mL). As a trial run, inject a small amount of the sample (300 μL) while using the chosen gradient, collecting the major peak (assumed to be the desired PNA oligomer; see Note 4). 5. The identity of the peak of interest is verified by MALDI-TOF MS, and the peak of interest is collected for all runs. 6. The combined pure PNA fractions (see Note 5) are lyophilized. 7. Store at 20  C until further use.

4

Notes 1. Polystyrene resins work just as well for up to 20-mer sequences. Resin will have different loading capacities and swelling properties depending on manufacturer. 2. Loading of the first monomer is performed at room temperature. 3. In order to verify the outcome of the synthesis, it is recommended to perform a “test cleavage” in which a small amount of the PNA (~1–2 mg) is released and concomitantly side-chain deprotected by treatment of a resin sample with 100 μL of the cleavage cocktail solution. Subsequently, dilution with MeCNH2O 50:50 for Fmoc-PNA (Fig. 2) or 100% eluent A for PNA allows for analysis by HPLC and MALDI-TOF MS. For FmocPNA, an elution gradient of 0–70% B during 10 min is used, while fully deprotected PNA (Fig. 3) is eluted with a gradient of 0–20% B during 10 min. 4. Preparative HPLC purification of crude mixtures (gradient of 0–15% B during 20 min) (Fig. 4). 5. Analytical HPLC and MALDI-TOF after final purification (Fig. 5).

Fmoc Manual and Microwave-Assisted PNA Synthesis

Fig. 2 Fmoc-PNA

11

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Ashif Yasin Shaikh et al.

Fig. 3 Fully deprotected PNA

Fmoc Manual and Microwave-Assisted PNA Synthesis

13

Fig. 4 Preparative HPLC purification of crude mixtures (gradient of 0–15% B during 20 min)

Acknowledgments This research was supported by the Department of Drug Design and Pharmacology and Center for Peptide-Based Antibiotics. We thank Prof. C.A. Olsen for providing access to the Biotage Syro Wave synthesizer.

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Fig. 5 Analytical HPLC and MALDI-TOF after final purification

Fmoc Manual and Microwave-Assisted PNA Synthesis

15

References 1. Nielsen PE, Egholm M, Berg RH, Buchardt O (1991) Sequence-selective recognition of DNA by strand displacement with a thyminesubstituted polyamide. Science 254:1497–1500 2. Dueholm KL, Egholm M, Behrens C, Christensen L, Hansen HF, Vulpius T, Petersen KH, Berg RH, Nielsen PE, Buchardt O (1994) Synthesis of peptide nucleic acid monomers containing the four natural nucleobases: thymine, cytosine, adenine, and guanine and their oligomerization. J Org Chem 59:5767–5773 3. Christensen L, Fitzpatrick R, Gildea B, Petersen KH, Hansen HF, Koch T, Egholm M, Buchardt O, Nielsen PE, Coull J, Berg RH (1995) Solid-phase synthesis of peptide nucleic acids. J Pept Sci 3:175–183 4. Koch T, Hansen HF, Andersen P, Larsen T, Batz HG, Otteson K, Ørum H (1997) Improvements in automated PNA synthesis using Boc/Z monomers. J Pept Res 49:80–88 5. Thomson SA, Josey JA, Cadilla R, Gaul MD, Hassman CF, Luzzio MJ, Pipe AJ, Reed KL, Ricca DJ, Wiethe RW, Noble SA (1995) Fmocmediated synthesis of peptide nucleic acids. Tetrahedron 51:6179–6194 6. Casale R, Jensen IS, Egholm M (1999) Synthesis of PNA oligomers by Fmoc chemistry. In: Nielsen PE (ed) Peptide nucleic acids, protocols and applications. Horizon Scientific Press, Norfolk, UK, pp 39–50 7. Kovacs G, Timar Z, Kupihar Z, Kele Z, Kovacs L (2002) Synthesis and analysis of peptide nucleic acid oligomers using Fmoc/acylprotected monomers. J Chem Soc Perkin Trans 1:1266–1270 8. Turner JJ, Williams D, Owen D, Gait MJ (2006) Disulfide conjugation of peptides to oligonucleotides and their analogs. Curr Protoc Nucleic Acid Chem. Chapter 4:Unit 4.28.1–4.28.21 9. Avitabile C, Moggio L, d’Andrea LD, Pedone C, Romanelli A (2010) Development of an efficient and low-cost protocol for the manual PNA synthesis by Fmoc chemistry. Tetrahedron Lett 51:3716–3718 10. Joshi R, Jha D, Su W, Engelmann J (2011) Facile synthesis of peptide nucleic acids and peptide nucleic acid-peptide conjugates on an automated peptide synthesizer. J Pept Sci 17:8–13 11. Fabani MM, Abreu-Goodger C, Williams D, Lyons PA, Torres AG, Smith KG, Enright AJ, Gait MJ, Vigorito E (2010) Efficient inhibition

of miR-155 function in vivo by peptide nucleic acids. Nucleic Acids Res 38:4466–4475 12. Pothukanuri S, Pianowski Z, Winssinger N (2008) Expanding the scope and orthogonality of PNA synthesis. Eur J Org Chem 2008:3141–3148 13. Pritz S, Wolf Y, Klemm C, Bienert M (2006) Modification of guanine residues in PNA-synthesis by PyBOP. Tetrahedron Lett 47:5893–5896 14. Brelpohl G, Knolle J, Langner D, O’Malley G, Uhlmann E (1996) Synthesis of polyamide nucleic acids (PNAs) using a novel Fmoc/ Mmt protecting-group combination. Bioorg Med Chem Lett 6:665–670 15. Will DW, Breipohl G, Langner D, Knolle J, Uhlmann E (1995) The synthesis of polyamide nucleic acids using a novel monomethoxytrityl protecting-group strategy. Tetrahedron 51:12069–12082 16. Chouikhi D, Ciobanu M, Zambaldo C, Duplan V, Barluenga S, Winssinger N (2012) Expanding the scope of PNA-encoded synthesis (PES): Mtt-protected PNA fully orthogonal to Fmoc chemistry and a broad array of robust diversity-generating reactions. Chem Eur J 18:12698–12704 17. Debaene F, Mejias L, Harris JL, Winssinger N (2004) Synthesis of a PNA-encoded cysteine protease inhibitor library. Tetrahedron 60:8677–8690 18. Bialy L, Diaz-Mochon JJ, Specker E, Keinicke L, Bradley M (2005) Dde-protected PNA monomers, orthogonal to Fmoc, for the synthesis of PNA-peptide conjugates. Tetrahedron 61:8295–8305 19. Svensen N, Dı´az-Mocho´n JJ, Bradley M (2008) Microwave-assisted orthogonal synthesis of PNA–peptide conjugates. Tetrahedron Lett 49:6498–6500 20. Huang Y-C, Cao C, Tan X-L, Li X, Liu L (2014) Facile solid-phase synthesis of PNApeptide conjugates using pNZ-protected PNA monomers. Org Chem Front 1:1050–1054 21. Lee H, Jeon JH, Lim JC, Choi H, Yoon Y, Kim SK (2007) Peptide nucleic acid synthesis by novel amide formation. Org Lett 9:3291–3293 22. Chantell CA, Fuentes G, Patel H, Menakuru M (2009) Low-cost, automated synthesis of a PNA-peptide conjugate on a peptide synthesizer. Am Biotechnol Lab 27:8–10 23. Wojciechowski F, Hudson RHE (2008) A convenient route to N-[2-(Fmoc)aminoethyl]glycine esters and PNA oligomerization using a

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bis-N-Boc nucleobase protecting group strategy. J Org Chem 73:3807–3816 24. Porcheddu A, Giacomelli G, Piredda I, Carta M, Nieddu G (2008) A practical and efficient approach to PNA monomers compatible with Fmoc-mediated solid-phase synthesis protocols. Eur J Org Chem 2008:5786–5797 25. Browne EC, Langford SJ, Abbott BM (2012) Peptide nucleic acid monomers: a convenient and efficient synthetic approach to Fmoc/Boc monomers. Aust J Chem 65:539–544 26. Hansen AM, Bonke G, Hogendorph WFJ, Bjo¨rkling F, Nielsen J, Nielsen PE, Kongstad KT, Zabicka D, Franzyk H (2019) Microwaveassisted solid-phase synthesis of antisense acpP peptide nucleic acid-peptide conjugates active against colistin- and tigecycline-resistant E. coli and K. pneumoniae. Eur J Med Chem 168:134–145 27. Sugiyama T, Kittaka A, Takemoto Y, Takayama H, Kuroda R (2002) Synthesis of

PNA using a Fmoc/Boc protecting group strategy. Nucl Acids Res Suppl (2):145–146 28. Debaene F, Da Silva JA, Pianowski Z, Duran FJ, Winssinger N (2007) Expanding the scope of PNA-encoded libraries: divergent synthesis of libraries targeting cysteine, serine and metallo-proteases as well as tyrosine phosphatases. Tetrahedron 63:6577–6586 29. Hansen AM, Bonke G, Larsen CJ, Yavari N, Nielsen PE, Franzyk H (2016) Antibacterial peptide nucleic acid–antimicrobial peptide (PNA-AMP) conjugates: antisense targeting of fatty acid biosynthesis. Bioconjugate Chem 27:863–867 30. Ivanova GD, Arzumanov A, Abes R, Yin H, Wood MJA, Lebleu B, Gait MJ (2008) Improved cell-penetrating peptide-PNA conjugates for splicing redirection in HeLa cells and exon skipping in mdx mouse muscle. Nucleic Acids Res 36:6418–6428

Chapter 2 A Robust Method for Preparing Optically Pure MiniPEG-Containing Gamma PNA Monomers Wei-Che Hsieh and Danith H. Ly Abstract We report the syntheses of chemical building blocks of a particular class of chiral PNAs, called miniPEGcontaining (R)-gamma PNAs (or (R)-MPγPNAs). The strategy involves the application of 9-(4-bromophenyl)-9-fluorenyl as a temporary, safety-catch protecting group for the suppression of racemization in the alkylation and reductive amination steps. The methodology is general and robust, ideally suited for largescale monomer productions with most synthetic steps providing excellent chemical yields without the need for purification other than a simple workup and precipitation. Key words Chiral PNAs, Large-scale monomer productions, Peptide nucleic acids, miniPEG

1

Introduction Peptide nucleic acids (PNAs) have emerged over the past two decades as a promising class of DNA and RNA mimics, in which the sugar phosphodiester backbone has been replaced by achiral N(aminoethyl)glycine units [1]. Among their many appeals, including tight and sequence-specific binding and resistance to enzymatic degradation [2, 3], they are relatively simple to chemically synthesize and structurally modify as compared to the natural, chiral-rich DNA/RNA counterparts. Since the initial report by Nielsen and colleagues in the early 1990s, considerable efforts have been devoted toward understanding the structure and function relationship of PNAs by making chemical modifications in the backbone [4–6] and in the carboxymethyl linker that connects the backbone to the nucleobases [7, 8]. Among them, installation of a chiral center at the gamma (γ)-backbone showed great potential because of its helical induction [9]. Depending on the nature of chirality, those that bear the stereochemistry as shown in Fig. 1 adopt a right-handed (RH) helical motif and hybridize to DNA or RNA with high affinity and sequence specificity [10] and are able to invade double helical B-form DNA

Peter E. Nielsen (ed.), Peptide Nucleic Acids: Methods and Protocols, Methods in Molecular Biology, vol. 2105, https://doi.org/10.1007/978-1-0716-0243-0_2, © Springer Science+Business Media, LLC, part of Springer Nature 2020

17

18

Wei-Che Hsieh and Danith H. Ly B O

O

O

FmocHN

O (R) N g b

a

O OH

1a-d O

NHBoc NH

B= N a

O

NHBoc

N

N

N

N b

N N

O N

HN O BocHN

c

N

N

d

Fig. 1 Chemical structure of MiniPEG gamma PNA

[11]. In contrast, those that contain an inverted stereochemistry adopt a left-handed (LH) helical sense and are orthogonal to DNA, RNA, and RH-γPNA in recognition [12]. Such a feature is in itself appealing for certain applications [13]. Of the various chemical groups that have been installed at this position, diethylene glycol (commonly referred to as “MiniPEG” or “MP”) has shown considerable improvements in water solubility and biocompatibility [14]. These newly endowed features of (R)MPγPNAs have been exploited in a number of biological and biomedical applications including diagnostics [15] and gene editing [16, 17]; however, the chemical building blocks from which they are assembled are daunting to prepare in large scale and with high optical purity [14]. Such an impediment has prevented (R)MPγPNAs from finding widespread applications in biology, biotechnology, and medicine. This chapter outlines a robust synthetic method for preparing optically pure (>99.5% ee) (R)-MP-gamma PNA monomers by employing a sterically hindered 9-(4-bromophenyl)-9-fluorenyl (BrPhF) as a temporary protecting group in the alkylation and reductive amination steps in the preparation of the backbone intermediate. The synthesis schemes are shown in Figs. 2, 3, and 4. Such a synthetic route is well suited for the production of (R)-MP-gamma PNAs for preclinical and clinical applications (Figs. 2–4). Compounds 2a–d, which are not shown and contained an inverted stereochemistry at the γ-backbone, were prepared in the same way starting from D-serine.

2

Materials Chemical reagents can be used directly from commercial sources without further purification, but solvents need to be dried and freshly distilled prior to use. Silica gels (60 A˚, 63–200 μm) and NH2-coated silica gel (100 A˚, 40–75 μm) are recommended for flash chromatography.

Preparing Optically Pure MiniPEG Gamma PNA Monomers

O HCl H2N

OMe 3 OH

BrPhFHN

2. [13], Pb(NO3)2 K3PO4, ACN; 96%

3. PTSA, MeOH 95% (3 steps)

OMe 5

N

O

OH

Br

O MPhFHN

2. Pd(OAc)2, XPhos, Cs2CO3, morpholine, toluene; 96%

= MPhF N

10

OTHP

= BrPhF

O

8

1. [17], NaH, TBAI, DMF; 77% [9]

O

1. DHP, PTSA, DCM; 83% [4]

1. LiOH, THF/H2O; [6] 2. CH3ONHCH3 HCl, DCC, DhBtOH, DMF, DCM; [7] BrPhFHN

19

O

OR

N O

Fig. 2 Synthetic route for gamma PNA (A)

Br

1. i. n-BuLi, ether ii. 9-Fluorenone; 87% [12]

Br

Br

2. AcBr, toluene, Quant. yield

Br 11

13

(B) HO

(Boc)2O, Mg(ClO4)2, DCM; 71% O

BnO

O

BnO 15

14 1. H2, Pd/C, MeOH; [16] 2. TsCl, Et3N, DMAP, DCM 90% (2 steps)

O

O O

TsO 17

Fig. 3 Synthetic route for gamma PNA

3

Methods Perform all TLC chromatography with normal phase silica gel unless otherwise noted. Glassware needs to be oven-dried, and all reactions are performed in an inert atmosphere (N2-filled balloon). The compounds are characterized by 1H- and 13C- using DMSOd6 or CDCl3 as solvent and high-resolution mass spectrometry (HRMS).

20

Wei-Che Hsieh and Danith H. Ly RO 1. LiAH, THF; [18] 10

BCH2CO2H, HBTU, DMF; 70-96%

MPhFHN

2. PTSA NH2CH2CO2Allyl, DIEA, NaBH(OAc)3, DCE; 82% (2 steps)

NH

19 O

O B

B RO

1. HOC(CO2H)3, ACN/H2O; [21a-d]

RO

2. Fmoc-Cl, NaHCO3, THF; O 82-91% (2 steps)

FmocHN

O N

MPhFHN

O

O N O

O

22a-d

20a-d B O

RO

Pd(PPh3)4, PhSiH3, DCM; 88-96%

FmocHN

N

O

R = (CH2CH2O)2tBu a, B = T; b, B = ABoc; OH c, B = CBoc; d, B = GBoc

1a-d

Fig. 4 Synthetic route for gamma PNA 3.1 Synthesis of O3(Tetrahydropyran-2yl)-L-Serine Methyl Ester Hydrochloride (4)

1. This procedure is adopted from Asahina’s work [18]. 2. To a 250 mL RB flask containing 100 mL of anhydrous CH2Cl2, add 19.5 g of L-serine methyl ester hydrochloride, 17.1 mL of 3,4-dihydro-1-H-pyran, and 0.476 g of p-toluenesulfonic acid monohydrate. Stir the mixture at RT for 20 h. 3. Monitor the reaction progress by TLC: The Rf value of the desired product is 0.6 and that of the starting material is 0.0 using MeOH/CH2Cl2 (10/90) eluents. 4. Upon consumption of the starting material, cool the reaction mixture in an ice bath for 5 min. Filter the precipitate using Grade-1 filter paper on Bu¨chner funnel and wash with chilled CH2Cl2 (50 mL) and hexane (100 mL). 5. Dry the precipitate in vacuo for 12 h to afford ~26 g of compound 4 as white powder, corresponding to ~83% yield. The crude material is used in the next step without further purification.

Preparing Optically Pure MiniPEG Gamma PNA Monomers

21

3.2 Synthesis of N-(9(4-Bromophenyl)-9Fluorenyl)-O3(Tetrahydropyran-2-yl)Serine Methyl Ester (5)

1. To a 500 mL RB flask containing 208 mL of anhydrous toluene, add 14.1 g of 9-(4-bromophenyl)-9-fluorenol (13) and stir in an ice bath for 10 min. 2. To this flask, dispense 61.9 mL of acetyl bromide and heat the mixture at 120
C in an oil bath under reflux. 3. Monitor the reaction progress by GC-MS. The reaction is typically complete within 2 h. 4. Concentrate the reaction mixture under reduced pressure. Dry the resulting residue under high vac overnight to afford compound 13 as yellowish syrup. The compound is carried to the next step without further purification (see Note 1). 5. In a dried 500 mL RB flask, add 110 mL of anhydrous acetonitrile, 8.0 g of serine ester (4), 55.3 g of Pb(NO3)2, and 35.5 g of K3PO4. Stir the resulting mixture at RT for 30 min (see Note 2). 6. Add 44 mL of anhydrous CH2Cl2 to the flask in step 5 and swirl a few times. This should dissolve compound 13. Transfer this solution via cannula into the suspension in step 4 and stir at RT. 7. Monitor the reaction progress by TLC. The Rf value of the desired product is 0.4 with EtOAc/hexane (15/85) as eluents. The reaction is complete within 24 h. 8. Add 10 mL of MeOH to the flask and swirl for a few min. Filter the reaction mixture with Celite pad on Grade-1 filter paper. Concentrate the filtrate under reduced pressure. 9. Purify the reaction mixture by flash chromatography on silica gel using EtOAc/hexane gradient from 8/92 to 15/85. This should give ~17 g of compound 5 as yellow syrup, corresponding to ~96% yield from 4.

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Wei-Che Hsieh and Danith H. Ly

3.3 Synthesis of 2-N(9-(4-Bromophenyl)-9Fluorenyl)-3-HydroxylN-Methoxy-NMethylpropanamide (8)

1. To a 500 mL RB flask cooled in an ice bath, add 200 mL of ethanol, 200 mL of water, and 10.0 g of methyl ester 5. Continue to stir in an ice bath for 5 min. 2. To this reaction mixture, add 3.21 g of lithium hydroxide monohydrate. 3. Remove the ice bath and continue to stir at RT. 4. Monitor the reaction progress by TLC. The Rf value of the hydrolyzed intermediate 6 is 0.0 with EtOAc/hexane (15/85) as eluents. 5. Upon completion (~12 h), neutralize the reaction with Dowex 50WX8 (see Note 3). 6. Filter the reaction mixture with Grade-1 filter paper on a Bu¨chner funnel. Concentrate the filtrate under reduced pressure, followed by co-evaporation with anhydrous toluene (3 100 mL), to afford compound 6 as white solid. The crude material is used in the next step without further purification (see Note 4). 7. To a 500 mL RB flask containing compound 6, add 150 mL of DMF and 50 mL of CH2Cl2 and stir in an ice bath for 5 min until all the material is dissolved. Then add 5.9 g of dicyclohexylcarbodiimide and 4.7 g of 3-hydroxy-1,2,3-benzotriazin-4 (3H)-one and stir at 0
C for 1 h. 8. In a separate 250 mL RB flask, add 150 mL of DMF, 50 mL of CH2Cl2, 2.8 g of N,O-dimethylhydroxylamine, and 5 mL of N,N-diisopropylethylamine, and stir at RT for 5 min. Transfer the solution via cannula to the reaction mixture in step 7. The reaction is typically complete within 2 h. 9. Monitor the reaction progress by TLC. The Rf value of compound 7 is 0.3 with EtOAc/hexane (15/85) as eluents. 10. Quench the reaction by adding 20 mL of methanol to the flask. Concentrate the reaction mixture under reduced pressure.

Preparing Optically Pure MiniPEG Gamma PNA Monomers

23

11. Dissolve the crude material with 100 mL of EtOAc and transfer the mixture to an extraction flask. Wash the combined organic layers with sat. NaHCO3 (3 50 mL) and brine (3 50 mL), and dry over Na2SO4. Concentrate the solution under reduced pressure to afford Weinreb amide 7 as yellowish foam. This compound is used in the next step without further purification. 12. To the flask containing compound 7 above, add 95.7 mL of methanol and 3.6 g of p-toluenesulfonic acid monohydrate. Stir the mixture at RT. 13. Monitor the reaction progress by TLC. The Rf value of the deprotected alcohol 8 is 0.4 with EtOAc/hexane (5/35) as eluents. The reaction is typically complete within 18 h. 14. Quench the reaction by adding 100 mL of sat. NaHCO3 into the flask. Remove solvents under reduced pressure to afford solid residue. 15. Dissolve the residue with 100 mL of EtOAc and wash with 100 mL of H2O. Dry the organic layer over Na2SO4, filter, and concentrate under reduced pressure to give the crude sticky material. 16. Purify the mixture by flash chromatography with EtOAc/hexane gradient from 40/60 to 85/15 to afford ~8 g of alcohol 8 as colorless syrup, corresponding to ~95% yield from compound 5. 3.4 Synthesis of 2-N(9-(4-Bromophenyl)-9Fluorenyl)-3-(2-(2(tert-Butoxy)Ethoxy) Ethoxy)-N-Methoxy-NMethylpropanamide (9)

1. To a 250 mL RB flask containing 83 mL of DMF, add 7.73 g of alcohol (8) and stir the mixture at 0
C in an ice bath for 10 min. To this flask, add 1.00 g of 60% NaH slowly (see Note 5).

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Wei-Che Hsieh and Danith H. Ly

2. Stir the reaction mixture at 0
C for another 30 min before adding 0.10 g of tetrabutylammonium iodide and 10.5 g of tosylate (17). Remove the flask from ice bath and continue to stir at RT. 3. Monitor the reaction progress by TLC: The Rf value of the alkylated product (9) is 0.4 using EtOAc/hexane (50/50) as eluents. 4. Upon completion (~12 h), add 20 mL of sat. NH4Cl to the flask. Remove solvents by rotovap. Dissolve the residue with EtOAc (100 mL) and wash with water (2 100 mL). Dry the organic layer over Na2SO4, and remove the solvent by rotovap. 5. Purify the residue by column chromatography on silica gel with EtOAc/hexane gradient from 22/78 to 53/47 to afford 7.80 g of alkylated compound (9) as colorless syrup with 77% yield. 3.5 Synthesis of 3(2-(2-(tert-Butoxy) Ethoxy)Ethoxy)-NMethoxy-N-Methyl-2N-(9-(4Morpholinophenyl)-9Fluorenyl)Propanamide (10)

1. Under N2 in the glove box, add 7.50 g of compound (9), 137.7 mg of Pd(OAc)2, 292.31 mg of XPhos, 20.0 g of cesium carbonate, and 24 mL of anhydrous toluene to a 50 mL pressure vessel. 2. Transfer the vessel to an atmospheric environment and purge with N2 gas by Schlenk line. After 2 min, add 1.29 mL of morpholine to the reaction vessel, seal the vessel with PTFE cap, and heat it to 80
C. 3. After 2 h, gradually cool the vessel to RT (~1 h). Monitor the reaction progress by TLC: The Rf value of the amination product (10) is 0.2 with EtOAc/hexane (50/50) as eluents (see Note 6).

Preparing Optically Pure MiniPEG Gamma PNA Monomers

25

4. Upon completion, filter the mixture through Celite pad with Grade-1 filter paper on Bu¨chner funnel. Concentrate the filtrate by rotovap and purify the resulting product by flash column chromatography on NH2-functionalized silica gel with EtOAc/hexane gradient from 18/72 to 63/37 to afford 7.27 g of amination product (10) as colorless syrup with 96% yield. 3.6 Synthesis of 9(4-Bromophenyl)-9Fluorenol (12)

Br

OH 12

1. To a 2.0 L RB flask with two necks containing 1.0 L of anhydrous ether with a N2 balloon, add 72.1 g of 1,4-dibromobenzene and stir the mixture at 50
C in ACN/dry-ice bath for 20 min. 2. To this flask, add 25 mL of 1 M n-butyllithium (1 M, 25 mL) dropwise over the course of 1 h via cannula. Stir the mixture for another 30 min at the same temperature. 3. To a separate 500 mL RB flask containing 400 mL of anhydrous ether, add 38.5 g of fluorenone and stir the mixture under N2. Transfer this fluorenone solution to the 2 L RB flask at 50
C. Allow the mixture to gradually warm to RT and continue to stir until the reaction is complete (~3 h). 4. Monitor the reaction progress by TLC: The Rf value of compound (12) is 0.4 with EtOAc/hexane (15/85) as eluents. 5. Upon completion, place the reaction vessel in an ice bath and stir for 10 min. To this flask, slowly add 200 mL of water. Extract the solution with ether (2 300 mL). Combine the organic layers and dry over MgSO4. Remove solvent by rotovap and purify the resulting yellow syrup by flash column chromatography on silica gel with EtOAc/hexane gradient from 1/49 to 1/9 to afford 62.6 g of product (12) as white solid with 87% yield. 3.7 Synthesis of ((2(2-(tert-Butoxy) Ethoxy) Ethoxy)Methyl) Benzene (15)

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1. To a 2 L RB flask containing 1.0 L of anhydrous CH2Cl2, add 157.0 g of ethylene glycol monobenzyl ether and 401.6 g of Boc anhydride. Stir the reaction mixture at 50
C in an ACN/dry-ice bath for 10 min, followed by the addition of 17.9 g of Mg(ClO4)2 (see Note 7). Allow the reaction mixture to warm to RT and reflux at 40
C. 2. Monitor the reaction progress by TLC: The Rf value of compound (15) is 0.6 with EtOAc/hexane (15/85) as eluents. 3. Upon completion (~36 h), quench the reaction by adding 200 mL of sat. NaHCO3 to the flask. Extract the aqueous solution with CH2Cl2 (2 200 mL). Combine the organic layers and wash with brine (200 mL), and dry over Na2SO4. Remove the solvent by rotovap. 4. Purify the resulting residue by flash column chromatography on silica gel with EtOAc/hexane gradient from 1/99 to 16/84 to afford 144.3 g of product (15) as colorless syrup with 71% yield. 3.8 Synthesis of 2-(2-(tert-Butoxy) Ethoxy)Ethyl-4MethylbenzeneSulfonate (17)

1. To a 100 mL glass beaker containing 50 mL of MeOH, add 144.3 g of tert-butyl ether (15). Stir the reaction mixture in an ice bath for 10 min, followed by the addition of 608.5 mg of Pd/C (see Note 8). 2. Place the beaker in a stainless high-pressure reactor and charge it with 100 psi of H2 gas. After 12 h, carefully bleed H2 gas in the hood. Purge the reactor with N2 gas (3 50 psi) before opening it. 3. Remove the beaker from the reactor and place it in an ice bath for 10 min. Filter the solution through a Celite pad with Grade-1 filter paper on Bu¨chner funnel. 4. Remove the solvent by rotovap to afford alcohol (16). Use the crude product in the next step without further purification. 5. To a 2 L RB flask containing the above alcohol (16), add 1.0 L of anhydrous CH2Cl2, 41.9 g of 4-dimethylaminopyridine, 79.7 mL of triethylamine, and 130.8 g of tosyl chloride at 0
C in an ice bath. Stir the reaction mixture at RT. 6. Monitor the reaction progress by TLC: The Rf value of product is 0.4 with EtOAc/hexane (22/78) as eluents.

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27

7. Upon completion (~12 h), quench the reaction with 200 mL of 10% citric acid. Extract the product with ether (200 mL), wash with brine (200 mL), and dry over Na2SO4. 8. Remove the solvent by rotovap and purify the product by flash column chromatography on silica gel with EtOAc/hexane gradient from 1/99 to 35/65 to afford 163.1 g of tosylate (17) as colorless syrup with 90% overall yield from tert-butyl ether (15) (see Note 9). 3.9 Synthesis of 3-(2(2-(tert-Butoxy)Ethoxy) Ethoxy-2-N-(9-(4Morpholinophenyl)-9Fluorenyl))-Serine-Ψ [CH2N]Gly-O-Allyl (19)

1. To a 50 mL RB flask containing 7 mL of anhydrous THF at 0
C, add 423.9 mg of amide (10) and 26.0 mg of lithium aluminum hydride. Stir the reaction mixture at the same temperature. 2. Monitor the reaction progress by TLC: The Rf value of product is 0.5 with EtOAc/hexane (50/50) as eluents. 3. Upon completion (~1 h), quench the reaction by adding 5 mL of sat. Na2SO4 into the flask. Extract the compound with ether (2 10 mL), combine and wash the organic layers with brine (20 mL), and dry over Na2SO4. Remove solvent by rotovap to afford aldehyde (18). This crude product is used in the next step without further purification. 4. Add 7 mL of anhydrous dichloroethane to the 50 mL RB flask containing aldehyde (18) above and chill it in an ice bath for 10 min. 5. To this flask, add 197.2 mg of glycine allyl ester toluenesulfonic acid salt, 239.0 μL of N,N-diisopropylethylamie, and 218.1 mg of sodium triacetoxyborohydride. Stir the reaction mixture at RT.

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6. Monitor the reaction progress by TLC. The Rf value of product is 0.4 with EtOAc/hexane (80/20) as eluents. 7. Upon completion (~20 h), quench the reaction by adding 10 mL of sat. NaHCO3 along with 10 mL of ether into the flask and stir. Extract the mixture with ether (2 10 mL), wash the combined organic layer with brine (20 mL), and dry over Na2SO4. 8. Remove solvent by rotovap and purify the resulting product by flash column chromatography on silica gel with EtOAc/hexane gradient from 24/76 to 99/1 to afford 370.1 mg of backbone (19) as colorless syrup with 82% yield from amide (10) (see Note 10). 3.10 A General Procedure for Coupling Nucleobase Acetic Acid with Backbone (19)

1. To a 25 mL RB flask containing DMF, add 1.0 equiv of backbone, N,N-diisopropylethylamine (2.0 equiv), nucleobase acetic acid (2.0 equiv) and HBTU (1.8 equiv). Stir the mixture at RT for 12 h before quenching with MeOH. 2. Remove solvents by rotovap. Take up the crude residue in EtOAc (20 mL), wash with sat. NaHCO3 (10 mL) and brine (10 mL), dry over Na2SO4, and concentrate in vacuo. 3. Purify the crude material by flash column chromatography on NH2-functionalized silica gel with MeOH/EtOAc gradient from 1/99 to 15/85. 4. (a) Synthesis of 3-(2-(2-(tert-butoxy)ethoxy)ethoxy)-2-N(9-(4-morpholinophenyl)-9-fluorenyl)-L-serine thymine allyl ester (20a): 96% yield; TLC (MeOH/EtOAc, 5/95) Rf ¼ 0.5. (b) Synthesis of 3-(2-(2-(tert-butoxy)ethoxy)ethoxy)-2-N(9-(4-morpholinophenyl)-9-fluorenyl)-L-serine adenine(Boc)

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allyl ester (20b): 70% yield; TLC (MeOH/EtOAc, 5/95) Rf ¼ 0.4. (c) Synthesis of 3-(2-(2-(tert-butoxy)ethoxy)ethoxy)-2-N(9-(4-morpholinophenyl)-9-fluorenyl)-L-serine cytosine(Boc) allyl ester (20c): 83% yield; TLC (MeOH/CHCl3, 5/95) Rf ¼ 0.5. (d) Synthesis of 3-(2-(2-(tert-butoxy)ethoxy)ethoxy)-2-N(9-(4-morpholinophenyl)-9-fluorenyl)-L-serine guanine(Boc) allyl ester (20d): 75% yield; TLC (MeOH/CHCl3, 10/90) Rf ¼ 0.2 on NH2-functionalized TLC plate. 3.11 A General Method to Convert MPhF-Protected Monomer Allyl Ester 20a–d to FmocProtected Monomer Allyl Ester 22a–d

1. To a 25 mL RB flask, add 1 equiv of MPhF-protected monomer allyl ester 20 and ACN (0.1 M) at 0
C and 3.6 equiv of 1 M citric acid. Allow the reaction mixture to gradually warm to RT and stir for 12 h. 2. Monitor the reaction progress by HRMS of the diluted reaction mixture in methanol. 3. Upon completion, transfer the flask to an ice bath and stir for 10 min before adding 14 equiv of NaHCO3 and 2.5 equiv of Fmoc-Cl and stir for another 3 h. Remove solvent by rotovap and purify the resulting residue by column chromatography with EtOH/CHCl3 gradient from 1/99 to 10/90 to afford Fmoc-miniPEG-γPNA monomer allyl ester (22). 4. (a) Synthesis of Fmoc-3-(2-(2-(tert-butoxy)ethoxy)ethoxy)-Lserine thymine monomer allyl ester (22a): 86% yield; TLC (MeOH/CHCl3, 5/95) Rf ¼ 0.5. (b) Synthesis of Fmoc-3-(2-(2-(tert-butoxy)ethoxy)ethoxy)-Lserine adenine(Boc) monomer allyl ester (22b): 85% yield; TLC (MeOH/CHCl3, 5/95) Rf ¼ 0.5. (c) Synthesis of Fmoc-3-(2-(2-(tert-butoxy)ethoxy)ethoxy)-Lserine cytosine(Boc) monomer allyl ester (22c): 91% yield; TLC (MeOH/CHCl3, 5/95) Rf ¼ 0.3. (d) Synthesis of Fmoc-3-(2-(2-(tert-butoxy)ethoxy)ethoxy)-Lserine guanine(Boc) monomer allyl ester (22d): 85% yield; TLC (MeOH/CHCl3, 5/95) Rf ¼ 0.1.

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3.12 A General Method for Converting Fmoc-Protected Monomer Allyl Ester 22a–d to the Final Monomer 1a–d

1. To a 25 mL RB flask, add 1 equiv of allyl ester 22, DCM (0.1 M), 0.2 equiv of Pd[PPh3]4, and 20 equiv of PhSiH3 under N2. Stir the reaction mixture at RT for 12 h. Remove solvent by rotovap and purify the crude product by column chromatography (A ¼ EtOAc, B ¼ ACN/MeOH/H2O ¼ 2/ 1/1, A/B ¼ 97/3 to 66/34) to afford final Fmoc-miniPEG-γ PNA monomers 1. 2. (a) Synthesis of Fmoc-3-(2-(2-(tert-butoxy)ethoxy)ethoxy)-Lserine thymine monomer (1a): 96% yield; TLC (EtOAc/ ACN/MeOH/H2O, 6/1/1/1) Rf ¼ 0.5. (b) Synthesis of Fmoc-3-(2-(2-(tert-butoxy)ethoxy)ethoxy)-Lserine adenine(Boc) monomer (1b): 90% yield; TLC (EtOAc/ ACN/MeOH/H2O, 6/1/1/1) Rf ¼ 0.4. (c) Synthesis of Fmoc-3-(2-(2-(tert-butoxy)ethoxy)ethoxy)-Lserine cytosine(Boc) monomer (1c): 92% yield; TLC (EtOAc/ ACN/MeOH/H2O, 6/1/1/1) Rf ¼ 0.5. (d) Synthesis of Fmoc-3-(2-(2-(tert-butoxy)ethoxy)ethoxy)-Lserine guanine(Boc) monomer (1d): 88% yield; TLC (EtOAc/ ACN/MeOH/H2O, 6/1/1/1) Rf ¼ 0.3. 3.13 Determination of Optical Purities

1. To a 10 mL RB flask, add 1.0 equiv of thymine monomers (1a and 2a), DMF (0.1 M), and 10 equiv of piperidine. 2. After 2 h, remove solvents under reduced pressure. Co-evaporate residue with toluene three times. 3. To this crude residue, add CH2Cl2 (0.1 M), 2 equiv of N,Ndiisopropylethylamine, and 1.1 equiv of (S)(+)-α-methoxy-α-(trifluoromethyl)phenylacetyl chloride (MTPA-Cl). Allow the reaction to stir at RT for 3 h before diluting with CH2Cl2. Wash the organic layer with water twice and then brine, and dry over Na2SO4. Remove solvent by rotovap. 4. Inject the crude mixture into HPLC/C18 column to analyze the enantiomeric excess (see Note 11).

Preparing Optically Pure MiniPEG Gamma PNA Monomers

31

5. For NMR analysis, purify the crude sample by column chromatography (A ¼ EtOAc, B ¼ ACN/MeOH/H2O ¼ 2/1/1, A/B ¼ 97/3 to 66/34).

Compound 23: TLC (EtOAc/ACN/MeOH/H2O, 6/1/ 1/1) Rf ¼ 0.5.

Compound 24: TLC (EtOAc/ACN/MeOH/H2O, 6/1/ 1/1).

4

Notes 1. Further extraction with water may result in debromination of the product, 9-(4-bromophenyl)-9-fluorene. 2. Pb(NO3)2 and K3PO4 are dried in 200
C oven for 12 h and gradually cooled to RT in a desiccator with DRIERITE desiccant and molecular sieve. 3. The resin is rinsed with methanol before use in order to remove the orange residue. 4. As the reaction proceeds, the hydrolyzed intermediate (6) may precipitate and encapsulate undissolved precursor ester (5) and slow the reaction progress. This problem could be addressed by warming the reaction mixture to 40
C in a water bath.

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5. Strong effervesce occurs! Add NaH in small portions. 6. Under 254 nm irradiation, the amination product is fluorescently blue, while the bromide starting material is dark. 7. Strong effervesce occurs! Add Mg(ClO4)2 in small portions. 8. During the addition, it is easy to catch fire due to the high reactivity of Pd/C. Chilling the flask in an ice bath will mitigate the risk. 9. For long-term storage, tosylate (17) must be placed at –78
C. 10. Purification of backbone (19) by column chromatography could be skipped if the excess by-product acetic acid is thoroughly removed by extraction. 11. HPLC is performed with a C18 column (dimensions 4.6 mm  250 mm) at a flow rate of 1 mL/min at 60
C oven temperature on Shimadzu UFLC system. The gradient is from 25% to 75% ACN/H2O with 0.1% TFA in 30 min. References 1. Nielsen PE, Egholm M, Berg RH, Buchardt O (1991) Sequence-selective recognition of DNA by strand displacement with a thyminesubstituted polyamide. Science 254 (5037):1497–1500 2. Nielsen PE (1999) Peptide nucleic acid. A molecule with two identities. Acc Chem Res 32:624–630 3. Demidov VV, Potaman VN, FrankKamenetskii MD, Egholm M, Buchard O, Sonnichsen SH, Nielsen PE (1994) Stability of peptide nucleic acids in human serum and cellular extracts. Biochem Pharmacol 48 (6):1310–1313 4. Nielsen PE, Haaima G (1997) Peptide nucleic acid (PNA). A DNA mimic with a pseudopeptide backbone. Chem Soc Rev 26(2):73–78 5. Kumar VA, Ganesh KN (2005) Conformationally constrained PNA analogues: structural evolution toward DNA/RNA binding selectivity. Acc Chem Res 38:404–412 6. Corradini R, Sforza S, Tedeschi T, Marchelli R (2007) Chirality as a tool in nucleic acid recognition: principles and relevance in biotechnology and in medicinal chemistry. Chirality 19:269–294 7. Hyrup B, Egholm M, Buchardt O, Nielsen PE (1996) A flexible and positively charged PNA analog with an ethylene-linker to the nucleobase: synthesis and hybridization properties. Bioorg Med Chem Lett 6:1083–1088 8. Hyrup B, Egholm M, Nielsen PE, Wittung P, Norden B, Buchardt O (1994) Structure-

activity studies of the binding of modified peptide nucleic acids (PNAs) to DNA. J Am Chem Soc 116(18):7964–7970 9. Dragulescu-Andrasi A, Rapireddy S, Frezza BM, Gayathri C, Gil RR, Ly DH (2006) A simple gamma-backbone modification preorganizes peptide nucleic acid into a helical structure. J Am Chem Soc 128:10258–10267 10. Rapireddy S, He G, Roy S, Armitage BA, Ly DH (2007) Strand invasion of mixed-sequence B-DNA by acridine-linked, gamma-peptide nucleic acid (gamma-PNA). J Am Chem Soc 129:15596–15600 11. Bahal R, Sahu B, Rapireddy S, Lee C-M, Ly DH (2012) Sequence-unrestricted, WatsonCrick recognition of double helical B-DNA by (R)-MiniPEG-gPNAs. Chembiochem 13:56–60 12. Sacui I, Hsieh W-C, Manna A, Sahu B, Ly DH (2015) Gamma peptide nucleic acids: as orthogonal nucleic acid recognition codes for organizing molecular self-assembly. J Am Chem Soc 137:8603–8610 13. Hsieh W-C, Martinez GR, Wang AH-J, Wu SF, Chamdia R, Ly DH (2018) Stereochemical conversion of nucleic acid circuits via strand displacement. Commun Chem 1:89 14. Sahu B, Sacui I, Rapireddy S, Zanotti KJ, Bahal R, Armitage BA, Ly DH (2011) Synthesis and characterization of conformationally preorganized, (R)-diethylene glycol-containing g-peptide nucleic acids with superior

Preparing Optically Pure MiniPEG Gamma PNA Monomers hybridization properties and water solubility. J Org Chem 76:5614–5627 15. Singer A, Rapireddy S, Ly DH, Meller A (2012) Electronic barcoding of a viral gene at the single-molecule level. Nano Lett 12:1722–1728 16. Bahal R, McNeer NA, Quijano E, Liu Y, Sulkowski P, Bhunia DC, Manna A, Greiner DL, Brehm MA, Cheng CJ, Lopez-Giraldez F, Beloor J, Krause DS, Kumar P, Gallagher PG, Braddock D, Saltzman WM, Ly DH, Glazer PM (2016) In vivo correction of anemia in b-thalassemic mice by yPNA-mediated gene editing with nanoparticle delivery. Nat Commun 7:13304

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17. Ricciadi AS, Bahal R, Farrelly JS, Quijano E, Bianchi AH, Luks VL, Putman R, LopezGiraldez F, Coskun S, Song E, Liu Y, Hsieh W-C, Ly DH, Stitelman DH, Glazer PM, Saltzman WM (2018) In utero nanoparticle delivery for site-specific genome editing. Nat Commun 9:2481 18. Asahina Y, Takei M, Kimura T, Fukuda Y (2008) Synthesis and antibacterial activity of novel pyrido[1,2,3-de][1,4]benzoxazine-6carboxylic acid derivatives carrying the 3-cyclopropylaminomethyl-4-substituted-1-pyrrolidinyl group as a C-10 substituent. J Med Chem 51:3238–3249

Chapter 3 Synthesis of Pyrrolidinyl PNA and Its Site-Specific Labeling at Internal Positions by Click Chemistry Boonsong Ditmangklo, Penthip Muangkaew, Kotchakorn Supabowornsathit, and Tirayut Vilaivan Abstract Pyrrolidinyl PNA with an α-/β-dipeptide backbone consisting of alternating nucleobase-modified Dproline and (1S,2S)-2-aminocyclopentanecarboxylic acid (also known as acpcPNA) is a class of conformationally constrained PNA that shows exceptional DNA hybridization properties including very high specificity and the inability to form self-pairing hybrids. In this chapter, details of the syntheses of acpcPNA as well as its monomers and a protocol for site-specific labeling with a fluorescent dye via click chemistry are reported. Key words AcpcPNA, Pyrrolidinyl PNA, PNA synthesis, Labeling, Click chemistry

1

Introduction Preorganization of PNA structure by constraining its conformational flexibility is a strategy frequently proposed to improve the binding affinity and specificity toward nucleic acid targets. This can be typically achieved by incorporating certain substituents or cyclic structures into the backbone of the PNA [1]. Despite many variations developed to date, few of these conformationally constrained PNA analogues actually give the expected beneficial effects [2–4]. Pyrrolidinyl PNA is a class of such conformationally constrained analogues of PNA that employed a pyrrolidine ring as a constrain element. Extensive structure optimization by combination of nucleobase-modified proline (a pyrrolidinyl PNA monomer, which is equivalent to a nucleoside in DNA) and a cyclic β-amino acid (a spacer, which is equivalent to a phosphate group in DNA) revealed several pyrrolidinyl PNA systems that showed excellent nucleic acid binding properties [5]. One of the most promising systems is acpcPNA which consists of a nucleobase-modified D-prolyl-(1S,2S)-2-aminocyclopentanecarboxylic acid backbone

Peter E. Nielsen (ed.), Peptide Nucleic Acids: Methods and Protocols, Methods in Molecular Biology, vol. 2105, https://doi.org/10.1007/978-1-0716-0243-0_3, © Springer Science+Business Media, LLC, part of Springer Nature 2020

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[6]. AcpcPNA binds to target DNA in exclusively antiparallel fashion and exhibits stronger binding affinity and greater specificity than conventional PNA. Mismatched acpcPNA-DNA hybrids typically give around 20
C lower melting temperature than complementary acpcPNA-DNA hybrid which is much better than DNA and conventional PNA. Moreover, acpcPNA exhibits other unique properties including the preference of binding to DNA over RNA and over self-pairing [7]. The inability of acpcPNA to form selfpairing hybrids suggests its potential applications in targeting double-stranded DNA targets by double duplex invasion without the requirement for base modification [8, 9]. The exceptional ability of acpcPNA to discriminate DNA sequence at a single-base resolution makes it potentially useful as a high-performance probe for DNA sequence determination [5]. Several acpcPNA-based DNA sensing platforms have been proposed, but the most promising one was the quencher-free linear acpcPNA probes such as the thiazole orange-labeled acpcPNA probe that can light up in the presence of specific DNA targets [10, 11]. Similar probes have been successfully used for real-time detection and quantitation of the nucleic acid targets in living cells [12]. Incorporation of 3-aminopyrrolidine-4-carboxylic (APC) acid into the backbone of acpcPNA in place of the usual ACPC spacer provides a convenient handle for post-synthetic modification at any desired positions in the PNA molecule [13]. This approach not only eliminates the tedious steps in the preparation of dye-functionalized monomers but also offers a more precise control of the position of the dyes without much affecting the overall structure and thus the basepairing behavior of the PNA. This chapter will describe in detail the syntheses of acpcPNA probes and a protocol for labeling of the PNA at a predefined site with a fluorescent dye via click chemistry. Protocols for the syntheses of acpcPNA monomers as well as spacers from readily available starting materials are also included. The overall scheme of the processes involved is summarized in Fig. 1.

2 2.1

Materials General

Use solvents and reagents at highest purity grade available from standard suppliers. Use deionized water for all chemical synthesis steps and MilliQ water (18.2 MΩ) for dissolution and purification of PNA. Prepare dry tetrahydrofuran (THF) by distilling reagent grade THF from sodium benzophenone ketyl. Commercially available anhydrous dimethylformamide (DMF) (75% is transcribed into RNA [5, 6]. Today, the functional importance of most RNA transcripts is still unknown, and it is fairly safe to predict that the discovery of many more regulatory RNAs is forthcoming. The recent discovery of a new role for RNA in CRISPR-Cas immunity strongly supports this notion [7–9]. The ability to selectively recognize, detect, and inhibit the function of such RNAs will be highly useful for both fundamental biology and practical applications in biotechnology and medicine. However, since most noncoding RNAs (ncRNAs)

Peter E. Nielsen (ed.), Peptide Nucleic Acids: Methods and Protocols, Methods in Molecular Biology, vol. 2105, https://doi.org/10.1007/978-1-0716-0243-0_9, © Springer Science+Business Media, LLC, part of Springer Nature 2020

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form well-organized double helical conformations, molecular recognition of such species without disrupting their native structure is a formidable challenge. Herein, we describe a method for sequence-specific recognition of structured RNA using peptide nucleic acids (PNAs) [10, 11] that form parallel triple helices with the double-stranded RNA (dsRNA) target. Because of the relatively uniform and polar surface, dsRNA presents little opportunity for hydrophobic shape selective recognition using small molecules [12–18]. Consequently, the most common targets for small molecules are RNA bulges and internal loops. However, binding to these structures is frustrated by conformational flexibility of non-helical RNA. As a result, many RNA binders rely on electrostatic interactions with the negatively charged phosphate backbone to boost the affinity at the expense of selectivity. Designing small molecules that selectively recognize RNA using hydrophobic or electrostatic interactions has been an involved and challenging process [12–18]. Despite dsRNA being an inherently difficult target, several groups have achieved remarkable progress in developing small molecules that bind to and modulate the function of riboswitches [19], mRNA splicing sites [20], RNA three-way junctions [21], toxic RNA repeat sequences [22–26], viral RNAs [27–30], and microRNAs [31–36]. Notwithstanding the significant progress, these studies also revealed that small molecules will not provide a general solution for targeting every RNA, which leaves a substantial gap in technology and room for new alternative approaches. The present chapter describes such an alternative approach that uses Hoogsteen hydrogen bonding in the major groove for sequence-specific recognition of dsRNA. Native RNA forms relatively stable triple helices via parallel binding of a pyrimidine-rich third strand to a purine-rich strand of the double helix at mildly acidic conditions [37]. The sequence selectivity derives from uridine recognition of adenosine-uridine base pairs (U∗A-U triplet) and protonated cytidine recognition of guanosine-cytidine base pairs (C+∗G-C triplet) via the Hoogsteen hydrogen bonding scheme (Fig. 1). However, practical applications of triple helical recognition of nucleic acids have been limited by low affinity of the third strand oligonucleotides and the requirement for long homopurine tracts, as only the T∗A-T (or U∗A-U in RNA) and C+∗G-C triplets can be used in recognition [37]. Moreover, because of a pKa ~ 4.5 cytosine is hardly protonated and does not form the C+∗G-C triplet at physiological pH of 7.4. In 2010, our group was the first to report the formation of highly stable and sequence-specific triple helices between short PNAs and dsRNA [38]. Over the following years, we optimized the PNA design for triple helical recognition of dsRNA sequences in vitro and in cells [39–42]. The most significant modification was the replacement of cytosine with the more basic (pKa ~ 6.7) heterocycle, 2-aminopyridine (M, Fig. 1) [43–49, 56], which enabled

Triplex-Forming PNA for Recognition of dsRNA

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Fig. 1 Hoogsteen triplets for recognition of purines and pyrimidines in dsRNA

PNA-dsRNA triplex formation at physiologically relevant conditions [39]. Surprisingly, M-modification also significantly enhanced PNA uptake in HEK293 cells, which was further improved by conjugation of M-modified PNA to lysine or arginine tripeptides [40, 41]. For the cellular uptake studies, we developed an economic procedure for fluorescent labeling of PNA on solid phase [50], which will be described in detail below. Remarkably, the Mmodified PNAs had unique selectivity for binding RNA, as the PNA-dsRNA triplex was significantly more stable than the PNA-dsDNA triplex of the same sequence [39–41]. Recent structural studies [56] showed that the unusual stability of PNA-dsRNA triplex was due to favourable PNA backbone amide to RNA phosphate hydrogen bonding. This unique ability of chemically modified PNAs to distinguish RNA from DNA highlights their relevance as specific probes of RNA-mediated processes. As mentioned earlier, the requirement for long homopurine tracts is a challenge for practical applications of triplex-forming PNA. Our lab demonstrated that the addition of two previously developed nucleobase modifications, P [51, 52] and E [53] (Fig. 1), allowed recognition of isolated pyrimidine inversions in short polypurine tracts of dsRNA [54]. The ability of nucleobasemodified PNAs (containing M, P, and E) to form strong and selective triplexes with dsRNAs has already been used in a novel fluorescent RNA detection method [55] and in modulating gene expression through stabilization of mRNA secondary structure [42].

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NH N

N

N

HN O N

FmocHN

N O

O OH

Fmoc-PNA-M(Boc)-OH

O

N

FmocHN

OH

O

O

O Fmoc =

NH OH

N

O O

Fmoc-A EEA -OH FmocHN

OH

Fmoc-PNA-P -OH

O FmocHN

O

N

FmocHN

Fmoc-PNA-E -OH

O

O O

O

N

O

O OH

Boc =

O

Fmoc-PNA-T -OH

Fig. 2 Structures of nucleobase-modified [39, 54] and standard T and AEEA (Link Technologies) monomers used for solid-phase PNA synthesis in current protocol

Herein we describe detailed procedures for synthesis and fluorescent labeling [50] of M-modified PNAs and their conjugates with cell-penetrating peptides, as exemplified by the TAT peptide. The protocols for PNA synthesis described below are for the Expedite 8909 DNA synthesizer using the Fmoc-/Boc-protected monomers (Fig. 2). The specific steps, reagents and their concentrations, and PNA deprotection conditions may differ significantly if other instruments or PNA synthesis chemistry is used. To illustrate the molecular recognition of dsRNA, we used isothermal titration calorimetry (ITC) to measure triple helical binding of a PNA nonamer to an RNA hairpin. We also describe a protocol for using the fluorescently labeled PNA-TAT conjugate to monitor PNA uptake in MCF-7 cells. Taken together, the methods described herein should be useful in designing sequence selective PNA probes for detection and functional interference with biologically relevant dsRNA.

2

Materials

2.1 Peptide Synthesis

1. Liberty Blue Automated Microwave Peptide Synthesizer. 2. Amino acids: Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, and Fmoc-Gln(Trt)-OH (natural L series) are commercially available (from NovaBiochem).

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3. Solid support for peptide/PNA synthesis: NovaSyn TG Sieber resin (functionalized at 0.2 mmol/g). 4. Reagents for peptide synthesis on Liberty Blue: (a) Deblocking solution (20% (v/v) piperidine in DMF): mix 20 mL of piperidine (biotech grade, Aldrich) and 80 mL of anhydrous N,N-dimethylformamide (DMF). (b) Activator solution (0.2 M N,N0 -diisopropylcarbodiimide (DIC) in DMF): dissolve 1.57 mL of DIC in 50 mL of anhydrous DMF. (c) Base solution (0.4 M ethyl cyanohydroxyiminoacetate (oxyma) in DMF): dissolve 2.84 g of oxyma in 50 mL of anhydrous DMF. 2.2

PNA Synthesis

1. Expedite 8909 (or equivalent) DNA synthesizer capable of PNA synthesis. 2. Commercial PNA monomer and AEEA spacer: Fmoc-PNA-TOH and Fmoc-AEEA-OH are commercially available (from Link Technologies). 3. HiLyte Fluor 488 carboxylic acid is commercially available (from AnaSpec). 4. Nucleobase-modified PNA monomers 2-pyrimidone (P), 3-oxo-2,3-dihydropyridazine (E), and 2-aminopyridine (M): our recent publications describe details of synthetic preparation of Fmoc-PNA-P-OH and Fmoc-PNA-E-OH [54] and FmocPNA-M-OH [39]. 5. Solid support for peptide/PNA synthesis: NovaSyn TG Sieber resin (functionalized at 0.2 mmol/g). This is a universal support that can be used to synthesize any PNA sequence. 6. Empty synthesis columns for the Expedite 8909 (commercially available from Glen Research). 7. Anhydrous N,N-dimethylformamide (DMF) for Wash A and B and reagent solutions on the Expedite 8909. 8. Reagents for PNA synthesis on Expedite 8909: (a) Deblocking solution (20% (v/v) piperidine in DMF): mix 20 mL of piperidine (biotech grade, Aldrich) and 80 mL of anhydrous N,N-dimethylformamide (DMF). (b) Activator solution (0.19 M O-(7-azabenzotriazol-1-yl)1,1,3,3-tetramethyluronium hexafluorophosphate (HATU) in DMF): dissolve 1.089 g of HATU in 15 mL of anhydrous DMF. (c) Base solution (0.2 M diisopropylethylamine (DIPEA) and 0.3 M 2,6-lutidine in DMF): mix 93.2 mL of anhydrous DMF, 3.3 mL of DIPEA (reagent grade distilled over CaH2), and 3.5 mL of lutidine (distilled over CaH2).

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(d) Capping solution (5% (v/v) acetic anhydride and 6% (v/v) 2,6-lutidine in DMF): mix (in the following order) 89 mL of anhydrous DMF, 6 mL of lutidine (distilled over CaH2), and 5 mL of acetic anhydride (reagent grade). 9. Reagents for cleaving the PNA from the solid support: (a) Cleavage Cocktail (20% (v/v) m-cresol in TFA): mix 0.8 mL of trifluoroacetic acid (TFA, HPLC grade) and 0.2 mL of m-cresol (reagent grade). (b) Anhydrous diethyl ether. 2.3

HPLC Purification

1. HPLC system consisting of gradient-capable pumps, column oven, and UV detector. 2. HPLC columns: (1) Supelco Discovery BIO Wide Pore C-18 (3 μm, 4.6 mm  150 mm) for PNA purification and (2) Waters XBridge Prep C-18 column for RNA purification. 3. Syringe filter, 13 mm with a 0.2 μm PTFE membrane. 4. Eluents for HPLC purification of PNA: (a) Mobile phase A (0.1% (v/v) formic acid in water): mix 1 mL of formic acid (HPLC grade) and 1 L of water (HPLC grade). Filter the solution through a 0.20 μm nylon membrane filter. (b) Mobile phase B (0.1% (v/v) formic acid in acetonitrile): mix 1 mL of formic acid (HPLC grade) and 1 L of acetonitrile (HPLC grade). Filter the solution through a 0.20 μm nylon membrane filter. 5. Eluents for HPLC purification of RNA: (a) Mobile phase C (0.1 M triethylammonium acetate in water, pH 7.0): add 14 mL of triethylamine (HPLC grade) to 0.8 L of water (HPLC grade) followed by 5 mL of acetic acid (HPLC grade). In a separate vessel, mix 1 mL of acetic acid (HPLC grade) and 9 mL of water (HPLC grade); then use this solution to titrate mobile phase C until a pH 7.0 is obtained. Complete the preparation of mobile phase C by bringing the volume to 1 L by adding water (HPLC grade). Filter the solution through a 0.20 μm nylon membrane filter. (b) Mobile phase D (40% (v/v) acetonitrile and 0.1 M triethylammonium acetate in water, pH 7.0): mix 400 mL of acetonitrile (HPLC grade) and 600 mL of mobile phase C (see step 5(a)). Filter the solution through a 0.20 μm nylon membrane filter. 6. Freeze-dry system with SpeedVac capability. 7. Rotary evaporator. 8. UV spectrometer for PNA and RNA quantification.

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2.4 Isothermal Titration Calorimetry

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1. Isothermal titration microcalorimeter suitable for small biological samples (cell volume 1 mL or smaller and minimum detectable heat 0.1 μJ or smaller), such as a Malvern MicroCal iTC200 instrument. 2. ITC buffer (2 mM MgCl2, 90 mM KCl, 10 mM NaCl, and 50 mM potassium phosphate, pH 7.4). Prepare Solution A: dissolve 203 mg of MgCl2·6H2O (ACS grade), 3.355 g of KCl (ACS grade), 292 mg of NaCl (molecular biology grade), and 4.355 g of K2HPO4 (molecular biology grade) in 500 mL of water (HPLC grade). Prepare Solution B: dissolve 203 mg of MgCl2 (ACS grade), 3.355 g of KCl (ACS grade), 292 mg of NaCl (molecular biology grade), and 3.402 g of KH2PO4 (molecular biology grade) in 500 mL of water (HPLC grade). Adjust the pH of Solution A to 7.4 by slowly adding Solution B.

3

Methods

3.1 Peptide Synthesis 3.2

PNA Synthesis

Follow the synthesis procedure in Liberty Blue User Guide.

1. Place 10 mg of Sieber resin (2 mmol/g) into an empty synthesis column to set up a standard 2 μmol scale PNA synthesis protocol on an Expedite 8909 synthesizer. 2. Dissolve all required PNA monomers in anhydrous N-methyl2-pyrrolidone (NMP) to reach the final concentration of 0.2 M. Use dry Expedite 8909 monomer vials to weigh the following amounts of PNA monomers: Fmoc-PNA-T-OH (203 mg), Fmoc-PNA-M(Boc)-OH (230 mg), Fmoc-PNAP-OH (196 mg), Fmoc-PNA-E-OH (202 mg), FmocAEEA-OH (154 mg), and Fmoc-Lys(Boc)-OH (178 mg). Add 1.9 mL of anhydrous NMP to each vial. Gentle warming may be required to dissolve PNA-P and PNA-E monomers. The total volume in each vial will be 2 mL, which will allow approximately 14 monomer couplings (note that ~0.3 mL will be consumed during priming the Expedite reagent lines). 3. If switching between standard nucleic acid reagents and PNA reagents, follow proper flushing procedures in the Expedite PNA synthesis manual. Load the reagents and monomers onto the Expedite 8909 synthesizer taking necessary precautions to minimize contact with atmosphere. 4. Prime the Expedite 8909 synthesizer with the reagents and monomers, install the synthesis columns (Expedite 8909 allows simultaneous synthesis of two sequences), and program the sequences to be synthesized. It is common to add one lysine

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residue to improve the solubility of PNA and several AEEA spacers to separate a bulky fluorescent dye from a PNA oligomer. 5. Start automated synthesis. 6. Collect the Deblocking solution at each Fmoc deprotection step. Dilute the collected solution to 10 mL with fresh Deblocking solution, and measure the absorbance at 300 nm using a UV spectrophotometer. Compare the absorbance after each deprotection step to determine the efficiency of monomer coupling. 7. After the synthesis is complete, remove the final Fmoc group as described in Expedite PNA synthesis manual. 3.3 Labeling PNA with HiLyte Fluor 488 (HF488) [50]

1. Dry column with completed PNA sequence on a solid support with a stream of nitrogen gas once final Fmoc group is removed (terminal amino group deprotected). 2. Prepare a solution of activated dye: mix 12.5 μL of the Base solution (0.2 M DIPEA and 0.3 M 2,6-lutidine in DMF), 12 μL of 0.19 M HATU in DMF, 7.6 μL of 0.33 M HiLyte Fluor 488 carboxylic acid in DMF, and 18 μL of DMF in a small vial (1–1.5 mL) with silicon septum, and vortex the resulting 50 μL of the mixture for 3 min at RT. Wrap the vial with aluminum foil to protect the dye from light. Make a small hole in one of the filters with 26–30G needle, and inject the reaction mixture in the middle of the column. Attach two 1 mL polyethylene syringes to the opposite sides of the column, and let assembly vortex for 36 h. Wash the column with DMF (5  1 mL) before PNA cleavage from a solid support and final deprotection. Note! HiLyte Fluor 488 is sold as a mixture of 50 - and 0 6 -carboxylic acids where 50 -isomer is a major compound. There are two identical PNA sequences with different HPLC retention times upon attachment of HiLyte Fluor 488 (see Fig. 3).

3.4 PNA Cleavage from Solid Support and Deprotection

1. Remove the synthesis column, and add one plastic syringe containing 0.6 mL of the Cleavage Cocktail to the column carefully adding the cocktail to the column and expelling any air. Place the other syringe on the other end, and move the solution back and forth through the column several times (see Note 1). Leave the synthesis column with the Cleavage Cocktail solution for 2 h, occasionally moving the solution back and forth through the column. 2. Collect the Cleavage Cocktail solution in a plastic 2 mL centrifuge vial. Use another 0.3 mL of the Cleavage Cocktail to treat

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Fig. 3 LC/MS (Shimadzu LCMS-2020) analysis of purified PNA with 50 -isomer (left) and PNA with 60 -isomer (right) with the respective retention times. PNA sequence is HF488 50 (60 )-AEEA-AEEA-K-MTMTMMTMM-TAT (AEEA is spacer, K is lysine, TAT is peptide with the following sequence: RKKRRQRRR). LC conditions: ES Industries Harmony C18 (5 μm, 4 mm  150 mm) column at 55  C, eluting with a linear gradient of 2–25% (v/v) of mobile phase B in mobile phase A over 20 min, with flow rate of 0.7 mL/min

the synthesis column for another 30 min. Collect the second Cleavage Cocktail solution in the same centrifuge vial. 3. Split the combined Cleavage Cocktail solutions equally into five plastic 2 mL centrifuge vials. Add 1.8 mL of anhydrous diethyl ether to each of the vials and mix vigorously. The PNA should precipitate (see Note 2). 4. Centrifuge for 15 min at 14,500 rpm (14,104  g). Carefully, remove the diethyl ether from the PNA precipitate with a pipette. Add another 1 mL of diethyl ether, mix vigorously, centrifuge, and remove the solvent with a pipette. 5. To each vial, add ca. 0.3 mL of HPLC-grade water. Combine all fractions into one vial and evaporate using a SpeedVac. The sample is ready for HPLC purification. 3.5 HPLC Purification of Synthetic PNA

1. Dissolve the PNA sample in 1 mL of HPLC water. Filter the sample through a 13 mm syringe filter. 2. Purify the PNA using a Supelco Discovery BIO Wide Pore C-18 column at 55  C, eluting with a linear gradient of 5–20% (v/v) of mobile phase B in mobile phase A over 40 min, with a flow rate of 1 mL/min. 3. Collect the fractions corresponding to the major peak as detected by UV absorbance monitored at 254 and 280 nm. 4. Using rotary evaporator, reduce the volume of the collected fractions by one-half of the original volume (to remove formic acid), and freeze-dry the sample. 5. Confirm the purity and identity of PNA by LC/MS or MALDI TOF mass spectrometry.

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3.6 Quantification of Synthetic PNA

1. Dissolve the dry PNA sample (from step 5 in Subheading 3.5) in 1 mL of water (HPLC grade). Take 50 μL of the sample and dilute to 1 mL (dilution factor of 20). 2. Measure the UV absorbance of PNA at 260 nm. If the absorbance is greater than 1, dilute the sample further or repeat step 1 and increase the dilution factor. 3. Calculate the molar extinction coefficient (ε(260) M1 cm1) of PNA by summing up the individual extinction coefficients of monomers according to the PNA sequence: 8560 for T, 2212 for M, 5984 for E, 2150 for P, and 26,170 for HiLyte Fluor 488. 4. Determine the amount (n) of nmols of PNA using the following formula: n ¼ A  106  20=ε: where A is the UV absorbance of PNA at 260 nm and ε is the molar extinction coefficient at 260 nm (see Note 3). 5. Freeze-dry the sample and dissolve in HPLC-grade water by adding 1 mL of water for each 240 nmol to make a 0.24 mM stock solution. Store the stock solution frozen at 20  C.

3.7 Preparation of the RNA Target

1. Dissolve the crude RNA sample (see Note 4) in 400 μL of deprotection buffer (as provided by the commercial vendor). Make sure the RNA pellet is completely dissolved by vortexing or vigorous shaking. 2. Incubate at 60  C for 30 min and evaporate the sample in a SpeedVac. 3. Dissolve the RNA sample in 1 mL of HPLC-grade water. Filter the sample through a 13 mm syringe filter. 4. Purify the RNA using a semipreparative reverse-phase HPLC using an XBridge Prep C-18 column at 60  C, eluting with a linear gradient of 5–15% of mobile phase D in mobile phase C over 40 min with a flow rate of 5 mL/min. 5. Collect the fractions corresponding to the major peak as detected by UV absorbance monitored at 254 and 280 nm. 6. Freeze-dry the collected fractions. Dissolve the purified RNA in 2 mL of HPLC-grade water and freeze-dry. Repeat the dissolve/freeze-dry cycle (in order to remove all triethylammonium acetate). 7. Quantify the RNA and prepare a 0.24 mM stock solution using the same procedure as described for PNA above in Subheading 3.6.

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3.8 Isothermal Titration Calorimetry

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1. Place 12.5 μL of RNA stock solution (0.24 mM) in a plastic 2 mL centrifuge vial and evaporate in a SpeedVac (see Note 5). 2. Dissolve the RNA sample in 300 μL of degassed ITC buffer. 3. Place 22.5 μL of the PNA stock solution (0.24 mM) into a plastic 2 mL centrifuge vial and evaporate in SpeedVac. 4. Dissolve the PNA sample in 60 μL of degassed ITC buffer. 5. Load the RNA sample (250 μL) into the sample cell of ITC instrument. Load HPLC-grade water into the reference cell of the ITC instrument. 6. Transfer the PNA sample (60 μL) into 200 μL microcentrifuge tube. Load the syringe with PNA sample (syringe loading is an automated process). 7. Program the experimental parameters in the MicroCal iTC200 software: temperature (25  C), reference power (5 μcal/s), initial delay (60 s), number of injections (16), injection (1.0 μL of PNA for the first injection, 2.45 μL of PNA for others), interval between injections (500 s), stirring speed (750 rpm), filter period (5 s), and feedback mode (high). The intervals between injections can be either increased or decreased depending on the rate of the binding event. 8. Start the ITC experiment and collect the titration data (Fig. 4). 9. Analyze the data using Malvern software MicroCal PEAQ-ITC Analysis. An example of graphical analysis is shown in Fig. 5. (see Note 6).

Fig. 4 ITC titration curve of Lys-NH-MTMTTMMMT-CONH2 triple helical binding to an RNA hairpin (25  C)

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Fig. 5 Analysis of ITC data for Lys-NH-TMMMTTMTM-CONH2 triple helical binding to an RNA hairpin. The results of this experiment were association constant (Ka) ¼ 15.1  107 M1, binding enthalpy (ΔH ) ¼ 306 kJ/mol, and binding stoichiometry (n) ffi 1

10. MicroCal PEAQ-ITC Analysis software gives the experimental association constant (Ka), binding enthalpy (ΔH), binding entropy (ΔS), free energy (ΔG), and binding stoichiometry (n) (Fig. 5) (see Note 6). 3.9 Confocal Fluorescence Microscopy

1. Plate cells (15  103 cells per well) on an 8-well Lab-Tek chambered coverglass (pre-coated with Collagen I) in 10% FBS-DMEM. 2. Culture cells overnight at 37  C, 10% CO2. 3. Discard medium and wash cells with 200 μL of OptiMEM (no FBS). 4. Add 300 μL of 1 μM PNA-HF488 conjugate in OptiMEM (no FBS), and incubate for 12–24 h at 37  C, 10% CO2. 5. Wash cells twice with 300 μL of fresh OptiMEM, and add 300 μL of 50 μg/mL 40 ,6-diamidino-2-phenylindole (DAPI) in OptiMEM; incubate for 1 h at 37  C, 10% CO2. 6. Wash cells twice with 300 μL of fresh OptiMEM, and add another 300 μL of OptiMEM without phenol red for observation of live cells. 7. Detect uptake of HF488-labeled PNA as green fluorescence measured at 488 nm (Fig. 6).

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Fig. 6 MCF-7 cells incubated with 1 μM HF488-PNA-TAT conjugate for 12 h (full sequence: HF488 50 (60 )AEEA-AEEA-K-MTMTMMTMM-RKKRRQRRR). Images (right to left) of DAPI staining cell nuclei in blue, localization of green HF488-PNA-TAT conjugate, merged DAPI and HF488-PNA-TAT, and differential interference contrast (DIC) imaged with DAPI visible (see Note 7)

4

Notes 1. This simple procedure uses one syringe to push the reagent into the column, while the other syringe is used to pull reagents out at the other end. The procedure is described in detail for DNA deprotection in Expedite 8909 user’s manual. Warning! TFA is highly corrosive and toxic; m-Cresol has a strong stench. Perform all operations in a fume hood and wear proper personal protection. 2. Lack of PNA precipitate indicates that the synthesis was not successful. While there may be many reasons why a multistep procedure may fail, the most common problems we have encountered are blocked reagent delivery lines on the Expedite 8909, impure reagents and PNA monomers, and old solvents that have absorbed moisture. 3. A successful 2 μmol scale synthesis may yield a couple of hundred nanomoles of PNA after the HPLC purification. 4. The exact method for RNA preparation may vary depending on the identity, length, source, and purity of the RNA sample. We describe our procedure for preparation of an RNA model target starting from crude synthetic RNA obtained from Dharmacon. 5. If poor signal to noise ratio is a problem, we recommend using double the amount of PNA and RNA. This will generally improve the data and may be necessary in some cases. 6. Blank (linear) model allows baseline subtraction only if the last data points of the titration curve represent PNA dilution heats only, i.e., the receptor is completely saturated, and there is no binding heat due to addition of more ligand. It is not uncommon that in cases of weak binding or complex equilibrium the receptor is not completely saturated at the end of titration curve. In such cases, it is necessary to run a blank run of the ligand (PNA) against the buffer solution. This is done in order

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to subtract out the heat of dilution from the actual binding heat. Perform the blank run as described in Subheading 3.6, except omitting the RNA receptor. 7. When comparing the uptake of the PNA conjugates, keep the imaging conditions such as photomultiplier gain/offset, laser intensities, and confocal aperture size the same.

Acknowledgments This work was supported by NIH grant GM071461 (to E.R.) and NSF grants CHE-1406433 and CHE-1708761 (to E.R.) and CHE-1708699 (to J.A.M.). References 1. Cech TR, Steitz JA (2014) The noncoding RNA revolution-trashing old rules to forge new ones. Cell 157:77–94 2. Lee JT (2012) Epigenetic regulation by long noncoding RNAs. Science 338:1435–1439 3. Yoon J-H, Abdelmohsen K, Gorospe M (2013) Posttranscriptional gene regulation by long noncoding RNA. J Mol Biol 425:3723–3730 4. Gorski SA, Vogel J, Doudna JA (2017) RNA-based recognition and targeting: sowing the seeds of specificity. Nat Rev Mol Cell Biol 18:215–228 5. Consortium IHGS (2004) Finishing the euchromatic sequence of the human genome. Nature 431:931–945 6. Djebali S, Davis CA, Merkel A, Dobin A, Lassmann T, Mortazavi A, Tanzer A, Lagarde J, Lin W, Schlesinger F, Xue C, Marinov GK, Khatun J, Williams BA, Zaleski C, Rozowsky J, Roeder M, Kokocinski F, Abdelhamid RF, Alioto T, Antoshechkin I, Baer MT, Bar NS, Batut P, Bell K, Bell I, Chakrabortty S, Chen X, Chrast J, Curado J, Derrien T, Drenkow J, Dumais E, Dumais J, Duttagupta R, Falconnet E, Fastuca M, FejesToth K, Ferreira P, Foissac S, Fullwood MJ, Gao H, Gonzalez D, Gordon A, Gunawardena H, Howald C, Jha S, Johnson R, Kapranov P, King B, Kingswood C, Luo OJ, Park E, Persaud K, Preall JB, Ribeca P, Risk B, Robyr D, Sammeth M, Schaffer L, See L-H, Shahab A, Skancke J, Suzuki AM, Takahashi H, Tilgner H, Trout D, Walters N, Wang H, Wrobel J, Yu Y, Ruan X, Hayashizaki Y, Harrow J, Gerstein M, Hubbard T, Reymond A, Antonarakis SE, Hannon G, Giddings MC, Ruan Y, Wold B, Carninci P,

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Chapter 10 In Vitro Cellular Delivery of Peptide Nucleic Acid (PNA) Takehiko Shiraishi, Mahdi Ghavami, and Peter E. Nielsen Abstract Cellular delivery methods are a prerequisite for cellular studies with PNA. This chapter describes PNA cellular delivery using cell-penetrating peptide (CPP)-PNA conjugates and transfection of PNA-ligand conjugates mediated by cationic lipids. Furthermore, two endosomolytic procedures employing chloroquine treatment or photochemical internalization (PCI) for significantly improving PNA delivery efficacy are described. Key words Cell-penetrating peptides, CPP, Photochemical internalization (PCI), PNA conjugates, Transfection

1

Introduction Unaided cellular uptake of synthetic antisense oligomers (AOs) is generally negligible. Therefore, cellular studies with AOs, including charge neutral peptide nucleic acid (PNA), require efficient and robust cellular delivery methods [1–3]. Cationic lipid formulations (lipoplexes) are routinely used as effective delivery vehicles for anionic oligonucleotides, whereas lipoplexes are only efficient with non-charged AOs if these are conjugated to a lipophilic or an anionic domain or are hybridized to a carrier oligonucleotide [4, 5]. However, cellular uptake of AOs can also be dramatically improved by chemical conjugation to cell-penetrating peptides (CPPs) [6] without interfering with their (antisense) activity and sequence specificity [7–9]. This chapter will describe two PNA cellular delivery methods by using either CPP-PNA conjugates or PNA-ligand conjugates mediated by cationic lipids. Furthermore, two endosomolytic procedures (chloroquine treatment and photochemical internalization (PCI) treatment) for improvement of AOs’ delivery efficacy are described.

Peter E. Nielsen (ed.), Peptide Nucleic Acids: Methods and Protocols, Methods in Molecular Biology, vol. 2105, https://doi.org/10.1007/978-1-0716-0243-0_10, © Springer Science+Business Media, LLC, part of Springer Nature 2020

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1.1 Cellular Delivery of PNA by Cell-Penetrating Peptide (CPP)

CPPs are short (6–30 amino acid residues) cationic or amphipathic peptides that can be conjugated or complexed with bioactive molecules and transport PNA into mammalian cells by primarily endocytic pathway(s) both in vitro and in vivo [10]. CPPs are promising vectors for a wide range of ligands and drugs because of their remarkable cell-penetrating feature as well as their relatively nontoxic nature (compared to liposome-based method). The cellular uptake efficiency (and uptake mechanism/pathway) is strongly dependent on the specific CPP used as well as the cargo molecule [11–14]. To date, many different CPPs have been tested for various AOs, including PNA, and they have significantly improved cellular uptake of AOs without interfering with their biological activity and sequence specificity [12–15]. Therefore, CPP-mediated delivery, in particular using chemical conjugation, is a promising strategy also for in vivo applications and drug discovery. However, the efficacy of CPP-mediated delivery is generally limited due to the accumulation of CPP conjugates in endosomal compartments without direct access to the cytosol/nucleus as these conjugates use endocytic pathways as main uptake routes [16]. Thus, relatively high concentrations (micromolar) of the conjugates are typically needed to obtain significant biological responses. Nevertheless, it is determined that the delivery efficacy of CPP conjugates can be improved by facilitating their release from the endosomal compartments. Improved endosomal release can be achieved by further chemical modification of CPP conjugates using fusogenic peptides [17, 18] or lipidic ligands such as cholesterol or fatty acids [19, 20] or by co-treatment with endosomolytic agents [21–24]. Routinely, CPP-AO cellular delivery procedures are conducted in serum-free conditions due to the degradation of oligonucleotides by nucleases and the interaction of CPPs with serum proteins that abundantly confines cellular delivery [25, 26]. However, recent outcomes show that CPP-PNA can be efficiently delivered in the presence of serum proteins (Ref: under preparation). This chapter describes both methods used in serum-free and serum-containing conditions for PNA cellular transfection.

1.2 Cellular Delivery of PNA Conjugates by Cationic Lipids

The most common nonviral transfection methods are employment of cationic lipids or cationic polymer-based reagents that are capable of complex formation (lipoplexes or polyplexes, respectively) with negatively charged AOs via electrostatic interactions [3]. These delivery approaches by cationic carriers are quite simple to perform and extremely useful for many in vitro studies although their application in vivo is still limited owing to their low in vivo efficacy, acute immune responses, and toxicity to cells [27]. In order to use lipoplex carriers for charge neutral PNAs, several approaches have been introduced in order to increase the affinity

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of the PNA oligomers for the cationic lipids and thus allow formation of the PNA/lipid complexes required for the transfection. The first approach exploited a PNA complementary oligodeoxynucleotide (DNA) to specifically form a PNA/DNA heteroduplex [4], thereby utilizing the anionic oligonucleotide as a carrier for a complexation with cationic lipids. This method is active even at low nanomolar concentrations and does not require further chemical modification of the PNA. However, very careful optimization of complementary DNA oligomers (both oligomer length and length of complementary region) is necessary for each individual PNA. More recently other approaches have been devised for complexing the PNA with cationic lipids using covalent PNA conjugates. These include use of lipidic ligands, such as cholesterol, cholic acid, or polyheteroaromates [7–9, 28], or negatively charged ligands such as bis-phosphonate lysine peptides, allowing complex formation between PNA and cationic lipids via hydrophobic or electrostatic interactions, respectively. Among these PNA conjugates, bisphophonate-PNAs showed the highest potency exhibiting subnanomolar (EC50) activity in mammalian cells. Nonetheless, other lipidic PNA conjugates are also active at low nanomolar concentrations, and importantly these PNA conjugates are more readily synthetically available. The present chapter describes the use of PNA conjugates for cellular delivery and antisense targeting in mammalian cells in culture including cellular transfection of PNA conjugates by cationic lipids Lipofectamine 2000 using a simple co-incubation protocol. 1.3 Improvement of PNA Cellular Delivery by Endosomolytic Treatment

The poor inherent cellular uptake of PNA can be significantly improved by conjugation to CPPs and can be further enhanced by co-conjugation to lipidic ligands such as fatty acids or cholesterol [19, 20]. However, the efficacy of these PNA conjugates is still limited by significant endosomal entrapment [3, 29, 30]. It has been shown that the bioavailability of CPP-PNA conjugates can be significantly improved by the use of auxiliary agents such as chloroquine, Ca2+ treatment, sucrose, or photosensitizers (photochemical internalization (PCI)) [21–24, 31] that promote release of endosomal contents to the cytosol via different mechanisms dependent on the particular endosomolytic treatments. Two endosomolytic treatments, namely, chloroquine (CQ, Fig. 1) treatment and PCI treatment, are described in the present protocol. CQ shows significant accumulation in acidic cellular compartments, such as endosomes/lysosomes, and presumably induces their disruption through a proton sponge effect resulting in an increase of osmotic pressure in them [32]. It can be easily used by simple supplementation to the transfection medium. PCI treatment consists of treatment with a lipophilic photosensitizer (e.g., AlPcS2a or TPPS2a,

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OH N HN

O

N

Cl

HO

NH

OH Cholate

Chloroquine

SO3SO3-

-O3S N N N

NH

N

SO3-

N

Al N

N

N

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AlPcS2a

TPPS2a

Fig. 1 Structures of chloroquine, cholate (conjugated to amino end of a PNA), and photosensitizers (disulfonated aluminum phthalocyanine (AlPcS2a), disulfonated tetraphenylporphyrin (TPPS2a))

Fig. 1) combined with an irradiation of the cells. Photosensitizer absorbs visible light and generates short-lived, reactive oxygen species (ROS, mainly singlet oxygen in this case) resulting in transient pore formation in the endosomal membrane through perturbation (oxidation) of lipids, thereby facilitating release of endosomal content into the cytosol. As mentioned above, release of the delivered cargo from the endosomal compartment is one of the main challenges for efficient and functional cellular delivery of antisense oligomers and in particular PNA. The methods described here can easily be applied to other AOs such as morpholino oligomers and also other cargoes (e.g., siRNA) to improve intracellular bioavailability by liberating cargoes from endosome/lysosome compartments.

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Materials Cell Culture

1. Growth medium: RPMI1640 supplemented with 10% fetal bovine serum (FBS) and 1% GlutaMax (Gibco). 2. Adherent cells in culture (i.e., HeLa pLuc705 cells). 3. Cell culture flask (T-25). 4. Multiple well plate (e.g., 24-well plate or 96-well plate). 5. Silicon cell scraper.

2.2

PNA Transfection

1. PNA conjugate solution at 200 μM (this solution is preferably in water but also could be in organic solvents such as DMSO, DMF in case of PNA conjugate with low water solubility). PNA conjugates were synthesized, HPLC-purified, characterized (MALDI-TOF mass), and stored at 4
C until use. Synthesis of PNA conjugates is described in Subheading 4. Synthesis of other PNA conjugates (e.g., cholic acid-PNA, cholesterolPNA, bis-phosphonate-PNA, etc.) is described in the literature [7–9, 28]. 2. OPTI-MEM medium (Invitrogen). 3. Cationic lipids (e.g., Lipofectamine 2000 (Invitrogen)). 4. Growth medium: RPMI1640 supplemented with 1% GlutaMAX (Gibco) and FBS (20% for supplementation and 10% for the replacement).

2.3 Improvement of PNA Cellular Delivery Efficacy by Endosomolytic Treatment

1. OPTI-MEM medium with chloroquine (CQ): 100 μM chloroquine (CQ) in OPTI-MEM. 2. Growth medium with a photosensitizer: RPMI1640 (10% FBS, 1% GlutaMAX) containing aluminum phthalocyanine (AlPcS2a, Frontier Scientific) (5 μg/ml) or tetraphenylporphyrin (TPPS2a, Frontier Scientific) (2 μg/ml). 3. Equipment (specially required): light tubes for photosensitizer irradiation, red light irradiation for AlPcS2a (fluorescence tube FO18 (Arcadia)) or blue light irradiation for TPPS2a (fluorescent light tube 40W/03 (Phillips) with maximum emission at 420 nm).

2.4

PNA Synthesis

1. 4-Mehylbenzhydryl 4). The impact of these changes on gene expression should be carefully analyzed. NGS experiments were performed at the Laboratory for Technologies of Advanced Therapies (LTTA) of Ferrara University. Sequencing was performed using Illumina NextSeq500 platform and NextSeq® 500/550 High-Output Kit v2 (75 cycles) (Illumina, FC-404-2005). 10. Alternative targeting for miR-145-5p inhibition. As discussed in Note 6, the treatment of target cells with miRNAs (in the case described in this article Calu-3 targeting with R8-PNA-a145) might lead to effects on many genes, on the basis of the fact that the 30 UTR region of several mRNAs might be targeted by miR-145-5p. In consideration of this possible limit, an alternative strategy to inhibit miR-145-5p is the PNA-based “miRNA masking” [56–58], which is obtained by treating cells with a PNA fully complementary to the CFTR 30 UTR binding site for miR-145-5p. In this case miR-145-5p is expected to be unable to bind to the CFTR mRNA, being on the contrary fully able to interact with other mRNAs carrying miR-145-5p binding sites exhibiting nucleotide sequence variability with respect to the CFTR miR-1455p binding sites.

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Fig. 4 Effects of the R8-PNA-a145 on miRNome profile analyzed by NGS sequencing. Calu-3 cells were cultured in the absence or in the presence of R8-PNA-a145 for 48 h, RNA was extracted and NGS performed (a). In the right part of the panel, a summary of the % of miRNA not modulated or up- and down-modulated is shown (b). Enlisted on the bottom are the down- or upregulated miRNAs with FC>4 (c).

11. Alternative approaches for delivery of PNAs. As reported in several papers, the application of PNAs, including miRNA targeting, is limited by their low uptake by cells [58]. Currently, no simple and efficient delivery systems and methods are available to solve this open issue. One of the most promising approaches is the modification of the PNA structure through the covalent linkage of polyarginine tails. However, alternative methods are available, including those described in a recent report on the delivery ability of a macrocyclic multivalent tetraargininocalix[4]arene used as non-covalent vector for anti-miRNA PNAs. High delivery efficiency, low cytotoxicity, maintenance of the PNA biological activity, and easy preparation of the transfection formulation are key issues exhibited by these delivery vectors [39].

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34. Borgatti M, Breda L, Cortesi R, Nastruzzi C, Romanelli A, Saviano M et al (2002) Cationic liposomes as delivery systems for doublestranded PNA-DNA chimeras exhibiting decoy activity against NF-kappaB transcription factors. Biochem Pharmacol 64:609–616 35. Abes R, Arzumanov A, Moulton H, Abes S, Ivanova G (2008) Arginine-rich cell penetrating peptides: design, structure-activity, and applications to alter pre-mRNA splicing by steric-block oligonucleotides. J Pept Sci 14:455–460 36. Torres AG, Threlfall RN, Gait MJ (2011) Potent and sustained cellular inhibition of miR-122 by lysine-derivatized peptide nucleic acids (PNA) and phosphorothioate locked nucleic acid (LNA)/20 -O-methyl (OMe) mixmer anti-miRs in the absence of transfection agents. Artif DNA PNA XNA 2(3):71–78 37. Bertucci A, Prasetyanto EA, Septiadi D, Manicardi A, Brognara E, Gambari R, Corradini R, De Cola L (2015) Combined delivery of temozolomide and anti-miR221 PNA using mesoporous silica nanoparticles induces apoptosis in resistant glioma cells. Small 11:5687–5695 38. Chen J, Tang Y, Liu Y, Dou Y (2018) Nucleic acid-based therapeutics for pulmonary diseases. AAPS PharmSciTech 19:3670–3680 39. Gasparello J, Manicardi A, Casnati A, Corradini R, Gambari R, Finotti A, Sansone F (2019) Efficient cell penetration and delivery of peptide nucleic acids by an argininocalix[4] arene. Sci Rep, vol 9, p 3036 40. Brognara E, Fabbri E, Bazzoli E, Montagner G, Ghimenton C, Eccher A et al (2014) Uptake by human glioma cell lines and biological effects of a peptide-nucleic acids targeting miR-221. J Neurooncol 118:19–28 41. Brognara E, Fabbri E, Bianchi N, Finotti A, Corradini R, Gambari R (2014) Molecular methods for validation of the biological activity of peptide nucleic acids targeting microRNAs. Methods Mol Biol 1095:165–176 42. Cheng CJ, Bahal R, Babar IA, Pincus Z, Barrera F, Liu C et al (2015) MicroRNA silencing for cancer therapy targeted to the tumour microenvironment. Nature 518:107–110 43. Brognara E, Fabbri E, Montagner G, Gasparello J, Manicardi A, Corradini R et al (2016) High levels of apoptosis are induced in human glioma cell lines by co-administration of peptide nucleic acids targeting miR-221 and miR-222. Int J Oncol 48:1029–1038 44. Ghidini A, Bergquist H, Murtola M, Punga T, Zain R, Stro¨mberg R (2016) Clamping of

PNAs Inhibiting MicroRNA Activity RNA with PNA enables targeting of microRNA. Org Biomol Chem 14:5210–5213 45. Beavers KR, Werfel TA, Shen T, Kavanaugh TE, Kilchrist KV, Mares JW et al (2016) Porous silicon and polymer nanocomposites for delivery of peptide nucleic acids as anti-microRNA therapies. Adv Mater 28:7984–7992 46. Gupta A, Quijano E, Liu Y, Bahal R, Scanlon SE, Song E et al (2017) Anti-tumor activity of miniPEG-γ-modified PNAs to inhibit microRNA-210 for cancer therapy. Mol Ther Nucleic Acids 9:111–119 47. Quijano E, Bahal R, Ricciardi A, Saltzman WM, Glazer PM (2017) Therapeutic peptide nucleic acids: principles, limitations, and opportunities. Yale J Biol Med 90:583–598 48. Manicardi A, Gambari R, de Cola L, Corradini R (2018) Preparation of anti-miR PNAs for drug development and nanomedicine. Methods Mol Biol 1811:49–63 49. Fabbri E, Tamanini A, Jakova T, Gasparello J, Manicardi A, Corradini R et al (2017) A peptide nucleic acid against microRNA miR-1455p enhances the expression of the cystic fibrosis transmembrane conductance regulator (CFTR) in Calu-3 cells. Molecules 23(1). pii: E71 50. Manicardi A, Fabbri E, Tedeschi T, Sforza S, Bianchi N, Brognara E et al (2012) Cellular uptakes, biostabilities and anti-miR-210 activities of chiral arginine-PNAs in leukaemic K562 cells. Chembiochem 13:1327–1337 51. Fabbri E, Manicardi A, Tedeschi T, Sforza S, Bianchi N, Brognara E et al (2011) Modulation of the biological activity of microRNA210 with peptide nucleic acids (PNAs). ChemMedChem 6:2192–2202

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Chapter 13 Detection of Microorganisms by Fluorescence In Situ Hybridization Using Peptide Nucleic Acid Ricardo Oliveira, Carina Almeida, and Nuno F. Azevedo Abstract Fluorescence in situ hybridization (FISH) is a 30-year-old technology that has evolved continuously and is now one of the most well-established molecular biology techniques. Traditionally, DNA probes are used for in situ hybridization. However, synthetic molecules are emerging as very promising alternatives, providing better hybridization performance and making FISH procedures easier and more efficient. In this chapter, we describe a universal FISH protocol, using nucleic acid probes, for the detection of bacteria. This protocol should be easily applied to different microorganisms as a way of identifying in situ relevant microorganisms (including pathogens) and their distribution patterns in different types of samples. Key words Microorganisms, PNA, FISH, Pure cultures, Enriched samples, Histological samples, Biofilms

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Introduction Initially applied in the medical and developmental biology domains, fluorescence in situ hybridization (FISH) is a 30-year-old technology that has continuously evolved. Nowadays, it has applications in a wide range of areas from the detection of pathogens in clinical samples [1], to the identification of novel biomarkers for cancer progression [2], the characterization of communities’ structure and diversity of natural habitats [3], the determination of gene presence and expression [4], and even the study of chromosomal stability in stem cell research [5]. FISH for microorganisms was first described in 1989 by DeLong et al., but it was during the early 1990s that it gained increasing importance as a novel molecular technique for detection, identification, and/or quantification of microorganisms based on hybridization of fluorescence-labelled probes with complementary target sequences of nucleic acids [6, 7]. This chapter is focused on the procedures applied to microbial cells. Although protocols for FISH might differ significantly, the traditional procedure typically involves four steps: (1) a fixation

Peter E. Nielsen (ed.), Peptide Nucleic Acids: Methods and Protocols, Methods in Molecular Biology, vol. 2105, https://doi.org/10.1007/978-1-0716-0243-0_13, © Springer Science+Business Media, LLC, part of Springer Nature 2020

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Fig. 1 Basic steps of FISH. In the first step, the sample is fixed to stabilize the cells and permeabilize the cell membranes. In step 2, the labelled oligonucleotide probe is added to the cells and allowed to hybridize to its target, normally the ribosomal RNA. Then, the excess of probe is washed away. And finally, the sample is ready for single-cell identification either by epifluorescence microscopy or flow cytometry. Depending on the purpose of the analysis, an initial enrichment step may be necessary to ensure greater sensitivity by FISH. (Adapted from [11])

and permeabilization step of the sample, (2) the probe hybridization with the target sequence, (3) the washing of unbound and excess probes, and, finally, (4) the observation of cells (Fig. 1). Depending on the type of sample, additional steps may be required to ensure higher sensitivity by FISH. Typically, a sample homogenization step and a pre-enrichment step are applied to increase the pathogen load of the contaminated samples. The pre-enrichment, in particular, is essential to guarantee that the pathogen load reaches the detection limit of the FISH technique (
105 CFU/ mL for unfiltered samples) [8]. In addition to increasing the number of microorganisms to detectable levels, it also increases the ribosomal content—as the microorganism will enter into a more metabolically active state, which is directly related to the fluorescence intensity. Thus this step might also allow the recovery of stressed or damaged cells [9]. Another advantage of the enrichment step is the dilution of interfering matrix components which can make the visualization of the samples by fluorescence microscopy or flow cytometry a difficult task (e.g., in food samples) [10]. The use of pre-enrichment steps is something quite common in detection procedures for food or blood sample analysis to increase the pathogen load and is usually less useful for studies that aim at studying the diversity and ecology of microbial population (as, in these cases, populations should not be disturbed).

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The fixation step uses fixative agents and permeabilizing agents, such as paraformaldehyde and ethanol, to stabilize cell morphology, disable proteolytic enzymes, and stabilize samples to withstand further processing. Also, it protects against microbial contamination and decomposition [12, 13]. Then, specific binding occurs between the oligonucleotide probes and the target sequence in the hybridization step. This step must be performed under stringent conditions to ensure the specific binding of the probe. For that, a number of parameters, such as the concentration of salts and denaturants (i.e., formamide, urea), hybridization time, and temperature, must be well defined/optimized previously [14]. In the end, the washing step uses a mixture of detergents to ensure that all unbound probes are removed from the sample and will not interfere with the visualization of the samples. The observation of the cells is the last step and can be performed by fluorescence microscopy or flow cytometry, depending on the objective of the technician (qualitative or quantitative analysis). These equipment are often connected to a computer that allows the analysis of the images/data obtained [10]. Traditionally, the probes target a 16S/23S rRNA sequence in members of the Bacteria/Archaea domain or a 18S/28S rRNA sequence in the Eukarya domain. The rRNA sequences are preferential targets due to their abundance in cells and due to the presence of regions of both high and low sequence variability, making it possible either to distinguish small groups of cells from a specific species or to perform coarser discrimination of cells belonging to different kingdoms [15]. The main advantage of FISH over other molecular techniques is that by targeting abundant structures in living cells, this technique allows the visualization of cells with a stable ribosomal content, which is associated with an active metabolic state, thus allowing the differentiation of viable but non-culturable cells. The major challenge continues to be the critical influence of the matrices in the procedure [16], but the FISH procedures have been shown to be less influenced by this component than PCR-based methods, as no enzymes (that can be inhibited) are involved in hybridization process. The properties of the probes are crucial for the success of the hybridization and, consequently, for the FISH procedure. Standard DNA and RNA probes are used since the beginning for in situ hybridization. However, it is possible to point out several limitations that affect the robustness of the procedures using this type of probes [9]. The solution has been introduced with synthetic molecules which have a higher affinity toward the target than the natural nucleic acids. Within this group of molecules, peptide nucleic acids (PNAs) are the ones that have gained more attraction. PNAs were published only 2 years after the emergence of the FISH technique [17]. However, only in the late 1990s, these types of probes were introduced in FISH studies for the detection of microorganisms

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Fig. 2 Comparative analysis of the use of PNA probes and DNA probes in FISH methodologies. The lack of electrostatic repulsion and the nonpolar characteristic, due to the uncharged backbone, and the synthetic nature of PNA probes are perhaps the main reasons for its advantages over traditional DNA probes

[18]. Since then, several studies have demonstrated the applicability of PNA probes in the detection and analysis of microorganisms in a broad spectrum of samples, such as biofilms [19–23], biopsy specimens [1], blood and urine samples [24, 25], food samples [26–30], water, and even the detection of antimicrobial resistance [31]. The most relevant difference between DNA and PNA probes is the “backbone” of the molecules (Fig. 2). The non-charged polyamide backbone of PNA molecules makes them less susceptible to repulsive forces and salt concentrations, contributing to a higher thermal stability between PNA and target duplex when compared to DNA and target duplex. This enables PNA probes to bind stronger to nucleic acids than DNA probes [32, 33]. The non-charged nature allows even its hybridization with nucleic acids in low salt concentrations and high temperatures, an important feature when the target sequence is part of complex structures like those found in rRNA [34]. Besides that, the efficacy and the signal intensity of FISH are often hindered by the choice of binding site on the target. DNA-FISH methods must target accessible binding sites, since these probes have limitations on displacing complex structures. PNA probes have proved that accessibility is not really an issue. This has been related with the fact that when using PNA, salts can be reduced to a minimum to destabilize the secondary structure of the rRNA [35]. Furthermore, as synthetic molecules, the PNAs also show greater resistance to the action of the nucleases/proteases, which increases the stability of the molecule during storage and the FISH procedure [36, 37]. The nonpolar classification of PNA probes has

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been regarded as another advantage in FISH procedures since it might increase the penetration ability, resulting in an enhanced diffusion into the cell through cell membranes/walls and/or even into other naturally occurring microstructures such as the biofilm matrix [38]. This chapter aims to allow any investigator to apply the PNA FISH technique in the detection of specific microorganisms in different types of samples (e.g., food samples, histological samples, or biofilms), providing the essential steps for an accurate procedure from the design of the specific probes to the visualization of the sample.

2 2.1

Materials Probe Design

1. Probe design software: the ARB Project (available from http:// www.arb-home.de/) or Primrose (described in https://www. ncbi.nlm.nih.gov/pmc/articles/PMC137075/). 2. rRNA sequence databases: ARB Silva (available from http:// www.arb-silva.de/), Ribosomal Database Project II (RDP-II) (available from https://rdp.cme.msu.edu/), or National Center for Biotechnology Information (NCBI, available from http://www.ncbi.nlm.nih.gov/BLAST/) databases for probe selection. 3. Analytical tools: ClustalW program (available from the European Institute of Bioinformatics, https://www.ebi.ac. uk/Tools/msa/clustalw2/); probeCheck (available from http://131.130.66.200/cgi-bin/probecheck/ probecheck.pl), Probe Match (available from https://rdp.cme. msu.edu/probematch/search.jsp), TestProbe (available from https://www.arb-silva.de/search/testprobe/), or BLAST (available from http://blast.ncbi.nlm.nih.gov/Blast.cgi).

2.2

FISH Procedure

1. PNA probe, HPLC purified and attached to a fluorochrome at the N-terminal (a linker might be included between the probe and the fluorochrome). 2. Coated microscope slides with wells for FISH. 3. Coverslips with a 0.15 mm thickness. 4. Incubator or hybridization chamber. 5. Coplin jar for slides. 6. Milli-Q or deionized water. 7. Phosphate-buffered saline (PBS, 137 mM NaCl; 2.7 mM KCl; 10 mM Na2HPO4∙2H2O; and 1.8 mM KH2PO4). 8. 4% (vol/vol) paraformaldehyde solution. Store at 2–8  C (see Note 1).

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9. 50% (vol/vol) ethanol solution. 10. Hybridization solution: 10% (wt/vol) dextran sulfate, 10 mM NaCl, 30% (vol/vol) formamide, 0.1% (wt/vol) sodium pyrophosphate, 0.2% (wt/vol) polyvinylpyrrolidone, 0.2% (wt/vol) Ficoll, 5 mM disodium EDTA, 0.1% (vol/vol) Triton X-100, 50 mM Tris–HCl, pH 7.5, and 200 nM PNA probe. Store at 2–8  C (see Note 2). 11. Washing solution: 5 mM Tris base, 15 mM NaCl, and 1% (vol/vol) Triton X, pH 10. Store at 2–8  C. 12. Nonfluorescent immersion oil or mounting media. 13. Epifluorescence microscope equipped with a 60 or 100 oil objective, a 100 W mercury lamp (or a LED light source), and at least one filter sensitive to the respective fluorochrome molecule attached to the PNA probe.

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Methods Perform all procedures at room temperature and under sterile conditions unless otherwise specified. In each experiment, two types of negative controls should be included: a nontarget control (with a nontarget microorganism) following the same procedure as any sample including the probe-specific hybridization solution to exclude false positives and a target control that receives the hybridization solution without probe, to assess the specificity.

3.1

Probe Design

Typically, the probes used in the FISH studies target a 16S rRNA sequence in members of the Bacteria or Archaea domain due to the large amount of curated sequences present in the 16S subunit (SSU) databases. However, with the emergence of highthroughput sequencing techniques, databases for large subunit sequences [LSU—23S] started to grow in size and quality, becoming quite common in research studies. The availability of highquality databases, such as ARB Silva, RDP-II, or NCBI, has led to the development of highly accurate probes. Several sequences of probes described for microbial detection are available in a database named probeBase (http://www.micro bial-ecology.net/probebase/), so an initial search for probe sequences should be performed here before deciding on the design of a new one. For microorganisms without described probes or for original studies, some specific software, such as the ARB Project or Primrose, coupled with the abovementioned rRNA databases, may be used to design suitable probes. When the use of these softwares is not possible, other general tools, such as ClustalW, might be used to align sequences and identify conserved regions. This is a timeconsuming, nonoptimal approach, as this is not a specific tool for searching potential target regions/probes.

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The design of probes typically involves (1) identification of potential sequences in the rRNA gene sequences from a set of targets and nontargets available in the databases mentioned above; (2) identifying a conserved region of the target sequences through alignment of sequences using software for this purpose; (3) design of the probe(s) for the selected sequences; (4) analysis of the characteristics and thermodynamic parameters of the possible probes; and (5) synthesis of the selected probe(s). In the process, it will be useful to determine some thermodynamic parameters as well as the theoretical sensitivity and specificity of the designed probes using the tools/software mentioned above. For this, you must follow the following criteria: 1. To identify potentially useful oligonucleotides for use as probes, different 16S/23S rRNA sequences from the microorganism(s) of interest as well as other phylogenetically similar microorganisms available in the rRNA databases should be selected. For example, for the design of a Salmonella probe, ten Salmonella sequences, including representative strains of each of the seven subspecies, and seven other strains of related species belonging to the family Enterobacteriaceae, could be used to start looking for conserved regions among Salmonella. After that, conserved regions in the target microorganisms, which are not present in the nontarget microorganisms, should be selected from the alignment data obtained by the software being used. Of notice, when using the ClustalW program, the conserved regions selected are in fact the target, so the reverse complementary should be used as probe. 2. The criteria for the selection of the most suitable PNA probe should include Gibbs free energy typically between 13 kcal/ mol and 20 kcal/mol, lack of self-complementary structures, and probe sequences of approximately 15 bases with Tm values in the range of 60–80  C (see Note 3). 3. After that, perform an in silico study using one of the tools mentioned above (probeCheck, TestProbe, or Probe Match) to evaluate the specificity and sensitivity of the probes. Compare the probe sequences against sequences of target microorganism and nontarget microorganisms using an available rRNA database mentioned above. Select only high-quality sequences with 1200 bp (for 16S rRNA sequences). The selected probe should differ by at least two mismatches from nontarget species. Using the results of the analysis, a simple way to estimate these parameters is as follows: l

Specificity ¼ (nTs/TnS) ∗ 100, where nTs represents the number of nontarget strains that did not react with the probe and TnS is the total of nontarget strains examined.

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Sensitivity ¼ (Ts/TTs) ∗ 100, where Ts represents the number of target strains detected by the probe and TTs are the total number of target strains present in the databases.

4. If your study is for an ex vivo study (e.g., histological samples), analyze the specificity of the probe against the organism of tissue origin (e.g., mouse, human). This study can be performed using BLAST. 3.2 Fluorescence In Situ Hybridization (FISH) Procedure

3.2.1 Preparation of Samples Pure Cultures or Enriched Samples

FISH can be carried out in cells adhered to surfaces (or to histological tissues) or in suspension. Routinely, the procedure on surfaces is the technique used in analytical laboratories [11]. However, the suspension procedure can be used to avoid autofluorescence in complex matrix samples [8] or when the purpose is to analyze the sample by flow cytometry [11, 39]. However, there are also pre-treatments that can be applied during the FISH procedure on surfaces which help to reduce the autofluorescence conferred by certain compounds of complex samples [27, 40]. The procedure on surfaces requires a preparation step to promote the adhesion of the cells to the surface, whereas for the procedure in suspension, the procedure begins in the fixation and permeabilization of the sample. Regarding the analysis of histological samples, the sample preparation involves deparaffinization and rehydration of the tissues. Nevertheless, each researcher must do an experimental planning taking into account the nature of the sample that is to be analyzed. 1. For the procedure on a surface, place one drop of PBS in a well on a slide (e.g., coated glass slide), and gently emulsify a small amount of biomass using a loop. For enriched/liquid samples, put 20 μL of cellular suspension directly in the slide. 2. Allow the smears to air-dry, or fix the sample by heat using standard procedures, and proceed to the fixation and permeabilization in the solid support.

Histological Samples

Histological slides of formalin-fixed, paraffin-embedded biopsy specimens should be deparaffinized and rehydrated: 1. Firstly, immerse the histological slides twice into xylol for 15 min. 2. Immerse the slides first into absolute ethanol for 5 min and then in decreasing concentrations of ethanol (95%, 80%, 70%, and 50%) for 5 min each. 3. Wash the histological slides with distilled water for 10 min, allow them to air-dry, and proceed to the fixation and permeabilization in the solid support (see Note 4).

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1. Wash the sample with PBS to remove non-adhered cells. If samples are intended to be analyzed in the biofilm growth support, then move to the fixation and permeabilization step (see Note 5). 2. If cells are to be removed from the growth support, sonicate the samples in PBS using specific protocols adapted to the samples/species under study (see Note 6). 3. Then proceed to the fixation and permeabilization in suspension, or place a 20 μL sample in a slide; allow to air-dry, or fix by heat using standard procedures, and proceed to the fixation and permeabilization in the solid support.

3.2.2 Fixation and Permeabilization Procedure for Cells Adhered to Surfaces (or to Histological Tissues)

Procedure in Suspension

1. Cover the sample with 4% (vol/vol) paraformaldehyde solution, and allow to incubate for 10 min. After incubation, remove excess paraformaldehyde with a paper towel. 2. Add 50% (vol/vol) ethanol, leave for 10 min and then remove the excess with a paper towel. Allow to air-dry for a few minutes. 1. Collect 1 mL of the sample for an Eppendorf, and centrifuge at 10,000  g for 10 min at 4  C. 2. Discard the supernatant, resuspend the pellet in 200 mL of 4% (vol/vol) paraformaldehyde, and allow to incubate for 1 h. Depending on the pellet, the volume of solution might be adapted. 3. Centrifuge at 10,000  g for 5 min at 4  C, discard the supernatant, and resuspend the pellet with 200 μL of 50% (vol/vol) ethanol. Allow to incubate for at least 30 min at 20  C (see Note 7).

3.2.3 Hybridization Procedure for Cells Adhered to Surfaces (or to Histological Tissues)

1. After sample fixation and permeabilization, place the slides (or other solid support) in moist and opaque container (see Note 8). 2. Apply 20–40 μL of hybridization solution containing your PNA probe (200 nM), cover with coverslip, and incubate at the appropriate hybridization temperature for the required time (see Note 9). 3. At the same time, place in the incubator (together with the opaque container) a Coplin jar filled with washing solution to heat. Alternative containers to the Coplin jars might be used for the wash solution, depending on the type of solid supports being used. 4. Remove the slides from the chamber, remove the coverslip, and immerse them in the preheated wash solution. Leave in the incubator for 30 min.

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5. Remove the samples from the Coplin jar, and allow to air-dry in a dark place or protected from light. After that the samples are ready to be analyzed under the microscope (see Note 10). Procedure in Suspension

1. After fixing and permeabilizing the samples as described above, centrifuge the samples at 10,000  g for 5 min, discard the ethanol, and wash the cells with 500 μL of PBS buffer. 2. Centrifuge at 10,000  g for 5 min, discard the PBS, and resuspend the pellet with 100 μL of the hybridization solution with the respective PNA probe (200 nM). 3. Protect the Eppendorf tubes from light with aluminum foil, and incubate for the required time at the optimized hybridization temperature for the probe (see Note 9). 4. Centrifuge sample again (10,000  g, 5 min) and discard the hybridization solution. Add 500 μL of wash solution, mix well, and incubate for 15 min at the same temperature used for hybridization. Repeat the wash one more time. 5. After two washing steps, centrifuge (10,000  g, 5 min), discard the wash solution, and resuspend the pellet in sterile deionized water. This suspension can be used directly for cytometry analysis, or, when this type of equipment is not available, a quantitative analysis can be done by filtering a known volume of the suspension as described in the next steps. 6. Filter a known volume of the suspension on a cellulose nitrate membrane (or other membrane suitable for fluorescence use), with 0.2 μM pore size. If the sample volume to be filtered is small, the sample might be previously diluted in a higher volume to assure a homogeneous distribution in the membrane. 7. Place the membrane on a slide, and the samples are ready to be analyzed under the microscope (see Note 10).

3.3 Analysis of the Samples by Epifluorescence Microscopy

1. Sample visualization is performed in the same way regardless of the type of sample and whether the sample was prepared in a solid support or suspension. 2. Add a drop of immersion oil or mounting medium, cover with a coverslip, and add another drop or immersion oil on top of the slide. Observe the samples in a fluorescence microscope using an appropriate filter for the fluorochrome coupled to the probe. 3. Evaluate samples using all the other filters to exclude artifacts in the images. 4. Acquire images with equal exposure times between samples, for fluorescence intensity comparisons. 5. Analyze the acquired images for fluorescence intensity or cell count, using an appropriate image analysis software.

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Notes 1. The dissolution of paraformaldehyde is usually difficult to perform and toxic and therefore should be prepared in a chemical hood. To improve its dissolution, water (approximately half of the intended solution volume) can be heated at 50–60  C, followed by the addition of paraformaldehyde and some drops of NaOH until the opaque white solution becomes clear. Afterward, add PBS 3 concentration (1/3 of the intended solution volume—for a 100 mL final solution, add 33.3 mL of 3 PBS), complete the final volume with distilled water, and adjust the pH to 7.2. 2. Formamide presents a possible risk of harm to unborn child and is irritating to the eyes, respiratory system, and skin. Therefore, wear suitable protective clothing and gloves. In addition, the handling of solutions with fluorescence probes should avoid light in order to avoid degradation. 3. Gibbs free energy (G) is a thermodynamic parameter that can be used as indicator of whether a reaction is thermodynamically favorable or not. In general, when ΔG is negative (ΔG < 0 kcal/mol), the hybridization reaction will be favorable or spontaneous, and the more negative the value the more favorable the reaction. However, for PNA probes, researchers tend to use probes with a ΔG between 13 kcal/mol and 20 kcal/mol. Actually a ΔG higher than 12 kcal/mol has been associated with noneffective probes, while values bellow 20 kcal/mol might compromise the specificity [41]. 4. In certain cases, the washing of the histological slides with distilled water may be replaced by immersion in a 1% (vol/vol) Triton X-100 solution for 20 min at 70  C in order to increase the permeability and consequently the efficiency of the process. 5. Some biofilm samples might be analyzed ex situ if the materials used in the formation of these biological structures, such as PVC and silicone, presented a strong autofluorescence signal. Also, some metal surfaces, such as steel and copper, might interfere and compromise the hybridization [23]. 6. The sonication step will need to be optimized for the type of sample and microorganism being analyzed, as too strong sonication conditions might kill the cells while too weak condition might not be effective on recovering all the cells from the surface. For some bacteria these conditions have already been optimized in previous works and should be used as a starting point.

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7. After this step, the samples can be stored for long periods (up to 6 months) under these conditions for subsequent continuation of the FISH procedure. 8. Simple opaque container can be prepared with petri dishes previously wrapped in aluminum foil and with moist absorbent paper inside. 9. The hybridization time and temperature require a prior optimization to ensure the best performance and will depend on the efficiency of the probe. Because there are no thermodynamic models specifically developed to predict the hybridization temperature of a probe used in a FISH procedure, the melting temperature is used as a reference. The hybridization temperature is quite often 10–15  C bellow the Tm. 10. Samples may be stored at 4  C in the dark for a maximum of 24 h before microscopy visualization.

Acknowledgements This work was financially supported by: the project UID/EQU/ 00511/2019—Laboratory for Process Engineering, Environment, Biotechnology and Energy—LEPABE funded by national funds through FCT/MCTES (PIDDAC); the project POCI-01-0145FEDER-030431, the project PTDC/DTP-PIC/4562/2014— POCI-01-0145-FEDER-016678, the project POCI-01-0145FEDER-029961, and the project POCI-01-0145-FEDER031011 funded by FEDER funds through COMPETE2020—Programa Operacional Competitividade e Internacionalizac¸˜ao (POCI) and by national funds (PIDDAC) through FCT/MCTES; and the project “LEPABE-2-ECO-INNOVATION”—NORTE-01-0145FEDER-000005, funded by Norte Portugal Regional Operational Programme (NORTE 2020), under PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). The authors also thank FCT for the PhD Fellowship SFRH/BD/138883/2018. References 1. Guimara˜es N, Azevedo NF, Figueiredo C et al (2007) Development and application of a novel peptide nucleic acid probe for the specific detection of Helicobacter pylori in gastric biopsy specimens. J Clin Microbiol 45:3089–3094. https://doi.org/10.1128/ JCM.00858-07 2. Bayani J, Squire JA (2007) Application and interpretation of FISH in biomarker studies. Cancer Lett 249:97–109. https://doi.org/ 10.1016/j.canlet.2006.12.030

3. Rogers SW, Moorman TB, Ong SK (2007) Fluorescent in situ hybridization and microautoradiography applied to ecophysiology in soil. Soil Sci Soc Am J 71:620. https://doi. org/10.2136/sssaj2006.0105 4. Dmochowski IJ, Tang X (2007) Taking control of gene expression with light-activated oligonucleotides. Biotechniques 43:161, 163, 165 passim

PNA Probes for the Detection of Microorganisms by FISH 5. Catalina P, Cobo F, Corte´s JL et al (2007) Conventional and molecular cytogenetic diagnostic methods in stem cell research: a concise review. Cell Biol Int 31:861–869. https://doi. org/10.1016/j.cellbi.2007.03.012 6. DeLong EF, Wickham GS, Pace NR (1989) Phylogenetic stains: ribosomal RNA-based probes for the identification of single cells. Science 243:1360–1363 7. Levsky JM, Singer RH (2003) Fluorescence in situ hybridization: past, present and future. J Cell Sci 116:2833–2838. https://doi.org/10. 1242/jcs.00633 8. Almeida C, Azevedo NF, Iversen C et al (2009) Development and application of a novel peptide nucleic acid probe for the specific detection of Cronobacter genomospecies (enterobacter sakazakii) in powdered infant formula. Appl Environ Microbiol 75:2925–2930. https://doi.org/10.1128/ AEM.02470-08 9. Cerqueira L, Azevedo NF, Almeida C et al (2008) DNA mimics for the rapid identification of microorganisms by fluorescence in situ hybridization (FISH). Int J Mol Sci 9:1944–1960. https://doi.org/10.3390/ ijms9101944 10. Rohde A, Hammerl JA, Appel B et al (2015) FISHing for bacteria in food – a promising tool for the reliable detection of pathogenic bacteria? Food Microbiol 46:395–407. https://doi. org/10.1016/j.fm.2014.09.002 11. Amann R, Fuchs BM (2008) Single-cell identification in microbial communities by improved fluorescence in situ hybridization techniques. Nat Rev Microbiol 6(5):339–348 12. Felix H (1982) Permeabilized cells. Anal Biochem 120:211–234 13. Thavarajah R, Mudimbaimannar VK, Elizabeth J et al (2012) Chemical and physical basics of routine formaldehyde fixation. J Oral Maxillofac Pathol 16:400–405. https://doi.org/10. 4103/0973-029X.102496 14. Azevedo N (2005) Survival of Helicobacter pylori in drinking water and associated biofilms. Dissertation for PhD degree in Chemical and Biological Engineering. University of Minho 15. Amann R, Ku¨hl M (1998) In situ methods for assessment of microorganisms and their activities. Curr Opin Microbiol 1:352–358. https://doi.org/10.1016/S1369-5274(98) 80041-6 16. Jasson V, Jacxsens L, Luning P et al (2010) Alternative microbial methods: an overview and selection criteria. Food Microbiol

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27:710–730. https://doi.org/10.1016/j.fm. 2010.04.008 17. Nielsen PE, Egholm M, Berg RH, Buchardt O (1991) Sequence-selective recognition of DNA by strand displacement with a thyminesubstituted polyamide. Science 254:1497–1500 18. Stender H, Mollerup TA, Lund K et al (1999) Direct detection and identification of Mycobacterium tuberculosis in smear-positive sputum samples by fluorescence in situ hybridization (FISH) using peptide nucleic acid (PNA) probes. Int J Tuberc Lung Dis 3:830–837 19. Azevedo NF, Vieira MJ, Keevil CW (2003) Establishment of a continuous model system to study Helicobacter pylori survival in potable water biofilms. Water Sci Technol 47:155–160 20. Juhna T, Birzniece D, Larsson S et al (2007) Detection of Escherichia coli in biofilms from pipe samples and coupons in drinking water distribution networks. Appl Environ Microbiol 73:7456–7464. https://doi.org/10.1128/ AEM.00845-07 21. Braganc¸a SM, Azevedo NF, Simo¨es LC et al (2007) Use of fluorescent in situ hybridisation for the visualisation of Helicobacter pylori in real drinking water biofilms. Water Sci Technol 55:387–393 22. Gia˜o MS, Wilks SA, Azevedo NF et al (2009) Comparison between standard culture and peptide nucleic acid 16S rRNA hybridization quantification to study the influence of physico-chemical parameters on Legionella pneumophila survival in drinking water biofilms. Biofouling 25:335–343. https://doi. org/10.1080/08927010902802232 23. Almeida C, Azevedo NF, Santos S et al (2011) Discriminating multi-species populations in biofilms with peptide nucleic acid fluorescence in situ hybridization (PNA FISH). PLoS One 6:e14786. https://doi.org/10.1371/journal. pone.0014786 24. Azevedo NF, Jardim T, Almeida C et al (2011) Application of flow cytometry for the identification of Staphylococcus epidermidis by peptide nucleic acid fluorescence in situ hybridization (PNA FISH) in blood samples. Antonie van Leeuwenhoek 100:463–470. https://doi.org/10.1007/s10482-011-95959 25. Almeida C, Azevedo NF, Bento JC et al (2013) Rapid detection of urinary tract infections caused by Proteus spp. using PNA-FISH. Eur J Clin Microbiol Infect Dis 32:781–786. https://doi.org/10.1007/s10096-012-18082

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26. Almeida C, Azevedo NF, Fernandes RM et al (2010) Fluorescence in situ hybridization method using a peptide nucleic acid probe for identification of salmonella spp. in a broad spectrum of samples. Appl Environ Microbiol 76:4476–4485. https://doi.org/10.1128/ AEM.01678-09 27. Almeida C, Sousa JM, Rocha R et al (2013) Detection of Escherichia coli O157 by peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) and comparison to a standard culture method. Appl Environ Microbiol 79:6293–6300. https://doi.org/10.1128/ AEM.01009-13 28. Machado A, Almeida C, Carvalho A et al (2013) Fluorescence in situ hybridization method using a peptide nucleic acid probe for identification of Lactobacillus spp. in milk samples. Int J Food Microbiol 162:64–70. https:// doi.org/10.1016/j.ijfoodmicro.2012.09.024 29. Almeida C, Cerqueira L, Azevedo NF, Vieira MJ (2013) Detection of Salmonella enterica serovar Enteritidis using real time PCR, immunocapture assay, PNA FISH and standard culture methods in different types of food samples. Int J Food Microbiol 161:16–22. https://doi.org/10.1016/j.ijfoodmicro. 2012.11.014 30. Rocha R, Almeida C, Azevedo NF (2018) Influence of the fixation/permeabilization step on peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) for the detection of bacteria. PLoS One 13:e0196522. https://doi.org/10.1371/journal.pone. 0196522 31. Cerqueira L, Fernandes RM, Ferreira RM et al (2011) PNA-FISH as a new diagnostic method for the determination of clarithromycin resistance of Helicobacter pylori. BMC Microbiol 11:101. https://doi.org/10.1186/14712180-11-101 32. Nielsen PE (2001) Peptide nucleic acid: a versatile tool in genetic diagnostics and molecular biology. Curr Opin Biotechnol 12:16–20

33. Perry-O’Keefe H, Rigby S, Oliveira K et al (2001) Identification of indicator microorganisms using a standardized PNA FISH method. J Microbiol Methods 47:281–292 34. Orum H, Nielsen PE, Jørgensen M et al (1995) Sequence-specific purification of nucleic acids by PNA-controlled hybrid selection. Biotechniques 19:472–480 35. Mendes L, Rocha R, Azevedo AS et al (2016) Novel strategy to detect and locate periodontal pathogens: the PNA-FISH technique. Microbiol Res 192:185–191. https://doi.org/10. 1016/j.micres.2016.07.002 36. Stender H, Fiandaca M, Hyldig-Nielsen JJ, Coull J (2002) PNA for rapid microbiology. J Microbiol Methods 48:1–17. https://doi.org/ 10.1016/S0167-7012(01)00340-2 37. Wagner M, Horn M, Daims H (2003) Fluorescence in situ hybridisation for the identification and characterisation of prokaryotes. Curr Opin Microbiol 6:302–309. https://doi.org/10. 1016/S1369-5274(03)00054-7 38. Drobniewski FA, More PG, Harris GS (2000) Differentiation of Mycobacterium tuberculosis complex and nontuberculous mycobacterial liquid cultures by using peptide nucleic acidfluorescence in situ hybridization probes. J Clin Microbiol 38:444–447 39. Amann RI, Krumholz L, Stahl DA (1990) Fluorescent-oligonucleotide probing of whole cells for determinative, phylogenetic, and environmental studies in microbiology. J Bacteriol 172:762–770 40. Stevens KA, Jaykus LA (2004) Bacterial separation and concentration from complex sample matrices: a review. Crit Rev Microbiol 30:7–24. https://doi.org/10.1080/ 10408410490266410 41. Yilmaz LS, Noguera DR (2004) Mechanistic approach to the problem of hybridization efficiency in fluorescent in situ hybridization. Appl Environ Microbiol 70:7126–7139. https:// doi.org/10.1128/AEM.70.12.7126-7139. 2004

Chapter 14 PNA Antisense Targeting in Bacteria: Determination of Antibacterial Activity (MIC) of PNA-Peptide Conjugates Lise Goltermann and Peter E. Nielsen Abstract Antisense PNA-peptide conjugates targeting essential bacterial genes have shown interesting potential for discovery of novel precision antibiotics. In this context, the minimal inhibitory concentration (MIC) assay is used to assess and compare the antimicrobial activity of natural as well as synthetic antimicrobial compounds. Here, we describe the determination of the minimal inhibitory concentration of peptide-PNA conjugates against Escherichia coli. This method can be expanded to include minimal bactericidal concentration (MBC) determination and kill-curve kinetics. Key words Antimicrobials, Minimal inhibitory concentration (MIC), Minimal bactericidal concentration (MBC), Antisense, Translational inhibition

1

Introduction The armory of effective antibiotics against the ever-growing group of pathogenic, multidrug-resistant bacteria is being depleted; new classes of antibiotics, which do not share these resistance mechanisms, are urgently needed. Surveillance studies of the rising population of multidrugresistant Gram-negative pathogens report an increasing spread of antibiotic resistance mechanisms such as antibiotic degrading enzymes, target mutations, increased efflux, or reduced influx through membrane modifications [1, 2]. Especially the worldwide emergence of carbapenem- and colistin-resistant strains is alarming as these have been considered the last line of treatment. As recently stated by the WHO, finding new treatment options against carbapenem-resistant Acinetobacter, Pseudomonas, and Enterobacteriaceae species is now considered of critical importance [3]. Gram-negative bacteria are characterized by an asymmetrical outer membrane consisting of a bilayer of phospholipids, which are decorated with lipopolysaccharides (LPS) facing the extracellular milieu. The LPS provides the bacterial cell with a negatively

Peter E. Nielsen (ed.), Peptide Nucleic Acids: Methods and Protocols, Methods in Molecular Biology, vol. 2105, https://doi.org/10.1007/978-1-0716-0243-0_14, © Springer Science+Business Media, LLC, part of Springer Nature 2020

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charged, inaccessible outer surface significantly restricting the passage of large or hydrophobic compounds. In addition, many Gramnegative species harbor efflux pumps, which can be upregulated in response to antimicrobial treatment. Antisense antimicrobials are particularly intriguing, as they offer a different mechanism of bacterial eradication, and can be rationally designed to target specific organisms through essential or resistance genes. Peptide nucleic acid (PNA)-based antisense antimicrobials constitute a promising new class of synthetic precision antibiotics [4]. The mRNA of essential species-specific bacterial genes can be targeted with PNA, via an antisense mechanism employing translation inhibition, thereby killing preferentially the pathogen and sparing the natural bacterial flora. Other chemistries such as phosphorodiamidate morpholino (PMO) [5], locked nucleic acid (LNA) [6], phosphorothioates [7], and 20 -O-methyl oligonucleotide (20 -OMe) [8] have also been studied for gene downregulation in bacteria. Since the PMOs share chemical properties as well as the mechanism of the antisense effect with the PNAs, many of the considerations on design presented below can be extended to the PMOs. In order to achieve a strong antimicrobial antisense effect, the PNA must bind tightly and specifically to its mRNA target. By probing the position of a selected mRNA for targeting with PNA, it was established that the location around the ribosome binding site and proximally upstream results in the highest antisense activity [9]. Although antisense oligomers may exert their gene-silencing effect via different mechanisms, the evidence suggests that PNAs (and PMOs) work primarily by inhibiting translation initiation and may to a lower degree (as a consequence of translation inhibition) promote mRNA degradation. Optimization of the target sequence, position, and length is key to achieving high antisense activity. It is important that carefully designed mismatch controls are always included to ensure that any effect on bacterial survival is caused by direct antisense action, and not by, e.g., a general toxicity mechanism. The mismatch PNA should have the same nucleobase content and should deviate as little as possible from the match construct. This is most conveniently achieved by switching two nonadjacent bases within the sequence, thereby lowering Tm, and thus targeting affinity sufficiently to avoid target binding. The sequence of both the match and mismatch construct should be checked against the genome of the target organism to reduce possible off-targets. The acyl carrier protein (acpP) gene has successfully been used as antisense target, while other genes such as ftsZ, murA, fabI [10], gyrA [11], and rpoA [12] have also been reported as effective antibacterial targets.

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In general, the PNA oligomer requires conjugation to a carrier, typically a cationic (non-antimicrobial) peptide, for transport across the bacterial envelope [13]. Most notably, the (KFF)3K peptide has been successfully used as carrier-peptide in a wide range of bacteria, but other carrierpeptides including a range of natural (low activity) cationic antimicrobial peptides as well as vitamin B12 have also been explored (Table 1). Effective carriers should show low (no) toxicity to bacteria and host cells and display low hemolytic activity. PNA alone and some carrier-peptide-PNA conjugates require the inner membrane transporter SbmA for translocation over the inner membrane. This in turn means that the outer membrane and LPS layer becomes the primary barrier for bacterial peptide-PNA uptake. For SbmA-independent conjugates, it seems that the inner membrane becomes the major barrier for uptake since perturbation Table 1 Overview of peptides, which have successfully been used as carrier-peptides for PNA antisense antibiotics into a variety of bacterial species Peptide/carrier

Organism

Reference

(KFF)3K-

Escherichia coli S. aureus ESBL-Klebsiella pneumoniae Mycobacterium smegmatis Streptococcus pyogenes Acinetobacter baumanii

[14]/[15] [16] [17] [18] [11] [19]

(KFF)3K-Ahx-

Pseudomonas aeruginosa

[20]

(R-Ahx)6-(β-Ala)-

E. coli Pseudomonas aeruginosa

[21] [20]

(R-Ahx-R)4-Ahx-(β-Ala)-

(ESBL/MDR)-E. coli Pseudomonas aeruginosa MDR-Salmonella enterica Shigella flexneri ESBL-Klebsiella pneumoniae Listeria monocytogenes

[21, 22] [20] [22] [22] [22] [12]

(RFF)3R-

Haemophilus influenzae

[23]

GRKKRRQRRRYK-(Tat)

Streptococcus pyogenes S. aureus S. epidermidis Listeria monocytogenes

[11] [24] [24] [12]

GKPRPYSPRPTSHPRPIRV- (Drosocin)

Escherichia coli

[25]

RAGLQFPVGRVHRLLRK- (Buforin)

Escherichia coli

[25]

Vitamin B12

Escherichia coli

[26]

Ahx: 6-aminohexanoic acid, β-Ala: β-alanine

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of the LPS layer has only minor effects on the efficacy of these conjugates. The above-mentioned restrictions to peptide-PNA uptake have also led to the discovery that 10–12-mer long PNAs are most efficient as antisense antimicrobial agents; surprisingly this effect seems little dependent of the carrier-peptide [21]. Antimicrobial susceptibility of bacteria toward antibiotics across strains and species is conventionally compared based on the minimal inhibitory concentration. International guidelines [27] determine bacterial inoculum, incubation time, setup, and read out in order to classify bacterial strains as either sensitive, tolerant, or resistant to the tested antibiotics. However, peptide nucleic acid (PNA) and the cationic peptides used as carriers, as well as antimicrobial peptides in general, are prone to adhere to polymer surfaces (in particular polystyrene), thereby reducing the actual concentration in solution. In addition, divalent cations compete with the cationic peptides for binding to the negatively charged bacterial surface. For those reasons, a modified MIC protocol has been established. This includes using low-bind plastics and non-cation-adjusted Mueller-Hinton Broth. There are options for additional modifications, and these are included as optional in the described protocol.

2 2.1

Materials PNA Preparation

1. NanoDrop spectrophotometer or similar instrumentation. 2. LoBind 1.5 ml tubes (SafeSeal Microcentrifuge Tubes, Cat. No. 11720, Sorenson, Bioscience, Inc.). 3. LoBind pipette tips (Axygen, Corning, Maxymum Recovery pipette tips, Cat. No. TR-222-C-L-R). 4. Milli-Q water.

2.2

Bacterial Culture

1. LB agar. 2. Non-cation-adjusted Mueller-Hinton (Sigma, Cat. No. 70192).

Broth

(nca-MHB)

3. Sterile growth tubes. 2.3 Minimal Inhibitory Concentration Measurement

1. Sterile 25 ml reagent reservoirs. 2. Sterile 96-well low-bind microtiter plate (Thermo Scientific, Nunc Cat. No. 260896, 96F straight w/lid). 3. Multichannel pipette (P200). 4. Optional: microtiter plate incubator/reader. 5. Optional: acetic acid. 6. Optional: BSA. 7. Optional: LB agar plate for MBC.

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3

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Methods

3.1 Peptide-PNA Conjugate Stock Preparation

1. Dissolve the peptide-PNA in Milli-Q water (use 50 μl pr 1 mg) (see Note 1). 2. Prepare one 10 and one 50 peptide-PNA dilution in MilliQ water. 3. Measure OD (260 nm) on 1 μl of each peptide-PNA dilution in a NanoDrop spectrophotometer. 4. Calculate the concentration using the specific extinction coefficient for the peptide-PNA conjugate (see Note 2). 5. Prepare peptide-PNA stocks as 20 stocks in a twofold dilution range calculating 10 μl stock for each MIC assay. 6. Optional: peptide-PNA stocks can also be prepared in a solution of 0.02% acetic acid and 0.4% BSA to reduce unspecific binding to the plastics (see Note 3).

3.2

Bacterial Growth

1. Streak the bacterial strain of choice onto LB agar and incubate over night at 37  C. 2. Pick a single bacterial colony and inoculate in 3–5 ml noncation-adjusted Mueller-Hinton Broth. Grow the culture with aeration at 230 rpm at 37  C for 18–20 h.

3.3 MIC Assay (Fig. 1)

1. Dilute the o.n. culture to an OD 595 nm of 0.5 in non-cationadjusted MHB (see Note 4). 2. Dilute this bacterial suspension 2500-fold (see Note 5).

Fig. 1 Left: Example of a MIC setup for one strain in duplicates with nine different peptide-PNA concentrations and a growth control. Each column represents a twofold dilution of the test conjugate. The MIC value can be read as the peptide-PNA concentration in well B/C5. Right: Example of growth curves obtained from the MIC experiment. The wells around the MIC value are plotted, and the MIC value is confirmed to be B/C5 by the growth curves

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3. Pour the final bacterial solution now containing 5  105 CFU/ ml into a solution basin, and, using a multichannel pipette, dispense 190 μl solution into a 96-well microtiter tray (see Note 6). 4. Pipette 10 μl of each 20 peptide-PNA stock into the wells. 5. Fill the wells surrounding the samples with 200 μl medium without bacteria to serve as sterility control. 6. Incubate the plate 18 h at 37  C with shaking (see Note 7). 7. The next day, determine the MIC value as the lowest concentration of peptide-PNA, which inhibits visible growth. 8. (Optional) To determine the minimal bactericidal concentration (MBC), use a pin replicator or multichannel pipette to transfer a droplet from each well onto LB agar directly after ending the MIC assay. Incubate the plate at 37  C for 18–20 h, and determine the lowest concentration of peptide-PNA conjugate that results in no regrowth on the LB agar. 3.4

Kill Curves

1. Set up a plate exactly as the MIC plate, but include only the PNA concentration corresponding to the MIC value for the match construct and the same concentration for the mismatch construct. 2. Before addition of the PNA, take out 10 μl bacterial solution (5  105 CFU/ml), make a dilution series of tenfold dilutions (in either 0.9% NaCl or nca-MHB) down to 1000-fold, and spot 10 μl of each dilution on an agar plate. Let the plate dry and incubate over night at 37  C. 3. Start the MIC assay, and take out 10 μl samples every hour for 4 h for plating as described in step 2 (see Note 8). 4. The next day, enumerate the CFU on each plate, and plot as a function of time.

4

Notes 1. Peptide-PNA can be very electrostatic; it is important to be careful when transferring the dry peptide-PNA between tubes. 2. Extinction coefficient ε260 nm for the PNA monomers: T ¼ 8.6  103; G ¼ 11.7  103; A ¼ 13.7  103; C ¼ 6.6  103 (L  mol1  cm1). Our prototype anti-acpP-PNA (CTC ATA CTC T) has an ε260 of (4  8.6 + 0  11.7 + 2  13.7 + 4  6.6)  103 L  mol1  cm1 ¼ 88.2  103 L  mol1  cm1. If the peptide contains functional groups with absorbance at 260 nm, the extinction coefficient of these groups must be added to that of the PNA.

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3. There is a modified MIC protocol optimized for peptides described by R.E.W. Hancock (http://cmdr.ubc.ca/bobh/ method/modified-mic-method-for-cationic-antimicrobial-pepti des/). This protocol uses the addition of BSA and acetic acid to reduce unspecific binding of the compounds to the plastics. The reader is referred to http://cmdr.ubc.ca/bobh/method/ modified-mic-method-for-cationic-antimicrobial-peptides/ for more information on this. 4. For E. coli MG1655, an OD 595 nm ¼ 1 corresponds to approximately 1.25  109 CFU/ml. When using a new bacterial strain, it is essential to check the correlation between OD and CFU/ml and adjust the dilutions correspondingly. This ensures that the MIC assay is always started with 5  105 CFU/ml. The first few times the MIC is performed, plate out a volume of the final bacterial solution used for the MIC to confirm that the concentration of bacteria is correct. 5. Suggested dilutions: three times 10 dilution using 500 μl bacteria into 4.5 ml non-cation-adjusted Mueller-Hinton Broth. Combine the last 5 ml with 7.5 ml nca-MHB for a total of 12.5 ml. 6. Depending on the incubator, some evaporation can occur. It is therefore advised not to use the outer rows and columns of the 96-well plate. Fill instead these with non-cation-adjusted Mueller-Hinton Broth. This reduces evaporation from the samples and also serves as a sterility control. 7. It is preferred to use a microtiter tray reader to monitor OD during the 18 h. This allows visualization of the growth curves. Alternatively plates can be incubated with linear shaking for 18 h in a normal incubator. We use a Genius Sunrise or BioTek ELx808, which measures OD 595 nm every 20 min with linear shaking between each measurement. 8. We typically see rapid bacterial killing with the peptide-PNA conjugates, reaching the lower detection limit within 2–4 h. The kill curve can be extended beyond 4 h if necessary or the sample intervals reduced to 30 min. References 1. Sidjabat H, Nimmo GR, Walsh TR et al (2011) Carbapenem resistance in Klebsiella pneumoniae due to the New Delhi metallo–βlactamase. Clin Infect Dis 52:481–484. https://doi.org/ 10.1093/cid/ciq178 2. Bonomo RA (2011) New Delhi metallo-β-lactamase and multidrug resistance: a global SOS? Clin Infect Dis 52:485–487. https://doi.org/10.1093/cid/ciq179

3. Tacconelli E, Carrara E, Savoldi A et al (2018) Discovery, research, and development of new antibiotics: the WHO priority list of antibioticresistant bacteria and tuberculosis. Lancet Infect Dis 18:318–327. https://doi.org/10. 1016/S1473-3099(17)30753-3 4. Good L, Nielsen PE (1998) Antisense inhibition of gene expression in bacteria by PNA targeted to mRNA. Nat Biotechnol

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16:355–358. https://doi.org/10.1038/ nbt0498-355 5. Geller BL, Deere JD, Stein DA et al (2003) Inhibition of gene expression in Escherichia coli by antisense phosphorodiamidate morpholino oligomers. Antimicrob Agents Chemother 47:3233–3239. https://doi.org/10.1128/ aac.47.10.3233-3239.2003 6. Wahlestedt C, Salmi P, Good L et al (2000) Potent and nontoxic antisense oligonucleotides containing locked nucleic acids. Proc Natl Acad Sci U S A 97:5633–5638. https:// doi.org/10.1073/pnas.97.10.5633 7. Harth G, Zamecnik PC, Tang JY et al (2000) Treatment of Mycobacterium tuberculosis with antisense oligonucleotides to glutamine synthetase mRNA inhibits glutamine synthetase activity, formation of the poly-L-glutamate/ glutamine cell wall structure, and bacterial replication. Proc Natl Acad Sci U S A 97:418–423. https://doi.org/10.1073/pnas.97.1.418 8. Hegarty J, Krzeminski J, Sharma A et al (2016) Bolaamphiphile-based nanocomplex delivery of phosphorothioate gapmer antisense oligonucleotides as a treatment for Clostridium difficile. Int J Nanomedicine 11:3607–3619 9. Dryselius R, Aswasti SK, Rajarao GK et al (2003) The translation start codon region is sensitive to antisense PNA inhibition in Escherichia coli. Oligonucleotides 13:427–433. https://doi.org/10.1089/ 154545703322860753 10. Goh S, Boberek JM, Nakashima N et al (2009) Concurrent growth rate and transcript analyses reveal essential gene stringency in Escherichia coli. PLoS One 4:e6061. https://doi.org/10. 1371/journal.pone.0006061 11. Patenge N, Pappesch R, Krawack F et al (2013) Inhibition of growth and gene expression by PNA-peptide conjugates in Streptococcus pyogenes. Mol Ther Nucleic Acids 2:e132. https://doi.org/10.1038/mtna.2013.62 12. Abushahba MFN, Mohammad H, Thangamani S et al (2016) Impact of different cell penetrating peptides on the efficacy of antisense therapeutics for targeting intracellular pathogens. Sci Rep 6:20832. https://doi. org/10.1038/srep20832 13. Eriksson M, Nielsen PE, Good L (2002) Cell permeabilization and uptake of antisense peptide-peptide nucleic acid (PNA) into Escherichia coli. J Biol Chem 277:7144–7147. https://doi.org/10.1074/ jbc.M106624200 14. Good L, Awasthi SK, Dryselius R et al (2001) Bactericidal antisense effects of peptide-PNA

conjugates. Nat Biotechnol 19:360–364. https://doi.org/10.1038/86753 15. Vaara M, Porro M (1996) Group of peptides that act synergistically with hydrophobic antibiotics against gram-negative enteric bacteria. Antimicrob Agents Chemother 40:1801–1805 16. Nekhotiaeva N, Awasthi SK, Nielsen PE, Good L (2004) Inhibition of Staphylococcus aureus gene expression and growth using antisense peptide nucleic acids. Mol Ther 10:652–659. https://doi.org/10.1016/j.ymthe.2004.07. 006 17. Kurupati P, Tan KSW, Kumarasinghe G, Poh CL (2007) Inhibition of gene expression and growth by antisense peptide nucleic acids in a multiresistant beta-lactamase-producing Klebsiella pneumoniae strain. Antimicrob Agents Chemother 51:805–811. https://doi.org/10. 1128/AAC.00709-06 18. Kulyte´ A, Nekhotiaeva N, Awasthi SK, Good L (2005) Inhibition of Mycobacterium smegmatis gene expression and growth using antisense peptide nucleic acids. J Mol Microbiol Biotechnol 9:101–109. https://doi.org/10.1159/ 000088840 19. Martı´nez-Guitia´n M, Va´zquez-Ucha JC, ´ lvarez-Fraga L et al (2020) Antisense inhibiA tion of lpxB gene expression in Acinetobacter baumannii by peptide–PNA conjugates and synergy with colistin. J Antimicrob Chemother 75(1):51–59. https://doi.org/10.1093/jac/ dkz409 20. Ghosal A, Nielsen PE (2012) Potent antibacterial antisense peptide-peptide nucleic acid conjugates against Pseudomonas aeruginosa. Nucleic Acids Ther 22:323–334. https://doi. org/10.1089/nat.2012.0370 21. Goltermann L, Yavari N, Zhang M et al (2019) PNA length restriction of antibacterial activity of peptide-PNA conjugates in Escherichia coli through effects of the inner membrane. Front Microbiol 10:427. https://doi.org/10.3389/ fmicb.2019.01032 22. Bai H, You Y, Yan H et al (2012) Antisense inhibition of gene expression and growth in gram-negative bacteria by cell-penetrating peptide conjugates of peptide nucleic acids targeted to rpoD gene. Biomaterials 33:659–667. https://doi.org/10.1016/j. biomaterials.2011.09.075 23. Otsuka T, Brauer AL, Kirkham C et al (2017) Antimicrobial activity of antisense peptidepeptide nucleic acid conjugates against non-typeable Haemophilus influenzae in planktonic and biofilm forms. J Antimicrob Chemother 72:137–144. https://doi.org/10. 1093/jac/dkw384

PNA Antisense Targeting in Bacteria 24. Abushahba MF, Mohammad H, Seleem MN (2016) Targeting multidrug-resistant Staphylococci with an anti-rpoA peptide nucleic acid conjugated to the HIV-1 TAT cell penetrating peptide. Mol Ther Nucleic Acids 5:e339. https://doi.org/10.1038/mtna.2016.53 25. Hansen AM, Bonke G, Larsen CJ et al (2016) Antibacterial peptide nucleic acid-antimicrobial peptide (PNA-AMP) conjugates: antisense targeting of fatty acid biosynthesis. Bioconjug Chem 27:863–867. https://doi.org/10. 1021/acs.bioconjchem.6b00013

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26. Ro´wnicki M, Wojciechowska M, Wierzba AJ et al (2017) Vitamin B12 as a carrier of peptide nucleic acid (PNA) into bacterial cells. Sci Rep 7:7644. https://doi.org/10.1038/s41598017-08032-8 27. Cockerill FR,Wikler MA, Alder J et al Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard—ninth edition. Accessed 8 Jan 2019

Chapter 15 In Vivo Administration of Splice Switching PNAs Using the mdx Mouse as a Model System Camilla Brolin, Ernest Wee Kiat Lim, and Peter E. Nielsen Abstract Duchenne muscular dystrophy (DMD) is the most common and severe form of muscular dystrophy and is caused by gene mutations that abolish production of functional dystrophin muscle protein. A promising new treatment exploits specifically targeted RNA-acting drugs that are able to partially restore the dystrophin protein. The mdx mouse model (animal model of DMD) serves as a good in vivo model for testing these antisense drugs. The simplest in vivo test, which circumvents the systemic circulation, is intramuscular administration of the compound. After 7 days it is possible to detect exon skipping by reverse transcriptase PCR, and newly synthesized dystrophin-positive fibers by immunohistochemistry and western blotting. All muscles, including the heart, are affected by the disease and must be treated. Therefore the use of antisense therapy for treatment of DMD requires systemic administration, and the model is also useful for systemic administration. Key words PNA, mdx, Exon skipping, Administration

1

Introduction More than 10 years ago, it was demonstrated that PNA could be used to modulate dystrophin pre-mRNA splicing in mdx mice, the animal model of Duchenne muscular dystrophy (DMD), by splicing out the exon harboring the nonsense mutation, and produce a shortened and partially functional dystrophin protein [1–3]. Clinically, this would turn a severe DMD patient into a much less affected patient with Becker muscular dystrophy (BMD). The mdx mouse is the most widely used animal model for DMD research but differs from DMD patients in many ways. Generally the mouse model displays a relatively mild phenotype compared to humans. Degeneration is a continuum in patients but appears as waves in the mdx mouse, which results in slow loss of muscle tissue and general muscle weakness in the mouse model, when compared to patients [4]. Nevertheless, the mdx mouse serves as a very good in vivo model system to test novel PNA and PNA conjugates for

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proof of concept. Efficacy, acute toxicity, and functional testing can be studied in the same mouse after systemic administration, the latter being beyond the scope of this chapter, but we refer TREATNMD for standard protocols [5]. PNA-mediated exon skipping can be detected at the mRNA level by reverse transcriptase PCR (RT-PCR), and newly synthesized dystrophin protein correctly located at the sarcolemma can be detected by immunohistochemistry staining and by Western blotting. The DMD gene is the largest known gene with 79 exons spanning 4 Mb, and it takes 16 h to generate a single complete transcript [6]. The fully spliced dystrophin is 14 kb long [7]. The exon skipping activity can be detected 2 days posttreatment, and dystrophin-positive fibers are detectable after 7 days [8] and peak at 8 weeks after a single treatment dose [2]. PNAs are like many antisense agents characterized by suboptimal pharmacokinetics (t½ for unmodified PNA is < 30 min), low cellular uptake, and accumulation in the liver and kidney [9–11]. By local intramuscular (i.m.) administration, it is possible to circumvent the systemic circulation and thereby screen activity of novel compounds in vivo with a simple procedure and without using high amounts of compound. Tibialis anterior (TA) is often used as it is easily accessible, easy to inject, and can readily be removed intact. When doing intramuscular injection, the volume injected and needle size are of importance, since it can physically distend the muscle causing swelling at the injection site associated with fiber damage and pain. In the present protocol, we inject the smallest volume possible with a 300 U insulin needle, 10 μL in TA. One of the disadvantages of intramuscular injections is uneven distribution of the compound in the muscle [8]. When promising PNA conjugate candidates are identified after intramuscular administration, the natural next step would be systemic administration. The use of antisense therapy for treatment of DMD requires systemic administration because all muscles, including the heart, are affected by the disease and must be treated. After systemic administration of the PNA conjugates, we observe the animals for clinical signs of acute toxicity. We have adapted and modified the scoring scheme for acute toxicity observations from Carina Vingsbo Lundberg (Statens Serum Institut, Denmark). In general for substances that are being administrated invasively to animals, it is recommended to have a pH of 4.5–8 (substances outside of the recommended pH range may cause tissue necrosis and vascular thrombosis) [12], room temperature, and normal osmolarity, and it should not give rise to local irritancy (see Note 1). Saline and isotonic glucose can be used as vehicle if pH adjustment of the injection solution is done prior to administration, but citrate buffer is also an option (see Note 2). In our experience, mice that are treated with PNA and various

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Fig. 1 In vivo activity of PNA administered intramuscularly. (a–d) Immunohistochemistry staining for dystrophin-positive fibers in TA muscles from mdx mice after IM administration of PNA dose of 10 μg/TA in mdx mice (scale bar ¼ 100 μm). (a) TA treated with D-lys4-PNA. (b) 25-mer PNA. (c) TA from negative control (injected with vehicle). (d) TA from C57 WT control. (e) RT-PCR to detect skipped dystrophin transcripts in TA muscles of treated mdx mice (n ¼ 2). An increase in the number of dystrophin-positive fibers can be detected (a, b) in mdx mice after i.m. administration of anti-dystrophin PNA compared to vehicle treated (c) and WT (d). Exon skipping was verified by RT-PCR (e). Consistent with the immunostaining results, detectable and comparable exon skipping levels were observed in mice treated with D-lys4-PNA and 25-mer PNA and as expected not in vehicle treated and WT muscles

PNA-peptide conjugates using doses of up to 25–50 mg/kg and taking the above precautions are in most cases returning to score 0 after 1–2 h, even if they may initially show that they are affected to score 1 or 2. The antisense activity is conveniently measured at the protein level by immunohistochemical staining of dystrophin and at the mRNA level by RT-PCR analysis of extracted RNA (Fig. 1).

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Materials 1. Mdx mice (C57BL/10ScSn-Dmdmdx/J, The Jackson Laboratory) and WT mice (C57BL/10ScSn) (see Note 3). 2. Anti-dystrophin PNA: H-GGCCAAACCTCGGCTTACCTNH2 (match), H-GGCCAACCCTCGGATTACCT-NH2 (mismatch). 3. Isotonic glucose (5%). 4. Citrate buffer pH (0.12 M citric acid, 0.24 M solution Na2HPO4, pH 7.2). 5. Insulin needles 0.5 mL (0.33 mm (29 G)  12.7 mm, 300 μL, BD Micro-Fine™ +). 6. Tissue-Tek® O.T.C.™ (Tissue-Tek). 7. SuperFrost Ultra Plus® microscope slides (Thermo Scientific). 8. Safe-lock tubes (2 mL). 9. Phosphate-buffered saline (PBS). 10. Fetal calf serum (FCS) (Sigma F9665). 11. Primary antibodies: rabbit anti-dystrophin (ab15277, Abcam) and rat anti-laminin alpha 2 (clone 4H8-2, Sigma-Aldrich). 12. Secondary antibodies: Alexa Fluor 594 anti-rabbit IgG (H+L) (molecular probes) and Alexa Fluor 488 goat anti-rat IgG (H +L) (molecular probes). 13. Maxima Reverse Transcriptase (Thermo Scientific). 14. RT primer: 50 -AGTCTGTAATTCATCTGGAG-30 . 15. 10 mM dNTP mix (Thermo Scientific). 16. PCR strip tubes. 17. Thermocycler. 18. DreamTaq DNA polymerase (Thermo Scientific). 19. Forward primer: 50 -ATCCAGCAGTCAGAAAGCAAA-30 . 20. Reverse primer: 50 -CAGCCATCCATTTCTGTAAGG-30 . 21. Agarose powder. 22. 1 TBE buffer (0.09 M Tris base, 0.09 M boric acid, 1 mM EDTA (pH 8)).

3

Methods

3.1 Preparation of Injection Solution

1. Calculate the administration dosages based on the weight of the animals recorded the day before the experiment to prepare the solution required in 200 μL injection solution.

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2. Dissolve the lyophilized PNA in Milli-Q water to obtain the stock solution. Aim for a high stock concentration of approximately 75 mg/mL based on the dry weight of the PNA. 3. Prepare injection solution by adding the required volume of the PNA stock solution to give the desired amount for the dose to the required injection volume of isotonic glucose or citrate (see Note 4). Prepare injection solution fresh, just before administration. 4. If using PNA in isotonic glucose, adjust pH of the injection solution using 0.5 or 1 M NaOH. Perform stepwise addition of 1 μL NaOH, vortex the solution, and test a droplet on a pH-indication strip. Repeat until the pH of the solution reaches pH 4 (if much higher, the PNA may precipitate). 3.2 I.m. Administration in Tibialis Anterior (TA)

1. Prepare syringes with 10 μL injection solution in an insulin syringe. 2. Anesthetize mouse/mice in a chamber via the inhalation of 2.5% isoflurane (Baxter, Deerfield, IL) in 1.5 L/min of oxygen. 3. Transfer the mouse to an inhalation mask with 2.5% isoflurane in 1.5 L/min of oxygen, and place the mouse on its back. 4. Remove the fur above from the ankle to the knee with a shaver/clipper to expose the TA muscle. 5. Hold the feet of the mouse, and stretch it (plantar flexion); place the needle parallel to the TA tendon, and move the needle slowly up in the muscle. When the needle is approximate in the middle, inject the 10 μL PNA solution.

3.3 I.v. Administration

1. Prepare syringes with 200 μL injection solution. 2. Place mouse/mice in a heat box for a short while to expose the tail veins. 3. Transfer the mouse to a restrainer and administer the PNA via a slow tail vein (i.v. injection). 4. When removing the cannula, compress the tail to avoid backflow of the solution and to prevent bleeding. 5. Observe mouse closely.

3.4 S.c. or i.p. Administration

1. Prepare syringes with 200 μL injection solution. 2. Restrain the mouse by hand and perform an s.c. or i.p. injection slowly. 3. Observe mouse closely.

3.5 Acute Toxicity Observation

We have adapted and modified the following scoring scheme for acute toxicity observations from Carina Vingsbo Lundberg (Statens Serum Institut, Denmark). We know from previously acute

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toxicity studies that if mice are affected after administration of PNA or PNA conjugates, they are, in most cases, back to score 0 after 1 h (see Note 5). 1. Remove nesting materials and cardboard house in the cage. 2. Observe the mice at the following time points after PNA administration: 0–2 min, 5 min, 15 min, 30 min, 45 min, 60 min, 120 min, and 24 h. Check for breathing abnormalities at the first two time points (rarely observed with PNA and PNA conjugates when dosing up to 25 mg/kg). 3. After 60 min put nesting materials and cardboard house back in the cage. 4. Check mice every hour until they are back to score 0. Score: Observations:

3.6 Removal of Tissue 7 Days or Longer After Treatment

0

Healthy

1

The mouse is a little affected (slightly slower movements, light piloerection)

2

The mouse is affected (changed activity, sitting still, but moves, when the cage lid is removed, possibly piloerection)

3

The mouse is clearly affected (moving only when it is pushed, halfclosed eyes, piloerection, hunchback, tucked up belly). The mouse is sacrificed if the score is 3 for more than 30 min

4

The mouse is very affected (moves very reluctantly although it is pushed, piloerection, eyes closed, cool) and is sacrificed immediately to avoid suffering

5

The mouse is motionless and cold (lying on the side) and is sacrificed immediately to avoid suffering

6

The mouse is dead

1. Euthanize the mouse/mice by cervical dislocation at desired time points. 2. Remove muscles of interest, e.g., tibialis anterior (TA), gastrocnemius (Gas), soleus (Sol), extensor digitorum longus (EDL), quadriceps (Quad), and diaphragm (Dia). 3. Snap-freeze muscle in liquid nitrogen-cooled isopentane. Ensure there are white dots/precipitates of frozen isopentane in the bottom and on the walls of the beaker before freezing the muscles; only then has the isopentane reached the optimal freezing temperature (~3–4 min). 4. Embed the tiny EDL and Sol muscles in Tissue-Tek before freezing them. If other muscles are embedded, they will freeze too slow and have a high risk of frost damage.

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5. Leave the muscles in the liquid nitrogen-cooled isopentane for minimum 30–60 s. 6. Transfer the frozen muscles to cryotubes, and keep them in liquid nitrogen or dry ice until all muscles are collected. 7. Store samples at 80  C until sectioning. 3.7

Cryosection

1. Transport muscles on dry ice to the cryostat (see Note 6). 2. Add a thin layer of Tissue-Tek on the frozen specimen disc and let it cool. Place the muscle in the correct orientation for transverse sections before it is completely frozen, and add a thin layer of Tissue-Tek around the muscle. Let it cool, and if needed, add another thin layer of Tissue-Tek. Leave for a few minutes to completely freeze. 3. Slice medial traverse sections at 10 μm from muscles. 4. Transfer sections onto a pre-warmed microscope slide. 5. Collect intervening sections for each muscle in two safe-lock tubes: one for RNA extraction to determine the extent of exon skipping by RT-PCR and one to quantify the amount of dystrophin by Western blotting, and store samples at 80  C (we refer to [13] for a WB protocol). 6. Store the slides at 80  C for future staining.

3.8 Dystrophin and Laminin Double-Staining of mdx Muscles

1. Take slides out of the 80  C cooler and let dry for about 5 min. 2. Draw a ring around with ImmEgde™ Pen to create a waterrepellent barrier. 3. Incubate sections with PBS for 5 min. 4. Block sections with 3% FCS in PBS for 30 min. 5. Incubate sections with primary anti-dystrophin antibody and anti-laminin alpha 2 (1:500) overnight at 4  C. 6. Wash sections with PBS 3  5 min on a Polymax shaker. 7. Incubate with secondary antibodies: Alexa Fluor® 594 anti-rabbit IgG (H+L) and Alexa Fluor® 488 goat anti-rat IgG (H+L) (molecular probes) diluted 1:500 for 2 h at RT, kept dark. 8. Wash sections with PBS 3  5 min on a Polymax shaker; keep sections dark. 9. Optional nuclei staining: incubate sections with DAPI (1:5000) in 5 min, followed by a PBS wash, 3  5 min on a Polymax shaker. 10. Mount coverslip with FluorSave™ reagent. 11. Observe sections using a fluorescence microscope (e.g., Nikon 20 Plan Apo VC N/A 0.75 mounted on a Nikon Eclipse 80i

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or a Nikon Ti-E epifluorescence microscope with motorized X/Y table). 12. Acquire images of the entire sections at 20 with a 5 Megapixel Andor Neo camera for fluorescence imaging using NIS-Elements Basic Research and Advanced Research (BR/AR) software (Nikon Instruments), and merge in software. 13. For quantification of dystrophin positive fibers, we refer to [14]. 3.9 RT-PCR Protocol for Detection of Exon Skipping

1. Add 1 mL of TRIzol reagent (Invitrogen) along with one stainless steel bead (4.8 mm diameter) into 2.0 mL safe-lock tube with intervening muscle section tissue. 2. Homogenize tissues using a homogenizer at 30 Hz for 5 min. Check to ensure that sample is completely homogenized. Repeat homogenizing step if necessary. 3. Perform TRIzol RNA extraction according to the protocol supplied by the manufacturer. 4. Quantify RNA using the NanoDrop and perform dilution to achieve a final concentration of 100 ng/μl. 5. Generate cDNA using 1000 ng of total RNA with 100 U Maxima Reverse Transcriptase (Thermo Scientific), 0.75 μM RT primer (50 -AGTCTGTAATTCATCTGGAG-30 ), and 0.2 mM dNTP. Cycling condition: 50  C for 30 min followed by 85  C for 5 min. 6. Perform PCR using 2 μL of Reverse Transcriptase reaction mix with 2 U DreamTaq DNA polymerase (Thermo Scientific), 0.2 mM dNTP, and the following primers and condition: 0.5 μM forward primer: 50 -ATCCAGCAGTCAGAAAG CAAA-30 . 0.5 μM reverse primer: 50 -CAGCCATCCATTTCTGTAAG G-30 . Cycling condition: 95  C for 30 s, 60  C for 30 s, and 72  C for 30 s for 32 cycles, followed by a final elongation at 72  C for 5 min. (For detection of low skipping percentages, increase the number of cycles to 38 cycles.) 7. Separate PCR products in a 2% agarose gel with 1 TBE buffer (pre-stained with ethidium bromide) at 6.66 V/cm for 20 min. 8. Quantify the skipped (121 bp) and unskipped (334 bp) bands (by scanning), and determine the extent of exon skipping.

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Notes 1. Only compounds that have been tested in vitro for toxicity with relevant methods are allowed to be injected into living animals. 2. PNA oligomers are typically purified by reversed-phase HPLC in a water acetonitrile gradient containing 0.1% TFA. Thus upon lyophilization, the product is obtained as an acidic TFA salt (because cytosine and adenine nucleobases are protonated), and upon dissolving in water, an acidic solution (at higher PNA concentrations) is adjusted down to pH 1–2. As such acidic solutions have high toxicity and thus cannot be used for injection in animals, pH adjustment to the PNA solutions must be performed before systemic administration (or alternatively a buffer with sufficient buffer capacity must be used). In our experience unmodified PNA can be pH adjusted up to around pH 4 (where cytosines are expected still to be protonated (pKa ~ 4.5) without precipitation of the PNA at injection concentrations. Since the pH of blood is tightly regulated at ~pH 7.4, it is possible that some (temporary?) precipitation takes place at the injection site after systemic administration of pH-adjusted, unmodified PNA. If conjugated to a peptide containing multiple lysines and/or arginines, the PNA solubility above pH 4 is improved. Also when a solution of unmodified PNA is pH adjusted; localized precipitation in the injection solution upon the addition of 1 M NaOH is frequently observed. Upon vortexing, the precipitate dispersed into the solution. When the pH is adjusted to above pH 4–4.5 in high dosages of injection solutions, PNA precipitation often occurs. Despite the above considerations, Gao and colleagues have reported a study in which very high dosages of unmodified PNA (5  100 mg/kg) were administrated without pH adjustment. Dystrophin protein was restored in bodywide skeletal muscles, and the mice had improved dystrophic pathology, and they did not observe any toxicity [15]. However, we have not been able to reproduce their data with a pH-adjusted injection solution, and we observed temporary acute toxicity with both adjusted and not adjusted injection solutions with unmodified PNA at the same dosages. 3. Obtain relevant permissions from authorities to perform animal experiments. 4. Always prepare 200 μL extra injection solution for the mice, due to loss in preparation of needles. 5. Include vehicle control mice for acute toxicity studies. 6. Include C57 WT control mice for RT-PCR, histology, and WB.

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References 1. Yin H, Lu Q, Wood M (2008) Effective exon skipping and restoration of dystrophin expression by peptide nucleic acid antisense oligonucleotides in mdx mice. Mol Ther 16(1):38–45. https://doi.org/10.1038/sj.mt.6300329 2. Yin H, Betts C, Saleh AF, Ivanova GD, Lee H, Seow Y, Kim D, Gait MJ, Wood MJ (2010) Optimization of peptide nucleic acid antisense oligonucleotides for local and systemic dystrophin splice correction in the mdx mouse. Mol Ther 18(4):819–827. https://doi.org/10. 1038/mt.2009.310 3. Ivanova GD, Arzumanov A, Abes R, Yin H, Wood MJ, Lebleu B, Gait MJ (2008) Improved cell-penetrating peptide-PNA conjugates for splicing redirection in HeLa cells and exon skipping in mdx mouse muscle. Nucleic Acids Res 36(20):6418–6428. https://doi.org/10.1093/nar/gkn671 4. Willmann R, Possekel S, Dubach-Powell J, Meier T, Ruegg MA (2009) Mammalian animal models for Duchenne muscular dystrophy. Neuromuscul Disord 19(4):241–249 5. TREAT-NMD Neuromuscular Network (2018, June 14) Experimental protocols for DMD animal models. http://www.treat-nmd. eu/research/preclinical/dmd-sops/ 6. Tennyson CN, Klamut HJ, Worton RG (1995) The human dystrophin gene requires 16 hours to be transcribed and is cotranscriptionally spliced. Nat Genet 9(2):184–190 7. Koenig M, Hoffman EP, Bertelson CJ, Monaco AP, Feener C, Kunkel LM (1987) Complete cloning of the Duchenne musculardystrophy (Dmd) Cdna and preliminary genomic organization of the Dmd gene in normal and affected individuals. Cell 50(3):509–517 8. Brolin C, Shiraishi T, Hojman P, Krag TO, Nielsen PE, Gehl J (2015) Electroporation enhanced effect of dystrophin splice switching PNA oligomers in normal and dystrophic muscle. Mol Ther Nucleic acids 4:e267. https:// doi.org/10.1038/mtna.2015.41 9. Mardirossian G, Lei K, Rusckowski M, Chang F, Qu T, Egholm M, Hnatowich DJ

(1997) In vivo hybridization of technetium99m-labeled peptide nucleic acid (PNA). J Nucl Med 38(6):907–913 10. McMahon BM, Mays D, Lipsky J, Stewart JA, Fauq A, Richelson E (2002) Pharmacokinetics and tissue distribution of a peptide nucleic acid after intravenous administration. Antisense Nucleic Acid Drug Dev 12(2):65–70. https://doi.org/10.1089/ 108729002760070803 11. Ganguly S, Chaubey B, Tripathi S, Upadhyay A, Neti PV, Howell RW, Pandey VN (2008) Pharmacokinetic analysis of polyamide nucleic-acid-cell penetrating peptide conjugates targeted against HIV-1 transactivation response element. Oligonucleotides 18 (3):277–286. https://doi.org/10.1089/oli. 2008.0140 12. Turner PV, Pekow C, Vasbinder MA, Brabb T (2011) Administration of substances to laboratory animals: equipment considerations, vehicle selection, and solute preparation. J Am Assoc Lab Anim Sci 50(5):614–627 13. Godfrey C, Muses S, McClorey G, Wells KE, Coursindel T, Terry RL, Betts C, Hammond S, O’Donovan L, Hildyard J, El Andaloussi S, Gait MJ, Wood MJ, Wells DJ (2015) How much dystrophin is enough: the physiological consequences of different levels of dystrophin in the mdx mouse. Hum Mol Genet 24 (15):4225–4237. https://doi.org/10.1093/ hmg/ddv155 14. Arechavala-Gomeza V, Kinali M, Feng L, Brown SC, Sewry C, Morgan JE, Muntoni F (2010) Immunohistological intensity measurements as a tool to assess sarcolemma-associated protein expression. Neuropathol Appl Neurobiol 36(4):265–274. https://doi.org/10. 1111/j.1365-2990.2009.01056.x 15. Gao X, Shen X, Dong X, Ran N, Han G, Cao L, Gu B, Yin H (2015) Peptide nucleic acid promotes systemic dystrophin expression and functional rescue in dystrophin-deficient mdx mice. Mol Ther Nucleic Acids 4:e255. https:// doi.org/10.1038/mtna.2015.27

Chapter 16 Near-Infrared In Vivo Whole-Body Fluorescence Imaging of PNA Ernest Wee Kiat Lim, Camilla Brolin, and Peter E. Nielsen Abstract Using near-infrared fluorophore Alexa Fluor 680 labeled peptide nucleic acids (PNAs) the biodistribution of such antisense agents can be analyzed in real time in live mice using in vivo imaging. Using the fluorescence intensity emitted from the mouse at different time points following administration, the systemic distribution and organ accumulation of PNA can be tracked. In addition, an estimation of the body half-life of the compound can be obtained by the change in fluorescence intensity over time. With this technique, the distribution of compounds can be monitored real time, while reducing the number of animals and amount of compounds required. Key words IVIS, Whole-body in vivo bioimaging, Biodistribution, Tissue accumulation

1

Introduction Whole-body in vivo bioimaging in small animals is a useful technique to obtain fast information about the time course of biodistribution (caused by the pharmacokinetic behavior), tissue accumulation, and route of excretion for novel compounds [1, 2]. Compared to conventional pharmacokinetic methods of blood sampling followed by, e.g., LC-MS analysis to determine the serum drug concentration or the use of radioactively labeled drug and scintillation counting, this noninvasive method requires fewer animals and less compound, while allowing the distribution of the compound to be monitored real time. Bioimaging studies require the conjugation of a fluorescent dye to the compound (in casu PNA). The addition of a fluorophore will to some extent alter the physicochemical properties and also increase the size of PNA by approximately 1000 Da. The fluorophore is conveniently attached to a terminal cysteine in the PNA via solution maleimide coupling chemistry (click chemistry is also an option). Fluorophores in the near-infrared (NIR) wavelength range are recommended, due to less tissue absorption of the light compared to

Peter E. Nielsen (ed.), Peptide Nucleic Acids: Methods and Protocols, Methods in Molecular Biology, vol. 2105, https://doi.org/10.1007/978-1-0716-0243-0_16, © Springer Science+Business Media, LLC, part of Springer Nature 2020

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the visible wavelength range [3]. We have chosen Alexa Fluor 680 (AF680) as a very photo stable and not too lipophilic fluorophore. In terms of proper interpretation of the data, it is critical that the fluorophore-labeled compound is stable for the duration of the experiment as only the fluorophore is monitored. One advantage of whole-body in vivo bioimaging is the need for only minute amounts of compound (~10 μg) compared to mg scale for conventional pharmacokinetic analysis methods, and it is therefore a highly convenient method to screen potential (PNA) drug hit and lead compounds. With the IVIS Spectrum in vivo imaging system, it is possible to use both epi-illumination (2D) and trans-illumination (3D) scanning modes. Epi-illumination images are acquired by illuminating the surface of the mouse and collecting the animals’ surface fluorescent signal, up to a depth of approximately 5 mm. The imaging procedure takes less than 1 min compared to full transillumination body imaging, which takes approximately 30 min and uses a trans-illumination excitation light source placed close to animal on the opposite side to the camera [4]. From unpublished in vivo bioimaging studies and from previous pharmacokinetic studies using radioactive-labeled unmodified PNA administrated i.v. or i.p., it is clear that PNA is excreted rapidly via the kidneys as intact molecules typically with an elimination half-life of less than 30 min [5–8]. The serum clearance profile for PNA and PNA conjugates is biphasic with a rapid clearance followed by slow elimination (T2½ ¼ 55.9 h) [7]. To monitor the first clearance phase, epi-illumination on both sides of the animal was chosen due to fast image acquisition and the possibility to image multiple time points within the first 30 min after administration (Figs. 1 and 2). Careful interpretation of the data is necessary, because 2D scanning only reflects the surface of the animal and not the deeper lying tissues. Semiquantitative determination of body half-life for AF680-labeled PNA based on 2D images gave a body half-life of ~ 20 min (n ¼ 3) (Figs. 1 and 2a) upon i.v. administration, which is quite similar to the previously reported serum half-life (0.64 h) [7] and (17  3 min) [5] after i.p. administration. Even though the signal will be much weaker, imaging of (part of) the tail may more closely reflect serum levels [9]. As the concentration of AF680-labeled PNA used for whole-body in vivo bioimaging (10 μM) is more than 100-fold lower than the concentration administered in some treatment studies (50 mg/kg (~1300 μM)), the pharmacokinetic parameters may not correctly represent the distribution profiles at treatment dosages. To obtain a more representative body kinetics profile, unlabeled PNA can be added to the AF680-labeled PNA to emulate the distribution at treatment dosages. The reason for adding unlabeled PNA instead of using higher concentrations of AF680-labeled PNA is to prevent saturation of the CCD camera which captures the fluorescence signal. Figure 1a illustrates the

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Fig. 1 Whole-body fluorescence images of NMRI-nu mice acquired at different time points after i.v. administration of AF680-labeled and unmodified PNA using the IVIS Spectrum CT. (a) Dorsal side and (b) ventral side. Mouse I: 10 μM AF680-labeled anti-dystrophin PNA (H-Cys(AF680)-GGC CAA ACC TCG GCT TAC CT-NH2). Mouse II: 10 μM AF680-labeled anti-dystrophin PNA and 260 μM of unmodified anti-dystrophin PNA (H-GGC CAA ACC TCG GCT TAC CT-NH2), corresponding to a treatment dose of 10 mg/kg. Mouse III: 10 μM AF680-labeled anti-dystrophin PNA and 650 μM of unmodified anti-dystrophin PNA, corresponding to a treatment dose of 25 mg/kg. Mouse IV: 10 μM of anti-dystrophin AF680-labeled PNA and 1300 μM of unmodified anti-dystrophin PNA, corresponding to a treatment dose of 50 mg/kg. From both dorsal and ventral images, a gradual decrease in whole-body fluorescence was observed over time for all treatment doses (mouse I–IV). Accumulation of fluorophore in the kidneys can be observed from the dorsal side, whereas accumulation in the bladder and liver can be observed from the ventral side. By increasing the dose, higher accumulation in the kidneys was observed. Using a treatment dose of 50 mg/kg, liver accumulation becomes evident in the ventral scans. Based on the images acquired via epi-illumination scan, it can be deduced that most of the administered compounds were removed from the system via renal clearance

dorsal distributions at treatment doses, showing a gradual decrease in fluorescence signal in the general body together with an increase in the kidney fluorescence over time (Fig. 1a); and from the ventral side, accumulation of PNA is visible in the bladder (Fig. 1b). By displaying the whole-body fluorescence versus time, body half-life’s (T1½) for the 10, 25, and 50 mg/kg treatment doses were determined as 24, 28, and 33 min, respectively, n ¼ 2–3 (Fig. 2a). Also the ratio between the fluorescence signals from the kidneys and the whole body increased over time for the treatment dose animals (Fig. 2b), demonstrating relatively slower elimination (relative accumulation) of PNAs in the kidney over time. Conjugation of the PNA to certain cell-penetrating peptides (CPP) or other delivery ligands can significantly increase the body half-life (unpublished data). To obtain a more precise localization of the AF680-labeled PNA in the deeper lying tissues, it is possible to use a 3D fluorescence scanning mode (also termed fluorescence imaging

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Fig. 2 Body profile kinetic of AF680-labeled and unmodified PNA at different dosages. Whole-body fluorescence from NMRI-nu mice injected with different additional doses of unmodified PNA was quantified using the Living Image® software. (a) Whole-body distribution profile constructed using ROI values measured from the dorsal side of the mouse including the kidneys. (b) Kidney fluorescence (ROI values from the kidneys) relative to whole-body fluorescence. (c) Heart, lungs, liver, kidneys, bladder, and TA muscle excised 48 h after injection. I: 10 μM AF680-labeled anti-dystrophin PNA. II: 10 μM AF680-labeled and 260 μM unmodified antidystrophin PNA, corresponding to a treatment dose of 10 mg/kg. III: 10 μM AF680-labeled and 650 μM unmodified anti-dystrophin PNA, corresponding to a treatment dose of 25 mg/kg. IV: 10 μM of AF680-labeled and 1300 μM unmodified anti-dystrophin PNA, corresponding to a treatment dose of 50 mg/kg. By constructing the whole-body fluorescence versus time curve, the body half-life of the AF680-PNA and the AF680-PNA with 10, 25, and 50 mg/kg treatment doses was calculated to be 21, 24, 28, and 33 min, respectively. The kinetics of the ratio of kidney to whole-body fluorescence reflects the extent of kidney accumulation in the mouse. If no accumulation in the kidneys takes place, the ratio over time should be constant. While the ratio of kidney to whole-body fluorescence of the AF680-PNA was observed to be constant, indicating little or no kidney accumulation, an increase in this ratio was observed in the mice treated with the AF680-PNA and unlabeled PNA. Through ex vivo scanning of the organs performed 48 h after injection, high levels of fluorescence were indeed also observed in the kidneys, as well as in the liver, supporting the observations from the dorsal and ventral scans. Epi-illumination imaging using AF680-PNA and AF680 with treatment doses suggests that at higher concentrations, the distribution profile changes

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Fig. 3 3D reconstruction after administration of 20 μM AF680-labeled and 260 μM of unmodified PNA, corresponding to a treatment dose of 10 mg/kg (from 18 to 53 min). (a) 3D reconstruction (red slice plane ¼ coronal (Z ); green slice plane ¼ transaxial (Y ); blue slice plane ¼ sagittal (X). PNA is distributed throughout the entire body (the head and limbs were not selected for imaging). (b) Z slice plane positioned around posterior region of the mouse and Y slice plane positioned around the kidney/liver. (c) Y slice plane positioned around the liver and kidney. (d) Z slice plane around the anterior region of the mouse and Y slice plane positioned around the bladder. (e) Y slice plane around the bladder region. The high fluorescence signals detected after PNA administration are localized to the liver, kidney, and bladder regions, corresponding to what we observe from the epi-illumination and ex vivo scans

tomography, FLIT). Some imaging systems (e.g. the IVIS Spectrum CT) have a built-in X-ray CT (computed tomography) to establish both the surface and tissue/bone anatomical context for image reconstruction. By combining FLIT with CT, it is possible to reconstruct a 3D distribution map of PNA within the body using the imaging software (Fig. 3). When FLIT imaging is performed at early time points after administration, it is important to consider the very short half-life of PNA and duration of the scan when interpreting the data. FLIT imaging performed at later time points can give useful information about biodistribution and tissue accumulation. In addition, information about biodistribution and tissue accumulation is accessible by subsequent ex vivo imaging of excised tissues/organs. This information can be used to compare the relative amount of drug (fluorescence) within tissues and between different tissues [1, 2]. Similar to conventional antisense oligonucleotides, PNA tends to accumulate mainly in the kidneys and liver in a dosedependent manner, as observed by high levels of fluorescence in

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these organs (Fig. 2c) [10, 11]. It is important to note that the imaging only monitors the position of AF680-PNA fluorophore per se and not necessarily the intact compound. Thus it is critical to obtain information of the stability/metabolism of the compound when interpreting the imaging data. In addition, the influence of the fluorophore (charge/lipophilicity) on the in vivo properties of the compound should also be taken into consideration, and it is advisable to confirm the results comparing at least two different fluorophore conjugates (see Note 1).

2

Materials Alexa Fluor 680 (AF680)-labeled PNA (e.g., H-Cys(AF680)-GGC CAA ACC TCG GCT TAC CT-NH2). Mice: nude mice are preferred (e.g., NMRI-nu mice: Rj: NMRI-Foxn1nu/nu from JANVIER LABS) as mouse fur/hair is effective at blocking, absorbing, and scattering light during optical imaging. Depilation is needed if conventional strains are used. Mice should be housed under pathogen-free conditions in a thermostatcontrolled environment with a 12:12 h light-dark cycle and provided with rodent chow diet and water ad libitum (see Note 2). IVIS® Spectrum CT in vivo imaging system (PerkinElmer). Vehicle: isotonic glucose (5%).

3

Methods

3.1 AF680-PNA Synthesis

1. Dissolve 1 mg CysPNA in 300 μL deoxygenated 10 mM Tris buffer pH 7.5. 2. Dissolve 0.5 mg Alexa Fluor 680-maleimide reagent in 300 μL deoxygenated DMSO. 3. Mix these two solutions and incubate overnight with stirring at room temperature. 4. Purify the product by reversed phase HPLC (RP18, water/ acetonitrile gradient 0–50% (containing 0.1% trifluoroacetic acid (TFA)). 5. Analyze by MALDI-TOF mass spectrometry.

3.2 Preparation of Injection Solutions (See Note 3)

1. Prepare the AF680-PNA stock solution by first dissolving the AF680-PNA (~1 mg) in a droplet of dimethyl sulfoxide (DMSO), followed by the addition of 100 μL Milli-Q water. 2. Determine the molar concentration of the AF680-PNA stock solution spectrophotometrically at A260 using the extinction coefficient of the compound.

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3. Based on the molar concentration obtained above, dilute the AF680-PNA stock with isotonic glucose to a final concentration of 10 μM to obtain the injection solution for the mice. The injection volume used for i.v., i.p., and s.c. administrations in each mouse is 200 μL (!2 nmol). Prepare an additional 100–200 μL of injection solution to account for dead space in the needle and loss during syringe preparation. 3.3 Preparation of Injection Solution with Treatment Dose (See Note 4)

1. Calculate the administration doses based on the weight of the animals recorded the day before or on the day of the experiment (e.g. to prepare a solution at 10 mg/kg, determine the concentration required in 200 μL injection volume based on the weight of the mouse). 2. Dissolve the lyophilized PNA in Milli-Q water to obtain the stock solution. Aim for a high stock concentration (e.g. 75 mg/mL based on the dry weight of the PNA). 3. Prepare injection solution by adding the required volume of AF680-PNA and unlabeled PNA from the stock to give the desired amount of 10 μM of AF680-labeled PNA (2 nmol) and unlabeled PNA (e.g. corresponding to 10 mg/kg administered) in the required injection volume (200 μL per mouse) of isotonic glucose. Prepare injection solution fresh, just before administration. 4. Adjust pH of the injection solution using 0.5 or 1 M NaOH. Perform stepwise addition of 1 μL NaOH, vortex the solution, and test a droplet on a pH-indicator strip. Repeat until the pH of the solution reaches at least pH 4 (see Note 5).

3.4 In Vivo Epifluorescence Imaging (See Note 6)

1. Set up the imaging software for epi-illumination imaging, and select the relevant filter pair set (see Note 7). 2. Anesthetize mouse/mice in a chamber via the inhalation of 2.5% isoflurane (Baxter, Deerfield, IL) in 100% oxygen with a delivery rate of 1.5 L/min. 3. Perform image acquisition of the pre-scan by placing the anesthetized mouse/mice with the dorsal side up on the animal stage in the imager under inhalational isoflurane anesthesia. Turn the mouse/mice around with the ventral side up, and repeat image acquisition to obtain the pre-scan of the ventral side. 4. Remove the mouse from the imager, and place it in a heat box for a short while to expose the tail veins. 5. Transfer the mouse to a restrainer and administer the AF680PNA via tail vein (i.v. injection). 6. Return the mouse/mice to the induction chamber for complete anesthesia before moving it back to the imager.

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7. Begin image acquisition of the dorsal and ventral sides at desired time points (e.g., 5, 10, 15, 20, 30, 60, 120, and 240 min, 24 h, and 48 h). Important: maintain positioning of the mouse/mice over the course of study. 8. Upon the acquisition of the last image, if desired, euthanize the mouse by cervical dislocation, and perform dissection to remove the heart, lungs, liver, spleen, kidneys, bladder, and other tissues/organs of interest for ex vivo organ/tissue scan. 9. Arrange organs on a black Lexan sheet and place it in the imager for image acquisition. 3.5 Post-scanning Data Processing

1. Using the imaging software, load all acquired dorsal/ventral scans from the same experiment as a group. 2. Perform image correction by subtracting the background fluorescence, and set the fluorescence levels of all images to the same scale. 3. Adjust the minimum fluorescence to a value where no signal can be observed on the pre-scan image and the maximum fluorescence to a value where the color contrast relative to the signal can be discriminated. 4. Save a snapshot of the “normalized” images. 5. Open all acquired dorsal images following the abovementioned steps. 6. Define region of interest (ROI) by demarcating the area occupied by the mouse. 7. Measure the fluorescence intensity of the defined ROIs for further biodistribution and kinetic analyses.

3.6 In Vivo 3D Fluorescence Imaging Tomography (FLIT)

1. Setup the imaging software for FLIT imaging and select the relevant filter pair set. 2. Anesthetize mouse in a chamber via the inhalation of 2.5% isoflurane (Baxter, Deerfield, IL) in 100% oxygen with a delivery rate of 1.5 L/min. 3. Perform image acquisition of the pre-scan by placing the anesthetized mouse with the dorsal side up on the animal stage in the imager under inhalational isoflurane anesthesia. 4. Remove the mouse from the imager, and place it in a heat box for a short while to expose the tail veins. 5. Transfer the mouse to a restrainer and administer the AF680PNA via tail vein (i.v. injection). 6. Return the mouse to the induction chamber for complete anesthesia, transfer it back to the imager with the dorsal side up, and begin 3D image acquisition.

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1. Using the imaging software, load the acquired 3D image. 2. Perform image correction by subtracting the background fluorescence. 3. Using the imaging data, generate a surface for 3D reconstruction, and select the fluorescence sequences to be used for the 3D reconstruction process. 4. Initiate the 3D reconstruction process. 5. Upon the completion of reconstruction, adjust the fluorescence voxels to obtain the desired image, and save the 3D view to a graphic file.

4

Notes 1. It is advisable to test two different fluorophores. In preliminary experiments we have tested Cy5.5. In general with the Cy5.5labeled peptide-PNAs, we observed higher accumulation in the liver compared to the AF680-labeled peptide-PNAs. The lipophilicity of Cy5.5 is reported to be higher than AF680, and therefore it would be predicted to more strongly alter the pharmacokinetics by nonspecific protein binding [12]. 2. Regular mouse chow contains chlorophyll that autofluoresces around 700 nm and can interfere with fluorescence signals from NIR dyes. This is most important when signals from AF680 are low and for oral administration studies. 3. To determine the exact dosage of PNA administered in the animals, we recommend calculating the concentration of the unlabeled PNA stock solution based on the absorbance measured spectrophotometrically at A260 and the compound’s extinction coefficient. We use the following extinction coefficients ε0260 (L  mol1  cm1) for the PNA monomers: [T ¼ 8.6; G ¼ 11.7; A ¼ 13.7; C ¼ 6.6]  103. This is to be able to take into account the varied presence of residual TFA in the lyophilized PNA from the HPLC purification (typically ~30% TFA). 4. To avoid photobleaching of AF680, perform the procedures with tubes wrapped with aluminum foil. 5. pH 4 for unmodified PNA, pH 6–7 for PNA conjugates to Arg/Lys-rich peptides. 6. Imager used here refers to the IVIS Spectrum CT. 7. The excitation/emission 675 nm/720 nm filter pair is used for scans with AF680 fluorophores.

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References 1. Vasquez KO, Casavant C, Peterson JD (2011) Quantitative whole body biodistribution of fluorescent-labeled agents by non-invasive tomographic imaging. PLoS One 6(6): e20594. https://doi.org/10.1371/journal. pone.0020594 2. Yokoi T, Otani T, Ishii K (2018) In vivo fluorescence bioimaging of ascorbic acid in mice: development of an efficient probe consisting of phthalocyanine, TEMPO, and albumin. Sci Rep 8(1):1560. https://doi.org/10.1038/ s41598-018-19762-8 3. Leblond F, Davis SC, Valdes PA, Pogue BW (2010) Pre-clinical whole-body fluorescence imaging: review of instruments, methods and applications. J Photochem Photobiol B 98 (1):77–94. https://doi.org/10.1016/j. jphotobiol.2009.11.007 4. Jen-Chieh Tseng PDKVDP, Ph.D. Optical imaging on the IVIS spectrum CT system: general and technical considerations for 2D and 3D imaging. https://www.perkinelmer.com/ CMSResources/Images/44-171013TCH_ 012007_01_IVIS-2D_3D_Imaging.pdf 5. McMahon BM, Mays D, Lipsky J, Stewart JA, Fauq A, Richelson E (2002) Pharmacokinetics and tissue distribution of a peptide nucleic acid after intravenous administration. Antisense Nucleic Acid Drug Dev 12(2):65–70. https://doi.org/10.1089/ 108729002760070803 6. Mardirossian G, Lei K, Rusckowski M, Chang F, Qu T, Egholm M, Hnatowich DJ (1997) In vivo hybridization of technetium99m-labeled peptide nucleic acid (PNA). J Nucl Med 38(6):907–913 7. Ganguly S, Chaubey B, Tripathi S, Upadhyay A, Neti PV, Howell RW, Pandey VN (2008) Pharmacokinetic analysis of polyamide nucleic-acid-cell penetrating peptide

conjugates targeted against HIV-1 transactivation response element. Oligonucleotides 18 (3):277–286. https://doi.org/10.1089/oli. 2008.0140 8. Hamzavi R, Dolle F, Tavitian B, Dahl O, Nielsen PE (2003) Modulation of the pharmacokinetic properties of PNA: preparation of galactosyl, mannosyl, fucosyl, N-acetylgalactosaminyl, and N-acetylglucosaminyl derivatives of aminoethylglycine peptide nucleic acid monomers and their incorporation into PNA oligomers. Bioconjug Chem 14(5):941–954. https://doi. org/10.1021/bc034022x 9. Vines DC, Green DE, Kudo G, Keller H (2011) Evaluation of mouse tail-vein injections both qualitatively and quantitatively on smallanimal PET tail scans. J Nucl Med Technol 39 (4):264–270. https://doi.org/10.2967/jnmt. 111.090951 10. Yu RZ, Geary RS, Levin AA (2006) Pharmacokinetics and pharmacodynamics of antisense oligonucleotides. In: Meibohm B (ed) Pharmacokinetics and pharmacodynamics of biotech drugs: principles and case studies in drug development. Wiley-VCH, Germany. https://doi.org/10.1002/9783527609628. ch4 11. Sazani P, Gemignani F, Kang SH, Maier MA, Manoharan M, Persmark M, Bortner D, Kole R (2002) Systemically delivered antisense oligomers upregulate gene expression in mouse tissues. Nat Biotechnol 20(12):1228–1233. https://doi.org/10.1038/nbt759 12. Ogawa M, Regino CA, Choyke PL, Kobayashi H (2009) In vivo target-specific activatable near-infrared optical labeling of humanized monoclonal antibodies. Mol Cancer Ther 8 (1):232–239. https://doi.org/10.1158/ 1535-7163.MCT-08-0862

Chapter 17 Poly(Lactic-co-Glycolic Acid) Nanoparticle Delivery of Peptide Nucleic Acids In Vivo Stanley N. Oyaghire, Elias Quijano, Alexandra S. Piotrowski-Daspit, W. Mark Saltzman, and Peter M. Glazer Abstract Many important biological applications of peptide nucleic acids (PNAs) target nucleic acid binding in eukaryotic cells, which requires PNA translocation across at least one membrane barrier. The delivery challenge is further exacerbated for applications in whole organisms, where clearance mechanisms rapidly deplete and/or deactivate exogenous agents. We have demonstrated that nanoparticles (NPs) composed of biodegradable polymers can encapsulate and release PNAs (alone or with co-reagents) in amounts sufficient to mediate desired effects in vitro and in vivo without deleterious reactions in the recipient cell or organism. For example, poly(lactic-co-glycolic acid) (PLGA) NPs can encapsulate and deliver PNAs and accompanying reagents to mediate gene editing outcomes in cells and animals, or PNAs alone to target oncogenic drivers in cells and correct cancer phenotypes in animal models. In this chapter, we provide a primer on PNA-induced gene editing and microRNA targeting—the two PNA-based biotechnological applications where NPs have enhanced and/or enabled in vivo demonstrations—as well as an introduction to the PLGA material and detailed protocols for formulation and robust characterization of PNA/DNA-laden PLGA NPs. Key words Peptide nucleic acid (PNA), Poly(lactic-co-glycolic acid) (PLGA), Nanoparticles (NP), Gene editing, Anti-microRNA (antimiR)

1

Introduction

1.1 Primer on PNA-Induced Gene Editing

Appropriately designed PNAs trigger repair activity after binding to their target sequences in genomic or episomal DNA, on the scale of those elicited by more overt, direct forms of DNA damage, presumably due to tight DNA binding and consequent helical distortion [1]. The repair-associated DNA syntheses, in turn, lead to DNA modification within [2, 3] or proximal [3] to the PNA binding site. Further, we have demonstrated that this exogenously induced but endogenously controlled DNA metabolism can result in gene disruption [2–5] due to stochastic repair events or precise gene modification [1, 6–17] when templated by a donor DNA

Peter E. Nielsen (ed.), Peptide Nucleic Acids: Methods and Protocols, Methods in Molecular Biology, vol. 2105, https://doi.org/10.1007/978-1-0716-0243-0_17, © Springer Science+Business Media, LLC, part of Springer Nature 2020

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Fig. 1 Gene editing by triplex-forming oligonucleotides. PNAs stimulate recombination of short (60 bp) DNA fragments into genomic DNA. Binding of the PNA subsequently produces a structural change within the dsDNA that activates cellular repair mechanisms, which are initiated by nucleotide excision repair (NER)

oligomer introduced with the PNA (Fig. 1). The precision of this latter application has been leveraged by our group to correct pathologic mutations in disease-related genes [6, 7, 9, 15–17], and introduce nonnatural but benign (and in some contexts, beneficial) genomic modifications [8, 10, 12] in normal genetic backgrounds (Fig. 1). A survey of the range of PNA designs deployed for gene modification has been covered elsewhere in recent reviews [18, 19]. Here, we summarize (Fig. 2) the PNA structural space explored for gene correction in our lab, in part because PLGA NP-assisted delivery of PNA has been demonstrated for this application in several reports [9, 10, 13–17]. As proof of principle for PNA-induced gene correction, we first reported that a bisPNA oligomer—a PNA structural variant [20, 21] possessing unique tethered domains designed to recognize the Watson-Crick and

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Fig. 2 PNA design variations applied for gene correction. (a) bisPNA, (b) tailclamp (tc) PNA, (c) tail-clamp gamma (γ) PNA, (d) pseudo-complementary (pc) PNA, (e) single-stranded (ss) γPNA

Hoogsteen faces of purine-rich DNA sequences—can stimulate recombination reactions between an episomal DNA target and a donor DNA oligomer [1] (Fig. 2a). In this demonstration, the bisPNA was directed toward a purine-rich binding site within the coding sequence for a reporter gene harboring an inactivating mutation and was tethered to or uncoupled from a donor DNA (Fig. 2a) designed to restore reporter activity by recombinationinduced transversion of the mutation [1]. Although of limited therapeutic utility, this work has had profound implications on subsequent applications of this gene editing technology by establishing foundational parameters for PNA/DNA design. Namely, the finding that a PNA/donor DNA reagent pair was more effective for gene correction when both oligomers were untethered from (instead of conjugated to) each other has simplified PNA/DNA preparation and design while also extending this technology to applications where the PNA binding and donor DNA target sites are relatively distal [1]. However, the requirement for simultaneous introduction of separated reagents reinforces the delivery challenge inherent in this strategy, since optimal results will require co-delivery of two components with significantly different chemical properties. The same bisPNA targeting strategy has been used to stimulate recombination reactions in the β-globin gene (HBB) [6, 9], in which pathologic mutations underlie the primary pathophysiology of β-thalassemia and sickle cell disease. Several bisPNA oligomers, directed to different purine stretches in intron 2 (IVS2) of HBB, were shown to be useful for stimulating recombination between the gene and a donor DNA designed to correct a thalassemiaassociated mutation at position 1 (hence IVS2-1) of the target

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intron [6]. In addition to demonstrating the feasibility of this correction paradigm in a genomic, endogenous, disease-relevant target, we also reported that gene correction frequencies were enhanced by chloroquine treatment subsequent to nucleofection—presumably due to lysosomal disruption [6] and ostensibly improved PNA/DNA bioavailability. This result again suggested that reagents capable of delivering PNA/DNA oligomers to specific intracellular compartments where they are active, or at least diverting them away from ineffective compartments, will be useful additions to this platform. Tail-clamp PNA (tcPNA) oligomers [22] have also been utilized to stimulate recombination reactions within HBB IVS2 [16] (Fig. 2b). In contrast to bisPNA oligomers, which feature WatsonCrick and Hoogsteen binding domains of equal length, this targeting modality incorporates into the PNA an extended Watson-Crick binding domain that enhances target duplex invasion [22]. Already demonstrated to significantly enhance donor recombination events in endogenous chromosomal targets significantly above those obtained with bisPNAs [8] (presumably due to enhanced duplex invasion and consequent helical distortion), we reported that tcPNA ligands directed proximal (~70–200 bp) to the location of another thalassemia-associated mutation in IVS2 were able to stimulate recombination-induced correction by apposite donor DNAs [16]. This targeting and correction modality was enhanced even further by the introduction of gamma (γ) PNA residues [23, 24] into the Watson-Crick binding domain of the tcPNAs, thus creating tcγPNAs [16] (Fig. 2c). When incorporated intermittently or completely into PNA oligomers, γPNA monomers—which feature chemical substituents in the γ position of the monomer backbone—impose conformational selection in the composite oligomers, the nature of which is determined by the stereochemistry at the γ position [23, 24]. Several reports from Ly and coworkers establish that γPNA monomers of appropriate stereochemistry can preorganize composite PNA oligomers into right-handed helices [23, 24] that are more effective for duplex DNA strand invasion [25], in addition to other benefits (such as solubility [24]) determined by the chemical nature of the γ substituents themselves. The judicious introduction of γPNA monomers into an already active tcPNA (in addition to other important improvements) led to an important demonstration of the therapeutic utility of our gene correction paradigm (see Subheading 1.3). Although less extensively explored, we have also demonstrated that pseudo-complementary (pc) PNA oligomers [26] are useful reagents for mediating PNA-induced gene correction [7] (Fig. 2d). By engaging both strands of target DNA duplexes [26], pcPNAs can overcome the targeting restriction imposed by bis/tcPNAs: the requirement for pronounced asymmetry in the strand distribution of purines and pyrimidines for effective invasion. (In the context of

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DNA repair induction, it is possible that dual-strand engagement yields stronger helical distortion and higher repair/recombination than single-strand targeting, although this theory is yet to be systematically evaluated.) We have reported that pcPNAs stimulate recombination of a donor DNA into HBB IVS2-1 at modest frequencies [7]. Here, as with bisPNA-induced editing, the observation that correction frequencies were marginally improved by chloroquine treatment [7] suggests that simultaneous delivery of all requisite co-reagents (2 pcPNAs + 1 donor DNA) will be an important challenge to address. Appropriately designed singlestranded (ss) PNAs—defined here as PNA oligomers for which target hybridization is mediated by only a single domain and engaging only one strand of the duplex—are also effective reagents for gene correction [13]. In this context, we have reported that ssPNAs can stimulate donor recombination into position 654 of HBB IVS2. Although requiring, in this example, γ-modifications [hence ssγPNA (Fig. 2e)] to achieve even modest levels of correction [13], this targeting modality offers the potential to overcome the sequence limitations of other targeting modalities, while significantly simplifying reagent design. 1.2 The Imperative of Nanoparticle-Mediated PNA/DNA Delivery for Gene Correction

While exploration of PNA variants useful for inducing gene correction should continue, even greater gains in editing efficacy— defined here as the yield of modified cells posttreatment—have been obtained by nanoparticle-assisted delivery of existing reagents. One demonstration was provided almost 10 years ago, in work by McNeer et al. [9], wherein bisPNA and donor DNA oligomers targeting HBB IVS2-1 were delivered by PLGA NPs or optimized nucleofections to primary human CD34+ cells—a population of interest in many gene-targeting therapeutic programs. Our results indicated that the nucleofection protocol itself decreased cell viability ~40%, 24 h after treatment—with toxicity increasing to 60% at 72 h. Introduction of the requisite PNA/DNA oligomers into the nucleofection cocktail was even more deleterious and increased nucleofection-associated toxicity to ~80% and 90% at 24 h and 72 h, respectively [9]. In contrast, essentially no toxicity was observed when the cells were exposed to PLGA NPs possessing no cargo (blanks) or PNA/DNA oligomers, at either time point. Further, while cells in all treatment groups, for either delivery method, showed a time-dependent attrition in CD34 expression—presumably due to spontaneous differentiation in culture—marker depletion was more rapid for nucleofected samples [9]. While the mechanisms of PLGA NP-mediated delivery of PNA/DNA oligomers are still being delineated, it is clear from our data that this transfection method is less inimical to cell viability than the transient membrane distortions created during nucleofection [27].

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Further, the intended modifications were detected in genomic DNA isolated from cells exposed to reagent-laden PLGA NPs at 3, 8, and 30 days posttreatment, demonstrating that these reagents, although benign, remain effective for delivering active PNA/DNA oligomers to the nucleus. Importantly, the induced modification frequencies detected in NP-treated cells were ~seven- fold higher than those in nucleofected cells. Conflated with the survival advantage (on day 3), this superiority translates to ~63-fold enhancement in editing efficacy mediated by improved delivery of PNA/DNA reagents [9]. While even more seminal demonstrations of gene correction/ modification by PNA/DNA-loaded PLGA NPs are described in the next section, it is worth examining some of the important considerations arising from the data summarized above: (1) even with its associated toxicity, nucleofection remains a transfection protocol of choice in many applications of gene editing, including those involving the nuclease-based reagents that produce higher modification frequencies than those induced by PNA [28]. (2) Consequently, for many applications, the realistic goals are to modify stem cells ex vivo, select for the modified population, and transplant into patients [29, 30]. Achieving any/all of these goals will require that treated cells survive long enough and in large enough quantities for additional manipulation posttreatment [29, 30]. (3) Accruing evidence that cells have potent mechanisms [31, 32] to detect and destroy exogenous nucleic acids entails that the transfection protocols for gene editing reagents must conceal them from such surveillance mechanisms long enough for the intended DNA metabolism to occur. The evidence suggests that reagent-laden PLGA NPs circumvent these challenges, as their introduction to cells in culture perturb neither cell survival, proliferation, differentiation capacity, nor lineage commitment [9]. Moreover, the reagents remain immunologically inert [11, 15–17], evading innate and/or humoral immune mechanisms, possibly because they physically (if transiently) conceal their cargo from surveillance pathways and release them in controlled amounts beneath detection thresholds. 1.3 In Vivo Demonstrations of PNA/DNA-Induced Gene Editing Enabled by PLGA NPs 1.3.1 Modification of CCR5

The biologically benign properties of PLGA NPs and editing precision achievable with PNA/DNA oligomers in vitro have incentivized the extension of this gene modification paradigm to proof-ofprinciple studies in humanized mouse models and therapeutic demonstrations in genetically engineered animal models that conservatively recapitulate human disease pathology. In early work, we demonstrated that PLGA NPs containing tcPNA and donor DNA oligomers previously designed and demonstrated to target and introduce stop codon mutations in human CCR5 [8]—mimicking a naturally occurring genotype [33] associated with R5-tropic HIV1 resistance—were able to mediate CCR5 modification in

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engrafted human cells in mice [11]. Although occurring at relatively low frequencies (0.4% in spleen; 0.05% in bone marrow), the targeted modifications were also detected in bone marrow cells from secondary recipient mice previously engrafted themselves with cells from NP-treated donor mice [11]. These results demonstrated that the initial NP treatments (in donor mice) were able to achieve targeted modifications in hematopoietic compartments populated by primitive stem cells that can persist, proliferate, and populate the hematopoietic system of nominally untreated recipients. Such demonstrations are crucial in the context of therapeutic gene editing, since, as alluded to above, the realistic goals of ex vivo manipulation and transplantation of autologous stem cells will be effective only if modifications occur in primitive cells without compromising their viability, proliferation, pluripotency, and engraftment—already a keen challenge for otherwise highly effective reagents [34, 35]. Expectedly, the genomic modification mediated by the reagent-laden NPs resulted in expression of an altered mRNA transcript in lung samples from treated humanized mice [11]. The functional relevance of this genomic modification and associated mRNA alteration was further demonstrated [11] by NP treatment of mice engrafted with peripheral blood mononuclear cells isolated from individuals heterozygous for the Δ32 mutation—the naturally occurring CCR5 deletion mutation conferring resistance to HIV-1. Following HIV-1 infection, mice receiving reagent-laden NPs showed T-cell retention at levels significantly higher than those receiving blank NPs, validating that PNA/DNAmediated editing enabled by PLGA NP delivery led to increased resistance to HIV1-mediated T-cell cytotoxicity [11]. While the therapeutic implications of such findings for AIDS prevention/ treatment are clear, the mechanistic implications are especially salient, as they suggest that NPs can deliver the active reagents to potentiate editing outcomes in circulating definitive CD4+ T-cells and/or primitive CD34+ cells in bone marrow (that can themselves differentiate into T-cells). 1.3.2 Gene Correction in Cystic Fibrosis Models

In vivo correction of F508del, the trinucleotide deletion mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that causes protein instability and is a predominant genotype responsible for cystic fibrosis (CF) lung disease [36] has been achieved by intranasal administration of NPs conveying apposite tcPNA and donor DNA oligomers [15]. The polymeric material deployed in this example was based primarily but not entirely on PLGA, and the resulting particles were further modified with a cellpenetrating peptide harboring a nuclear-localization sequence— adjustments which were shown to enhance delivery of plasmid DNA to primary human lung cells in vitro and improve PNA/ DNA-mediated correction in a reporter model in vivo [14].

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Intranasal administration of reagent-laden NPs to CF mouse models resulted in correction frequencies of ~6% in the nasal epithelium and ~1% in the lung [15], modification frequencies at least an order of magnitude higher than those reported by us for previous demonstrations in vitro. Moreover, the increased efficacies, mediated in part by iterative NP treatments, did not coincide with any increases in inflammatory cytokines or histological changes in modified tissues [15], suggesting that in vivo editing efficacies can be enhanced for these reagents in a manner that remains innocuous to recipients. Importantly, the elevated genotypic correction was functionally relevant, as nasal potential difference readings (an indication of chloride efflux, the primary function of CFTR) for CF mice homozygous for the F508del mutation receiving loaded NPs approached the wild-type range, in contrast to mice receiving blank NPs [15]. 1.3.3 Gene Correction in β-Thalassemia Models

Correction of a β-thalassemia-associated polymorphism in a mouse model of the disease represents our clearest demonstration of the therapeutic utility of PNA-mediated gene editing. Delivered in PLGA NPs, appropriately designed tcγPNA and donor DNA oligomers were shown to mediate reversion of mutant HBB alleles in vivo without genotoxic or deleterious immunological outcomes [16]. NP-mediated delivery, as extrapolated from the detection of allele correction potentiated by the nucleic acid encapsulants, was achieved in total bone marrow cells, including those possessing markers characteristic of primitive progenitors [16], which are difficult to transfect [37] but remain imperative for any modality of gene therapy or engineering in the context of β-thalassemia [29]. Importantly, successful transfection of and allele correction in hematopoietic components populated by primitive stem cells led to remediation of various hematological and anatomical disease phenotypes [16], demonstrating that modest correction frequencies (~4%), if directed toward the right cell populations by the delivery vehicle, can be therapeutically effective. This latter point has been emphasized in our most recent demonstration of therapeutic PNA-mediated correction in utero, wherein PNA/DNA-loaded PLGA NPs delivered intravenously to mouse fetuses mediated genotypic and phenotypic correction of β-thalassemia in resulting pups, with the therapeutic effects persisting into adulthood [17]. We showed that this delivery route, at the appropriate gestational age in mice, could direct NPs to the fetal liver [17], a prominent site of hematopoietic stem cell (HSC) expansion during murine hematopoiesis. Coupled with other factors, such as high basal expression levels of repair factors relevant to PNA-induced editing in HSCs [16], this targeting of NPs to cell populations amenable to correction and relevant for the disease led to allele modification frequencies (~6%) higher than those (~4%) obtained by iterative administration in adult models [16], despite requiring a single, smaller NP dose [17].

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1.4 In Vivo Applications of AntimiR PNAs Enabled by PLGA NPs 1.4.1 Primer on AntimiR PNAs

1.4.2 NP-Mediated Delivery of AntimiR-155 PNA

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MicroRNAs (miRNAs) are short (22 nt long) RNAs that are endogenously expressed and regulate mRNA expression [38]. Following transcription, as well as nuclear and cytoplasmic processing, a mature miRNA binds to an RNA-induced silencing complex (RISC). The miRNA in turn acts as a molecular guide, targeting complementary mRNA for degradation or translational repression [39]. Though once poorly understood, miRNAs have now been implicated in diverse processes including embryological development, cellular differentiation, and cancer. These broad roles have led to a boom in miRNA therapeutics, including ones based on PNA technology [40]. Yet rather than replenish therapeutic miRNAs (miRNA mimic), PNAs have been primarily used to suppress the effects of aberrantly expressed, oncogenic miRNAs. The first reported use of a PNA as an antimiR was by Fabani and Gait, who synthesized PNAs complementary to miR-122. Using in vitro models of hepatocellular carcinoma, they demonstrated that PNAs with four terminal lysine residues could sufficiently inhibit miR-122 activity in cells [41]. Using a terminal cysteine to conjugate the PNA to R6-penetratin, they further showed that cell-penetrating peptides (CPPs) could be used to deliver antimiR PNAs into cells, resulting in lower levels of endogenous miR-122 and increased expression of target genes [41]. The use of CPPs to deliver antimiR PNAs was further expanded by Oh et al., who systematically evaluated CPPs to deliver PNAs targeting miR-21 [42]. Using reporter plasmids transfected into cells, the group found that TAT-modified peptides were more effective than R6-penetratin in delivering antimiR-21 PNA [42]. While both of these examples demonstrated the effectiveness of antimiR PNAs, their clinical translatability was limited by the need for high doses in vitro (1 μM) [41, 42]. To overcome these limitations, our group has recently focused on encapsulating antimiR PNAs into PLGA NPs, which have a history of being effective in vitro and in vivo at significantly lower doses [43]. miR-155 is a critically important oncogenic miRNA, which has been shown to be upregulated in solid tumors of the lung, liver, kidney, gliomas, and pancreas, as well as B cell lymphoma and lymphoid leukemia [40]. Molecularly, miR-155 targets the SH2 domain-containing inositol 50 -phosphatase 1 (SHIP1) protein, reducing its expression through translational inhibition [44]. The resulting reductions in SHIP1 have now been implicated in the onset of acute myeloid leukemia (AML), as well as large B cell lymphoma [44]. Another group has also shown that miR-155 expression is essential for the survival of malignant lymphocytes in a mouse model of lymphoma [45]. Using PNAs targeting mature miR-155, we developed a method to encapsulate antimiR PNAs into PLGA NPs [43]. Although traditional oligonucleotide encapsulation is

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improved by the use of counterions to condense RNA- or DNA-based molecules, the use of charge-neutral PNAs, with slightly hydrophobic characteristics, enabled efficient loading into PLGA without complexation [43]. The use of polymeric NPs, rather than CPPs, provided further advantages by creating a reservoir of antisense molecules that are released over time after administration, extending inhibitory effects beyond the initial dose. Using a mouse model of miR-155-dependent lymphoma, antimir-155 PNA NPs inhibited miR-155 in vitro and in vivo [45]. NP-mediated depletion of miR-155 also significantly reduced tumor volumes when administered locally or systemically [45]. Importantly, the use of PLGA NPs densely loaded with PNA provided a critical step toward clinical translatability, reducing the in vivo dose from two doses at 50 mg/kg [46] to one dose at 1.5 mg/kg [45]. As interest in antimiR PNAs continues to expand, we are investigating novel peptides as well as peptide-modified NPs to enhance in vivo delivery [45, 47]. 1.4.3 NP-Mediated Delivery of PNA AntimiR-210

Although miRNA expression is regulated by several factors, miR-210 is partly expressed in response to hypoxia, a hallmark of the tumor microenvironment [48]. Molecularly, miR-210 targets the succinate dehydrogenase complex subunit D (SDHD), resulting in mitochondrial dysfunction and aberrant sensing of cellular O2. Consequently, reductions in SDHD lead to increased expression of hypoxia-inducible factor 1α (HIF-1α), which leads to further upregulation of miR-210 [49]. Our group has shown that miR-210 also directly targets the 30 UTR of RAD52 leading to reduced homology-dependent repair (HDR) activity and greater genomic instability in hypoxic cells [50]. Given its central role in cancer progression, we developed a strategy to target miR-210 using PNAs encapsulated in PLGA NPs [51]. Unlike our previous work targeting miR-155, which made use of standard PNAs [45], we developed chemically modified versions with diethylene glycol substitutions at the γ position of each PNA monomer (mpγPNA) [51]. Using these modified monomers, we synthesized mpγ-modified antimiR-210 PNA (mpγP210). As expected, mpγP210 was preorganized into a right-handed helical structure, with superior target hybridization relative to a chemically unmodified PNA (P210). Local administration of mpγP210-loaded NPs in a xenograft model effectively reduced miR-210 in vivo and increased miR-210 target protein (ISCU) expression. Importantly, in vivo administration of mpγP210 NPs significantly reduced tumor volumes, demonstrating for the first time that γPNAs have superior in vivo antimiR effects relative to unmodified controls. As before, the use of densely loaded PLGA NPs, with modifications in PNA chemistry, allowed for further reductions in antimiR dose to 0.8 mg/kg [51]. We are currently exploring several alternative

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NPs to enhance the in vivo effects of PNA, including polymer blends of PLGA with cationic poly(β-amino esters) (PBAE) [14, 15, 52] and a novel block copolymer of poly(lactic acid) and hyperbranched polyglycerol (PLA-HPG) [53]. 1.5 PLGA NPs as Vehicles for Delivery of Bioactive PNA/DNA Reagents

Major benefits of polymeric carrier systems include their potential biodegradable, biocompatible, and controlled release properties in addition to well-defined chemistries and physical characteristics. Many synthetic polymers have versatile chemistries that are controllable through synthesis. Polymer physicochemical properties can be designed and modified (e.g., composition, molecular weight, polydispersity) according to desired specifications or applications. Examples of widely used biodegradable synthetic polymers are poly(lactic acid) (PLA), poly(glycolic acid) (PGA), and the copolymer PLGA. The latter is an aliphatic polyester composed of lactic acid and glycolic acid in fixed ratios [54]. These materials degrade slowly via bulk hydrolysis in aqueous environments, providing the benefit of sustained release of cargo. PLGA degradation products are lactic and glycolic acid, which are eliminated via natural mechanisms such as the citric acid cycle [55]. We have primarily used PLGA-based formulations composed of a 50:50 lactic acid/ glycolic acid ratio to encapsulate PNA-based therapeutics. PLGAbased delivery systems have been used for a wide variety of therapeutic agents [56], including nucleic acids such as siRNA, miRNA, and PNA alone [45, 57, 58]. Therapeutics encapsulated in PLGA NPs have demonstrated enhanced activity in several disease applications due in part to the protection from cargo degradation, increased biological half-life, and reduced side effects offered by NP encapsulation [59]. Notably, PLGA is a major component in drug delivery devices that have been approved by the FDA; its safety after introduction by a variety of routes of administration is well known, increasing the potential for clinical translation of new therapeutics [60]. In the context of PNA-based gene editing (introduced in Subheading 1.1), PLGA NPs serve as nontoxic and efficient delivery vehicles for PNA oligomers and/or co-reagents (e.g., donor DNA) [9]. PLGA PNA/donor DNA NPs for gene editing applications can be formulated using a water-oil-water double emulsion solvent evaporation technique (Fig. 3). Using this method, PNA and DNA oligomers are usually encapsulated in a 2:1 molar ratio, although this can be adjusted as desired. A detailed protocol is provided in Subheading 3. Briefly, nucleic acids in an aqueous phase are emulsified with polymer dissolved in organic solvent (i.e., the oil phase). Energy in the form of sonication is added to the system to promote the formation of polymer droplets. Next, the first emulsion is emulsified with a surfactant (e.g., poly(vinyl alcohol)) in water and sonicated again. The second emulsion is then diluted into a larger aqueous volume and stirred over several hours

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Fig. 3 Schematic of PLGA PNA/DNA NP formulation protocol and NP characterization

to evaporate the organic solvent. After the hardening phase, NPs are collected and washed by centrifugation at high speeds prior to lyophilization and storage. Following formulation, PLGA NPs are characterized; NP hydrodynamic diameter and surface charge are determined by dynamic light scattering (DLS) and zeta potential measurements, respectively, and NP size and surface morphology are determined using scanning electron microscopy (SEM). PNA/DNA NPs are spherical in morphology with diameters ranging from ~150 to 300 nm and exhibit a negative surface charge (illustrated in Subheading 3.3, Fig. 4). These formulations are biocompatible in vitro and ex vivo and are well-tolerated following in vivo administration. While PLGA-based formulations have many desirable properties for drug delivery, the reproducible formulation of NPs can be challenging, with variety being introduced by equipment, batch variation in reagents, and subtle differences in the method of emulsification. However, the detailed annotated protocol we provide here is designed to support a high degree of reproducibility in PLGA NP formulation.

2

Materials

2.1 Instruments and General Laboratory Equipment

1. 750 W ultrasonic processors with temperature controller (Cole-Parmer®) or comparable model. 2. Sterile 150 mL flat-bottom beaker with stir bar. 3. Sterile 18  150 mm disposable test tubes. 4. Stir plate (Corning) or comparable model. 5. Standard pipettes (P1000, P200, and pipette controller for large volumes). 6. Water bath sonicator (Branson Ultrasonics).

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7. Zetasizer Nano ZS (Malvern Instruments) or comparable model. 8. Disposable square polystyrene cuvettes (Malvern Instruments). 9. Disposable folded capillary cell (Malvern Instruments). 10. Scanning electron microscopy pin stub (Agar Scientific). 11. Carbon conductive Scientific).

double-sided

adhesive

tape

(Agar

12. XL-30 scanning electron microscope (FEI, Hillsboro, Oregon) or similar instrument. 2.2

Chemicals

1. 50:50 poly(DL-lactide-co-glycolide), ester terminated, inherent viscosity 0.55–0.75 (dL/g) (LACTEL absorbable polymers, Birmingham, AL). 2. Poly(vinyl alcohol) (PVA), average molecular 30,000–70,000 (Sigma-Aldrich, St. Louis, MO).

weight

3. Dichloromethane (Sigma-Aldrich, St. Louis, MO). 4. Trehalose (Sigma-Aldrich, St. Louis, MO). 5. 1 TE buffer (10 mM Tris–HCl, 1 mM EDTA, pH 7.4). 6. Quant-iT™ OliGreen™ ssDNA Assay Kit (Invitrogen). 2.3 Surfactant Solutions

1. 5% PVA (w/v): dissolve 5 g of PVA in 100 mL of diH2O. Stir overnight or until PVA is fully dissolved (see Note 1). 2. 0.3% PVA (w/v): dissolve 300 mg of PVA in 100 mL diH2O. Stir overnight or until PVA is fully dissolved (see Note 1).

2.4

Nucleic Acids

1. PNAs can be synthesized using standard solid-phase techniques and purified as previously described [61]. Purified PNAs should be diluted to a 1 mM stock concentration prior to nanoparticle formulation. 2. Donor DNA can be purchased from Midland Certified Reagent Company Inc. (Midland, Texas, U.S.A.) or similar vendor. Donor DNA should be diluted to a 1 mM stock concentration prior to nanoparticle formulation (see Note 2).

3

Methods A detailed graphical representation of the methods below has been described in Fig. 3 to be used as a quick reference when formulating PNA/donor DNA NPs. When formulating NPs containing either PNA or donor DNA alone, follow the protocol below, adding the PNA or donor DNA alone dropwise to the polymer solution.

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3.1 Nanoparticle Formulation

1. Dissolve 50 mg of PLGA in 1 mL of DCM in a 18  150 mm test tube in a chemical fume hood.

3.1.1 Polymer Preparation (Day 1)

2. Cover the top of the tube with aluminum foil and parafilm, tightly securing the foil around the edges. 3. Mark the level of the solvent on the test tube. Allow the polymer to dissolve overnight. If evaporation occurs, add DCM the next day to the previously marked level.

3.1.2 PNA and Donor DNA Encapsulation (Day 2)

1. Heat 100 nmole of PNA (100 μL of 1 mM stock) and 50 nmole of donor DNA (50 μL of 1 mM stock) separately to 65  C for 10 min using a heating block. In this case, the final ratio of PNA/DNA/PLGA starting material will be 100 nmole:50 nmole:50 mg or 2 nmole:1 nmole:1 mg (see Notes 3 and 4). 2. Prepare a work area with a vortex, P1000 pipette, P200 pipette, as well as solvent compatible tips in a chemical fume hood. 3. Pipette 2 mL of 5% PVA into a disposable 18  150 mm test tube. 4. Pipette 25 mL of 0.3% PVA into a 150 mL flat-bottom beaker with a stir bar. Place the beaker on a stir plate and set the stir speed to 360 rpm. 5. Remove the parafilm and aluminum foil covering the PLGA solution. Add DCM to the previously marked level if any has evaporated. 6. While mixing the PLGA solution using a vortex, quickly add and mix the PNA with the donor DNA solution. Add the resulting mixture dropwise to the PLGA. This will form the first water-in-oil emulsion (w/o). 7. Quickly sonicate the solution using an ultrasonic processor set for 10 s with an amplitude of 38%. Pause after each sonication step, and allow the solution to cool on ice for approximately 5 s. Repeat each step two more times for a total of three sonication steps. 8. Mix the 5% PVA solution by vortexing. While vortexing, add the first w/o emulsion to the 5% PVA solution dropwise. 9. Quickly sonicate the resulting w/o/w emulsion as described above (step 7). 10. Directly transfer the final w/o/w emulsion to the 0.3% PVA solution. 1–5 mL of the stirring 0.3% PVA solution may be used to dilute the w/o/w emulsion prior to transfer. 11. Allow the solution to stir for 3 h as the DCM evaporates and NPs harden.

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1. After 3 h, transfer the hardened NPs to a sterile 50 mL Falcon tube. 2. Centrifuge the NPs for 15 min at 16,100  g. 3. Discard the supernatant, and resuspend the NP pellet in 5 mL of water using a water bath sonicator, and vortex until the pellet is fully resuspended. 4. Add 15 mL of diH2O to the resuspended pellet, and centrifuge for 15 min at 16,100  g. 5. Repeat steps 3 and 4 for a total of three centrifugation steps. 6. Following the final centrifugation, discard the supernatant, and resuspend the pellet in 4–5 mL of diH2O. A weight ratio of 1:1 trehalose/PLGA may be added as a cryoprotectant (see Note 5). 7. Transfer the resuspended NPs to 1.7 mL pre-weighed Eppendorf tubes, equally dividing the final volume among 10–15 tubes, depending on NP yield. 8. Flash freeze the NP aliquots with liquid nitrogen for 5 min. 9. Lyophilize the samples for 72 h.

3.2 Nanoparticle Characterization 3.2.1 Nanoparticle Diameter

1. Prepare a 0.05 mg/mL solution of NPs in diH2O, taking care to rigorously resuspend the nanoparticles through water bath sonication and vortex (see Note 6). 2. Load 1 mL of the sample into the square polystyrene cuvette with a pipette, taking extra care to avoid introducing air bubbles. 3. Insert sample and allow temperature to equilibrate for 3 min. 4. Perform three independent size (hydrodynamic diameter) measurements, taking note of the correlation data to ensure measurement stability.

3.2.2 Nanoparticle Zeta Potential

1. Prepare a 0.05 mg/mL solution of NPs in diH2O as above (see Note 6). 2. Load 1 mL of the sample into a disposable folded capillary cell, taking care to avoid introducing bubbles. 3. Insert sample and allow temperature to equilibrate for 2 min. 4. Perform three independent zeta potential measurements.

3.2.3 Nanoparticle Surface Morphology

1. Place double-sided carbon tape on an SEM pin stub (see Note 7). 2. Using a metal or disposable spatula, spread a thin layer of lyophilized NPs across the tape. 3. Sputter coat the sample with gold for 30 s. 4. Image gold-coated NPs using an XL-30 scanning electron microscope (FEI) or similar instrument.

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3.2.4 Nanoparticle Loading

1. Dissolve 2 mg of NPs in 0.5 mL of DCM overnight. 2. Add 0.5 mL of TE buffer to the dissolved NPs. 3. Mix vigorously by vortex and spin the sample at 12,000 rpm at 4  C. 4. Repeat steps 2 and 3 for a total of two volumes of 1 TE buffer. 5. Measure the OD at 260 nm of the combined 1 mL fraction. For quantification of donor DNA alone, use the Quant-iT™ OliGreen™ ssDNA Assay Kit (Invitrogen) according to the manufacturer’s protocol (see Note 8).

3.3 Representative Results

Using the methods above, one can reasonably expect to obtain PLGA NPs loaded with PNA and donor DNA molecules. While diameter measurements may vary depending on the methods used to quantify this parameter, we have found that the diameter of our PLGA PNA/DNA NPs are typically between 250 and 290 nm as measured by DLS. As DLS is a diffusion-based measurement, it is particularly sensitive to the effects of particle concentration, as well as salt concentration used in the buffer [62]. Therefore, while particle concentrations and buffers can be changed to explore parameters, such as size stability over time, it is critical to accurately report nanoparticle concentration and buffer conditions to ensure reproducibility. Figure 4a highlights a typical distribution of nanoparticle sizes as measured by DLS. In this example, NPs were resuspended in diH2O at a concentration of 0.05 mg/mL. Here, the average NP diameter is approximately 280 nm. The zeta potential, or surface charge of NPs, is likewise sensitive to buffer conditions and in particular pH [63]. Therefore, as above, it is critical to carefully report the buffer used to measure zeta potential to ensure reproducibility. Figure 4b highlights a typical distribution of zeta potential values for PLGA PNA/DNA NPs as measured in diH2O at a concentration of 0.05 mg/mL. In this example, the zeta potential of our NPs is approximately 23 mV, which is typical of such preparations. The use of SEM to study NP morphology, though seemingly straightforward, may be complicated by differences in sputter coating methods and in the materials used to coat NPs. In particular, the length of time during which NPs are coated will lead to variations in coating thickness, which can create imaging artifacts or result in poor resolution. Though several options are available for coating NPs, PLGA PNA/DNA NPs have been primarily imaged by SEM following gold-palladium coating. Figure 4c is a typical SEM image of NPs. As seen in the figure, NPs are spherical and homogeneous, with little to no observable surface defects. Loading of PNA and donor DNA is typically measured using absorbance readings at 260 nm, which are normalized to NP mass

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Fig. 4 Typical characteristics of PLGA NPs encapsulating donor DNA and PNA. (a) Dynamic light scattering measurement of NP diameter. (b) NP surface charge as measured by zeta potential. (c) SEM image of NPs. Scale bar is equal to 2 μm. (d) Total nucleic acid loading (PNA and donor DNA molecules) as measured by absorbance at 260 nm

and volume used for extraction (OD/mg/mL) [15]. In recent work, we have also used the Quant-iT™ OliGreen™ ssDNA Assay Kit (Invitrogen) to quantify loading of donor DNA alone. By using this kit, as well as fluorescently labeled PNAs, it is possible to precisely quantify loading of donor DNA and PNA independently, though use of fluorescently tagged PNA may alter loading of the PNA or donor DNA molecules [15]. As in Fig. 4d, typical OD/mg/mL values range between 0.4 and 0.6. So far, we have found that NPs with loading below 0.4 OD/mg/mL do not successfully edit genes in vitro.

4

Notes 1. Dissolving PVA requires vigorous stirring. Use of a stir bar and stir plate set to maximum speed is highly recommended. It is preferable to dissolve PVA slowly overtime, rather than use high heat to accelerate the process. 2. The typical length of donor DNA is 60 nucleotides. 50 and 30 ends can contain three phosphorothioate internucleotide

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linkages to prevent degradation. Donor DNA is purified by reversed-phase high-performance liquid chromatography (HPLC). 3. Typically, PNA is added to donor DNA seconds prior to formation of the first water in oil emulsion. Mixing nucleic acids several minutes ahead of time is not recommended, as PNA/DNA complexes may precipitate out of solution. 4. Typically, the ratio of PNA to donor DNA to starting material of PLGA is kept at 2 nmole PNA:1 nmole DNA:1 mg PLGA. These ratios may be adjusted to further improve PNA and donor DNA loading. 5. The mass of trehalose added to the final NP suspension is based on NP yield, not starting mass of PLGA. Prepare empty NPs to determine typical yields prior to formulating NPs with the addition of cryoprotectant. 6. When characterizing NPs by DLS and zeta potential, it is critical to report NP concentration and buffer selection, as these factors greatly influence the values obtained. While DLS and zeta values are typically reported for NPs after lyophilization, it is generally recommended to perform these measurements prior to drying, as a quality control step. 7. The addition of cryoprotectant may introduce artifacts when imaging by SEM. To avoid this, a small sample of nanoparticles (100 μL) can be separately frozen and lyophilized prior to the third centrifugation step. Alternatively, cryoprotectant can be removed from the final product following three washes with diH2O. The washed NPs can then be air-dried on a glasscovered SEM stub and processed and coated as described. 8. Loading of PNA and donor DNA may vary based on sequence, with typical loadings between 0.4 and 0.6 OD/mg/mL observed in a majority of PLGA NPs.

Acknowledgments This work was supported by the NIGMS Medical Scientist Training Program T32GM07205 (to E.Q.); National Institutes of Health grants R01HL125892, R01AI112443, and UG3HL147352 (to W.M.S. and P.M.G.); and institutional training grant 5T32GM007223-43 (to E.Q.). A.S.P. was supported by NIH National Research Service Awards (NRSAs): T32 (GM86287) training grant and F32 (HL142144) individual postdoctoral fellowship. S.N.O was supported by UG3HL147352 (to P.M.G. and W.M.S.).

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Disclosures/Competing interests: E.Q., A.S.P., W.M.S., and P.M.G. are consultants for Trucode Gene Repair, Inc. W.M.S. and P.M.G also have equity interests in Trucode Gene Repair, Inc. References 1. Rogers FA, Vasquez KM, Egholm M et al (2002) Site-directed recombination via bifunctional PNA–DNA conjugates. Proc Natl Acad Sci U S A 99(26):16695–16700 2. Faruqi AF, Egholm M, Glazer PM (1998) Peptide nucleic acid-targeted mutagenesis of a chromosomal gene in mouse cells. Proc Natl Acad Sci U S A 95(4):1398–1403 3. Rogers FA, Lin SS, Hegan DC et al (2012) Targeted gene modification of hematopoietic progenitor cells in mice following systemic administration of a PNA-peptide conjugate. Mol Ther 20(1):109–118 4. Kim K-H, Nielsen PE, Glazer PM (2006) Sitespecific gene modification by PNAs conjugated to psoralen. Biochemistry 45(1):314–323 5. Kim K-H, Nielsen PE, Glazer PM (2007) Sitedirected gene mutation at mixed sequence targets by psoralen-conjugated pseudo-complementary peptide nucleic acids. Nucleic Acids Res 35(22):7604–7613 6. Chin JY, Kuan JY, Lonkar PS et al (2008) Correction of a splice-site mutation in the beta-globin gene stimulated by triplex-forming peptide nucleic acids. Proc Natl Acad Sci U S A 105(36):13514–13519 7. Lonkar P, Kim K-H, Kuan JY et al (2009) Targeted correction of a thalassemia-associated β-globin mutation induced by pseudocomplementary peptide nucleic acids. Nucleic Acids Res 37(11):3635–3644 8. Schleifman Erica B, Bindra R, Leif J et al (2011) Targeted disruption of the CCR5 gene in human hematopoietic stem cells stimulated by peptide nucleic acids. Chem Biol 18 (9):1189–1198 9. McNeer NA, Chin JY, Schleifman EB et al (2011) Nanoparticles deliver triplex-forming PNAs for site-specific genomic recombination in CD34+ human hematopoietic progenitors. Mol Ther 19(1):172–180 10. McNeer NA, Schleifman EB, Cuthbert A et al (2012) Systemic delivery of triplex-forming PNA and donor DNA by nanoparticles mediates site-specific genome editing of human hematopoietic cells in vivo. Gene Ther 20:658 11. Schleifman EB, McNeer NA, Jackson A et al (2013) Site-specific genome editing in PBMCs with PLGA nanoparticle-delivered PNAs

confers HIV-1 resistance in humanized mice. Mol Ther Nucleic Acids 2:e135 12. Chin JY, Reza F, Glazer PM (2013) Triplexforming peptide nucleic acids induce heritable elevations in gamma-globin expression in hematopoietic progenitor cells. Mol Ther 21 (3):580–587 13. Raman B, Elias Q, Nicole AM et al (2014) Single-stranded γPNAs for in vivo site-specific genome editing via Watson-Crick recognition. Curr Gene Ther 14(5):331–342 14. Fields RJ, Quijano E, McNeer NA et al (2015) Modified poly(lactic-co-glycolic acid) nanoparticles for enhanced cellular uptake and gene editing in the lung. Adv Healthc Mater 4 (3):361–366 15. McNeer NA, Anandalingam K, Fields RJ et al (2015) Nanoparticles that deliver triplexforming peptide nucleic acid molecules correct F508del CFTR in airway epithelium. Nat Commun 6:6952 16. Bahal R, Ali McNeer N, Quijano E et al (2016) In vivo correction of anaemia in β-thalassemic mice by γPNA-mediated gene editing with nanoparticle delivery. Nat Commun 7:13304 17. Ricciardi AS, Bahal R, Farrelly JS et al (2018) In utero nanoparticle delivery for site-specific genome editing. Nat Commun 9(1):2481 18. Quijano E, Bahal R, Ricciardi A et al (2017) Therapeutic peptide nucleic acids: principles, limitations, and opportunities. Yale J Biol Med 90(4):583–598 19. Ricciardi AS, Quijano E, Putman R et al (2018) Peptide nucleic acids as a tool for site-specific gene editing. Molecules 23(3):632 20. Coull J, Deuholm KL, Christensen L et al (1995) Efficient pH-independent sequencespecific DNA binding by pseudoisocytosinecontaining bis-PNA. Nucleic Acids Res 23 (2):217–222 21. Griffith MC, Risen LM, Greig MJ et al (1995) Single and bis peptide nucleic acids as triplexing agents: binding and stoichiometry. J Am Chem Soc 117(2):831–832 22. Bentin T, Larsen HJ, Nielsen PE (2003) Combined triplex/duplex invasion of doublestranded DNA by “tail-clamp” peptide nucleic acid. Biochemistry 42(47):13987–13995

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cloning and characterization of complementary DNA. Science 245(4922):1066–1073 37. Aluigi M, Fogli M, Curti A et al (2006) Nucleofection is an efficient nonviral transfection technique for human bone marrow–derived mesenchymal stem cells. Stem Cells Dev 24(2):454–461 38. Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116 (2):281–297 39. Ha M, Kim VN (2014) Regulation of microRNA biogenesis. Nat Rev Mol Cell Biol 15 (8):509–524 40. Rupaimoole R, Slack FJ (2017) MicroRNA therapeutics: towards a new era for the management of cancer and other diseases. Nat Rev Drug Discov 16(3):203–222 41. Fabani MM, Gait MJ (2008) miR-122 targeting with LNA/20 -O-methyl oligonucleotide mixmers, peptide nucleic acids (PNA), and PNA-peptide conjugates. RNA 14 (2):336–346 42. Oh SY, Ju Y, Park H (2009) A highly effective and long-lasting inhibition of miRNAs with PNA-based antisense oligonucleotides. Mol Cells 28(4):341–345 43. Cheng CJ, Saltzman WM (2012) Polymer nanoparticle-mediated delivery of microRNA inhibition and alternative splicing. Mol Pharm 9(5):1481–1488 44. Xue H, Hua LM, Guo M et al (2014) SHIP1 is targeted by miR-155 in acute myeloid leukemia. Oncol Rep 32(5):2253–2259 45. Babar IA, Cheng CJ, Booth CJ et al (2012) Nanoparticle-based therapy in an in vivo microRNA-155 (miR-155)-dependent mouse model of lymphoma. Proc Natl Acad Sci U S A 109(26):E1695–E1704 46. Fabani MM, Abreu-Goodger C, Williams D et al (2010) Efficient inhibition of miR-155 function in vivo by peptide nucleic acids. Nucleic Acids Res 38(13):4466–4475 47. Cheng CJ, Bahal R, Babar IA et al (2015) MicroRNA silencing for cancer therapy targeted to the tumour microenvironment. Nature 518(7537):107–110 48. Kulshreshtha R, Ferracin M, Wojcik SE et al (2007) A microRNA signature of hypoxia. Mol Cell Biol 27(5):1859–1867 49. Puissegur MP, Mazure NM, Bertero T et al (2011) miR-210 is overexpressed in late stages of lung cancer and mediates mitochondrial alterations associated with modulation of HIF-1 activity. Cell Death Differ 18 (3):465–478 50. Crosby ME, Kulshreshtha R, Ivan M et al (2009) MicroRNA regulation of DNA repair

PLGA PNA Nanoparticle Formulations gene expression in hypoxic stress. Cancer Res 69(3):1221–1229 51. Gupta A, Quijano E, Liu Y et al (2017) Antitumor activity of miniPEG-gamma-modified PNAs to inhibit MicroRNA-210 for cancer therapy. Mol Ther Nucleic Acids 9:111–119 52. Bahal R, Sahu B, Rapireddy S et al (2012) Sequence-unrestricted, Watson-Crick recognition of double helical B-DNA by (R)-miniPEG-gammaPNAs. Chembiochem 13 (1):56–60 53. Seo YE, Suh HW, Bahal R et al (2019) Nanoparticle-mediated intratumoral inhibition of miR-21 for improved survival in glioblastoma. Biomaterials 201:87–98 54. Luo D, Woodrow-Mumford K, Belcheva N et al (1999) Controlled DNA delivery systems. Pharm Res 16(8):1300–1308 55. Saltzman WM (2001) Drug delivery: engineering principles for drug therapy. Oxford University Press, Oxford 56. Kapoor DN, Bhatia A, Kaur R et al (2015) PLGA: a unique polymer for drug delivery. Ther Deliv 6(1):41–58 57. Woodrow KA, Cu Y, Booth CJ et al (2009) Intravaginal gene silencing using biodegradable polymer nanoparticles densely loaded

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Chapter 18 Preparation of Conjugates for Affibody-Based PNA-Mediated Pretargeting Mohamed Altai, Anzhelika Vorobyeva, Vladimir Tolmachev, Amelie Eriksson Karlstro¨m, and Kristina Westerlund Abstract Affibody molecules are small engineered scaffold proteins suitable for in vivo tumor targeting. Radionuclide molecular imaging using directly radiolabelled affibody molecules provides excellent imaging. However, affibody molecules have a high renal reabsorption, which complicates their use for radionuclide therapy. The high renal reabsorption is a common problem for the use of engineered scaffold proteins for radionuclide therapy. Affibody-based PNA-mediated pretargeting reduces dramatically the absorbed dose to the kidneys and makes affibody-based radionuclide therapy possible. This methodology might, hopefully, solve the problem of high renal reabsorption for radionuclide therapy mediated by other engineered scaffold proteins. Key words Affibody molecules, Scaffold protein, PNA, Pretargeting, Peptide synthesis, Sortase A, Lu, 68Ga, 111In, 57Co

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Introduction The concept of targeted radionuclide therapy of cancer is based on molecular recognition of cell-surface proteins, which are aberrantly expressed by malignant cells. Delivery of cytotoxic radionuclides by monoclonal antibodies binding to such abnormalities with a high specificity should provide a lethal absorbed dose to the tumor but spare healthy tissues. Unfortunately, this concept worked so far only for radiosensitive hematologic malignancies, such as lymphomas, but failed to show effect in the case of solid tumors [1]. One of the major obstacles in efficient antibody-based targeted radionuclide therapy is the long residence of immunoglobulins in blood, which causes unacceptably high exposure of normal tissues. Particularly dangerous is the irradiation of a radiosensitive bone marrow [1]. As a possible solution, a pretargeting approach has been suggested (see, e.g., reviews [2–4]). In the pretargeting, recognition of a cancer-associated target and delivery of a cytotoxic radionuclide

Peter E. Nielsen (ed.), Peptide Nucleic Acids: Methods and Protocols, Methods in Molecular Biology, vol. 2105, https://doi.org/10.1007/978-1-0716-0243-0_18, © Springer Science+Business Media, LLC, part of Springer Nature 2020

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Fig. 1 (1) Cancer cells express a tumor-specific molecular target (green). (2) A primary targeting agent containing a target-binding part (blue) and a recognition tag (red) is injected. (3) The primary agent binds to the tumor-associated target and gets cleared from blood. (4) After clearance of the non-bound primary agent from blood, a radiolabelled secondary agent with high affinity to the recognition tag is injected. (5) The radiolabelled secondary agent binds to the recognition tag of the primary agent bound to the cancer cells

are separated in time. In the first step, a cancer-targeting antibody bearing a recognition tag (primary targeting agent) is injected. When the primary agent has been localized in a tumor and cleared from blood and unspecific compartments, a secondary (effector) probe is injected. This secondary probe carries a radionuclide and can bind the recognition tag (Fig. 1). Typically, the secondary probe is selected to have a rapid localization in tumors and to be excreted promptly from circulation. Such an approach enables the combination of the specificity of antibodies to cancer-related antigens with the favorable biodistribution properties of the effector molecules to avoid excessive exposure of the bone marrow. The major mechanisms of interaction of primary and secondary probes are (1) interaction of avidin and biotin; (2) interaction of engineered bispecific antibodies with a cancer-associated target and a radiolabelled hapten; (3) the bioorthogonal inverse electron demand Diels-Alder click reaction between trans-cyclooctene and tetrazine derivatives; and (4) hybridization of complementary oligonucleotides [4]. While interaction of DNA or RNA is highly specific, their oligonucleotide derivatives are rapidly degraded by nucleases in blood. To solve this issue, several nucleic acid analogues have been proposed for the use in vivo, such as L-DNA analogues [5], phosphorodiamidate morpholino oligomers (MORFs) [6], and peptide nucleic acids (PNAs) [7]. In peptide nucleic acid, the deoxyribose phosphate backbone of nucleic acids is replaced by N-(2-aminoethyl)-glycine derivatives [8]. The resulting PNA binds to complementary DNA or RNA with extraordinary strength and specificity [8, 9]. Importantly, PNAs are both nontoxic and nonimmunogenic [10, 11]. The pioneering work performed by Hnatowich and co-workers demonstrated the feasibility of

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PNA-PNA hybridization in vivo [7, 12, 13]. A complete PNA-mediated antibody-based pretargeting was demonstrated by Leonidova and co-workers [14]. During the last decade, a new type of targeting agent based on engineered scaffold proteins (ESPs) and single domain antibodies (sdAbs) was actively evaluated for molecular imaging [15]. These targeting agents are appreciably smaller than antibodies (molecular weight in the range of 4–20 kDa compared to 150 kDa for IgG antibodies), which offers advantages of much higher extravasation rate. Unbound agents pass readily the glomerular membranes, which results in a fast clearance from blood and in low background levels. Clinical studies demonstrated that both sdAbs and an ESP called affibody molecule enabled high-contrast imaging within 4 h after injection [15]. However, ESPs undergo an efficient renal reabsorption after glomerular filtration. In the case of residualizing radiometal labels, the reabsorbed activity is retained in the kidneys. Such promising therapeutic radionuclides as 177Lu, 161Tb, 225Ac, and 227Th cannot be used for ESP-mediated targeting because the renal absorbed dose would be higher than the tumor dose. To avoid the issue, we have evaluated the feasibility of using the pretargeting approach for radionuclide therapy using affibody molecules [16, 17]. Affibody molecules are small (7 kDa) ESPs, which are based on the scaffold of the domain B of the protein A. A number of affibody molecules binding to cancer-associated targets with affinity in the range from pM to nM have been selected [18]. Two features of affibody molecules make them attractive primary agents for pretargeting: (1) internalization of the majority of affibody molecules after binding to cancer-expressing cells is slow, and (2) clearance of unbound affibody molecules is rapid. The primary HP1 and secondary HP2 15-mer probes were designed to provide high specificity and high melting point and to avoid self-complementarity [19] (Fig. 2). The GGG tag was included at N-terminus of the HP1 probe for sortase A-mediated ligation to an affibody molecule. Sortase A is a cysteine transpeptidase first isolated from the bacterium Staphylococcus aureus in which it is responsible for covalently anchoring proteins to the cell wall. Recombinant variants of the enzyme, lacking the N-terminal membrane-anchoring domain, have become important and versatile tools used in biotechnology for covalent and site-specific peptidepeptide conjugation. Several mutants of sortase A with enhanced kinetics and activity have been generated which require lower amounts of enzyme and shorter reaction times than WT sortase A (reviewed in [20]). One such improved variant, P94S/D160N/ K196T-sortase A or sortase A3∗ [21], was used to create an affibody-PNA chimera capable of binding both to the HER2 receptor and to the secondary effector probe. A DOTA chelator was introduced into HP1 to enable labelling with radiometals and facilitate pharmacokinetics studies. The effector probe, HP2, was

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Fig. 2 Hybridization probes (a) HP1 and (b) HP2. The probes are synthesized with C-terminal amide modifications, as indicated in the figure. Amino acids are shown in upper case one letter code in bold (G, S, E, and Y), while the PNA bases are shown as lower case letters (a, c, g, and t)

dually functionalized with a DOTA chelator for radiometal complexing and a tyrosine for direct radioiodination [19]. The probes formed a structured duplex with a very high melting temperature (Tm ¼ 86–88  C). The ZHER2:342-SR-HP1 chimera bound to HER2 with an affinity of 212 pM. Feasibility of affibody molecule-based PNA-mediated pretargeting has been demonstrated using the anti-HER2 affibody molecule [17]. We have shown that radiolabelled ZHER2:342-SR-HP1 chimera accumulated in tumor xenografts in mice in a HER2specific manner and that the tumor uptake of 125I- and 111Inlabelled HP2 is HP1-mediated. Further, the labelling of HP2 with the beta-emitting therapeutic radionuclide 177Lu has been optimized, and the biodistribution of 177Lu-HP2 has been evaluated in mice [22]. An interesting observation was that the biodistribution of HP2 depends on the chemical nature of the radiometal label. 177Lu-HP2 had more rapid blood clearance and had lower uptake in many tissues compared with 111In-HP2. In addition, the renal uptake of 177Lu-HP2 could be further reduced by co-injection of lysine or Gelofusine. In the following preclinical study [23], the timing and dosing of both primary ZHER2:342-SRHP1 and 177Lu-HP2 injections were optimized to provide an estimated absorbed dose to tumor exceeding the dose to the kidneys more than fivefold. Experimental therapy demonstrated that

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ZHER2:342-SR-HP1-based 177Lu-HP2-mediated treatment extends significantly ( p < 0.05) the median survival of treated mice bearing HER2-expressing SKOV-3 xenografts (66 days) compared to control animals treated with the same amount of phosphate-buffered saline (37 days) and the same amount and activity of 177Lu-HP2 only (32 days) or ZHER2:342-SR-HP1 only (37 days). We have evaluated if the labelling of ZHER2:342-SR-HP1 and HP2 with the generator-produced positron-emitting radionuclide 68Ga would enable assessment of HER2 expression and 177Lu-HP2 accumulation in tumors using positron-emission tomography (PET) [24]. The use of both 68Ga-ZHER2:342-SR-HP1 and 68Ga-HP2 provided clear discrimination between tumors with high and low HER2 expression in mice. However, the tumor uptake of 68GaHP2 correlated better with the uptake of 177Lu-HP2 making the 68 Ga-HP2 the preferable theranostics counterpart. Our pretargeting system has a modular design and includes the following: l

l

l

Recombinantly or synthetically produced target-specific affibody molecule carrying an LPXTG sequence for fusion with HP1 using the sortase A enzyme. The use of sortase A enables in principle fusion of HP1 with any ESP or dsAb engineered with the LPXTG sequence. Primary hybridization probe HP1 carrying a GGG sequence for the sortase A-mediated fusion with the targeting moiety. A versatile DOTA chelator is incorporated into HP1, which enables labelling of the chimera with a variety of imaging radionuclides (e.g., 57Co,111In, or 68Ga). This might be used for development of companion diagnostics or optimization of the pretargeting process. DOTA chelator which enables labelling of the HP2 probe with a variety of promising therapeutic radionuclides, such as highenergy beta emitter 90Y, low-energy beta emitters 177Lu and 161 Tb, or alpha emitters 212Bi, 225Ac, and 227Th. Incorporation of tyrosine into the secondary HP2 probe enables its labelling with the low-energy beta emitter 131I for therapy, single-photon emitter 123I, or positron emitter 124I for single photon emission computed tomography (SPECT) or PET imaging, respectively, and with the long-lived 125I for preclinical development.

We hope that the design of this pretargeting system enables its application with a variety of targeting ESPs and enables selection of an optimal radionuclide depending on the tumor size, growth rate, or retention of primary probe in tumors. This chapter contains descriptions of general solid-phase synthesis of the PNA-based hybridization probes; HP1 and HP2 and their conjugation to chelators; sortase A-mediated affibody-PNA ligation; purification of hybridization probes; labelling of probes with 111In, 57Co,

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Lu, and 68Ga; and evaluation of in vitro pretargeting specificity test (see Note 1). A brief description of expression and purification of affibody molecules and sortase A3∗ is also provided.

2

Materials

2.1 Manual Solid-Phase Synthesis of the PNA-Based Hybridization Probes, HP1 and HP2

1. Fmoc/Bhoc-protected PNA monomers (PolyOrg Inc., USA). 2. Linker: Fmoc-8-amino-3,6-dioxaoctanoic (Sigma-Aldrich).

acid

(AEEA)

3. Chelator: DOTA-tri(tBu)-ester (CheMatech, France). 4. Fmoc-protected amino acid monomers (Iris Biotech and Novabiochem). 5. Fmoc-Lys(Mtt)-OH (Iris Biotech). 6. Activator: benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) (Sigma-Aldrich). 7. Synthesis resin: Rink amide ChemMatrix resin; loading, 0.54 mmol/g (Biotage, Sweden). 8. Dichloromethane (DCM). 9. N,N-Dimethylformamide (DMF). 10. N-Methyl-2-pyrrolidone (NMP). (a) N,N-Diisopropylethylamine (DIEA). 11. 2,6-Lutidine (Sigma-Aldrich). 12. Acetic anhydride (Fluka). 13. Piperidine (Sigma-Aldrich). 14. Trifluoroacetic acid (TFA) (Alfa Aesar). 15. Scavenger: triisopropylsilane (TIS), (Sigma-Aldrich). 16. Diethyl ether (Merck). 17. 98% ethanol (EtOH). 18. Kaiser test solutions: (a) Reagent A: mix 40 g phenol in 10 mL warm absolute EtOH. Dissolve 65 mg KCN in 100 mL water. Dilute 2 mL of the KCN solution in 100 mL pyridine. Mix the phenol and the KCN/pyridine solutions. (b) Reagent B: 5% (w/v) ninhydrin in EtOH. 19. Base solution amino acid coupling: 1.6 M DIEA in NMP. 20. Base solution PNA coupling: 0.2 M 2,6-lutidine + 0.2 M DIEA in NMP. 21. Fmoc deprotection solution: 20% piperidine in DMF. 22. Capping (5:6:89).

solution:

acetic

anhydride/2,6-lutidine/NMP

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23. 4-Methyltrityl (Mtt) deprotection solution: TFA/TIS/DCM (1:2:97). 24. Cleavage cocktail: TFA/TIS/Milli-Q water (95:2.5:2.5) . 25. Diethyl ether/Milli-Q water (50:50). 2.2 Sortase A-Mediated Ligation

1. Lyophilized ZHER2:342-SR-H6 protein (affibody with a C-terminal sortase A motif). 2. Lyophilized HP1 (GGG-PNA), synthesis crude. 3. Sortase A storage buffer: 50 mM Tris base, 150 mM NaCl, 10% glycerol, pH 7.5. 4. Sortase A3∗ (500 μM stock solution stored at 80  C in sortase A storage buffer. Thaw on ice. 5. 10% DMSO in Milli-Q water. 6. 10 sortase A ligation buffer: 500 mM Tris base, 1.5 M NaCl, 100 mM CaCl2, pH 7.5. 7. 10 mM Ni(II) acetate in Milli-Q water. 8. Immobilized metal-ion affinity chromatography (IMAC) resin: TALON metal affinity resin (Takara, USA). 9. IMAC binding buffer without imidazole: 25 mM NaH2PO4, 150 mM NaCl, pH 7. 10. 15 mL polypropylene centrifuge tubes. 11. Disposable polypropylene columns with porous frits for gravity flow applications (e.g., empty PD-10 columns; GE Healthcare).

2.3 Final RP-HPLC Purification of HP2 and ZHER2:342-SR-HP1

HP2 (DOTA-PNA) and ZHER2:342-SR-HP1 (affibody-PNA chimera) are purified on a semi-preparative Zorbax C18 RP-HPLC column (300SB-C18, 9.4  250 mm2, 5 μm pore size; Agilent) on an Agilent 1200 system equipped with a diode array detector. An elevated column temperature of 70  C is used for improved separation, and the detector is set to monitoring absorbance at 220, 260, and 280 nm. 1. A buffer: 0.1% TFA in Milli-Q water. 2. B buffer: 0.1% TFA in acetonitrile (CH3CN).

2.4 Preparation of Solutions for Labelling

1. Ion-exchange resin Chelex 100 in sodium form (Sigma). 2. Disposable 0.4 μm filters. 3. Disposable syringes. 4. Disposable polypropylene 20 mL vials. 5. Freeze-dried DOTA-PNA conjugate. 6. Ammonium acetate. 7. Ascorbic acid.

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8. Sodium hydroxide. 9. 30% ultrapure HCl (Merck, 1.01514.02509). 10. Ultrapure water (Fluka Analytical, 95305-500ML). 11. Deionized water for HPLC. 12. TFA (trifluoroacetic acid). 13. CH3CN (acetonitrile). 2.5 Labelling of DOTA-PNA with 111In or 57Co

1. 0.2 M ammonium acetate, pH 5.5 (stored over Chelex 100). 2.

111

In chloride in 0.05 M hydrochloric acid or 57Co chloride in 0.05 M hydrochloric acid.

3. DOTA-PNA conjugate. 4. Dose calibrator for measuring activities in a range of 1 MBq– 10 GBq. 5. Siliconized Eppendorf tubes (1.7 mL). 6. Instant thin layer chromatography (ITLC) strip (e.g., Tec-Control Chromatography 150-771 strips, Biodex, Shirley, NY, USA, or ITLC-SG chromatography paper, Agilent, Santa Clara, CA, USA). 7. ITLC chamber. 8. 0.2 M citric acid as mobile phase for ITLC chromatography. 9. PhosphorImager or TLC scanner or γ-spectrometer for measurement of radioactivity distribution along ITLC strip. 10. 2% BSA (bovine serum albumin) in PBS solution. 2.6 Labelling of DOTA-PNA with 177Lu

1. 1 M ascorbic acid, pH 5.5 (stored over Chelex 100). 2.

177

Lu chloride in 0.05 M hydrochloric acid.

3. DOTA-PNA conjugate. 4. Dose calibrator for measuring activities in a range of 1 MBq– 10 GBq. 5. Siliconized Eppendorf tubes (1.7 mL). 6. Instant thin layer chromatography (ITLC) strip (e.g., Tec-Control Chromatography 150-771 strips, Biodex, Shirley, NY, USA, or ITLC-SG chromatography paper, Agilent, Santa Clara, CA, USA). 7. ITLC chamber. 8. 0.2 M citric acid as mobile phase for ITLC chromatography. 9. PhosphorImager or TLC scanner or γ-spectrometer for measurement of radioactivity distribution along ITLC strip. 10. 2% BSA (bovine serum albumin) in PBS solution.

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2.7 Labelling of DOTA-PNA with 68Ga

1.

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Ge/68Ga generator (e.g., from Eckert & Ziegler Strahlenund Medizintechnik AG, Berlin, Germany, or Cyclotron Co., Obninsk, Russia).

2. 0.1 M HCl. 3. NAP-5 column (GE Healthcare). 4. 1% BSA (bovine serum albumin) in PBS solution. 5. PBS. 6. 1.25 M sodium acetate buffer, pH 3.6 (stored over Chelex 100). 7. DOTA-PNA conjugate. 8. Dose calibrator for measuring activities in a range of 1 MBq– 10 GBq. 9. Siliconized Eppendorf tubes (1.7 mL). 10. Tetrasodium (Na4EDTA).

salt

of

ethylenediaminetetraacetic

acid

11. Instant thin layer chromatography (ITLC) strip (e.g., Tec-Control Chromatography 150-771 strips, Biodex, Shirley, NY, USA, or ITLC-SG chromatography paper, Agilent, Santa Clara, CA, USA). 12. ITLC chamber. 13. 0.2 M citric acid as mobile phase for ITLC chromatography. 14. PhosphorImager or TLC scanner or γ-spectrometer for measurement of radioactivity distribution along ITLC strip. 2.8 Labelling of DOTA-Affibody-PNA Conjugate with 68Ga

1.

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Ge/68Ga generator (e.g., from Eckert & Ziegler Strahlenund Medizintechnik AG, Berlin, Germany, or Cyclotron Co., Obninsk, Russia).

2. 0.1 M HCl. 3. NAP-5 column (GE Healthcare). 4. 1% BSA (bovine serum albumin) in PBS solution. 5. PBS. 6. 1.25 M sodium acetate buffer, pH 3.6 (stored over Chelex 100). 7. DOTA-affibody-PNA conjugate. 8. Siliconized Eppendorf tubes (1.7 mL). 9. Tetrasodium (Na4EDTA).

salt

of

ethylenediaminetetraacetic

acid

10. Instant thin layer chromatography (ITLC) strip (e.g., Tec-Control Chromatography 150-771 strips, Biodex, Shirley, NY, USA, or ITLC-SG chromatography paper, Agilent, Santa Clara, CA, USA).

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11. ITLC chamber. 12. 0.2 M citric acid as mobile phase for ITLC chromatography. 13. PhosphorImager or TLC scanner or γ-spectrometer for measurement of radioactivity distribution along ITLC strip. 2.9 Quality Control Using HPLC

1. HPLC system equipped with a radioactivity detector. 2. Semi-preparative Zorbax C18 column 9.4  250 mm2, 5 μm pore size; Agilent).

(300SB-C18,

3. HPLC buffer A: 0.1% TFA in water. 4. HPLC buffer B: 0.1% TFA in CH3CN/water (80:20). 2.10 Evaluation of Pretargeting In Vitro Specificity

1. Cells (adherent) expressing the studied target. 2. Complete cell culture media for cell incubation (according to supplier’s recommendation). 3. Serum-free media. 4. Disposable cell culture dishes (diameter 3 cm). 5. Cell counter. 6. Humidified cell incubator providing 5% CO2 in atmosphere. 7. Trypsin-EDTA solution (0.25% trypsin, 0.02% EDTA in buffer) or other appropriate solution for cell detachment. 8. Primary probe (affibody molecules, mAb, or scaffold protein) conjugated to a PNA hybridization probe. This is called the primary targeting agent. 9. Radiolabelled complementary PNA hybridization probe. This is called the secondary agent. 10. Blocking agent (agent binding to the same epitope as the primary agent). It might be a non-labelled primary agent or its parental targeting vector. 11. Test tubes (maximum volume 3 mL). 12. γ-Spectrometer for activity measurement.

3

Methods

3.1 Expression and Purification of Recombinant Proteins

1. A detailed description on the construction of the plasmids coding for ZHER2:342-SR-H6 and sortase A3∗ can be found elsewhere [19, 22]. The synthetic genes are cloned into pET-derived expression vectors with different antibiotic resistance, kanamycin and ampicillin for ZHER2:342-SR-H6 and sortase A3∗, respectively, and transformed into BL21 Star (DE3) chemically competent E. coli cells.

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2. Sortase A3∗ and ZHER2:342-SR-H6 are expressed in E. coli and purified under native conditions using standard protocols for His-tagged proteins [19, 25, 26]. 3. Sortase A3∗ is concentrated to 500 μM in sortase A storage buffer using an Amicon Ultra concentrator with a 10 K molecular weight cutoff (Amicon Ultra-15, Merck Millipore, Ireland) and stored in 250 μL aliquots at 80  C (see Note 2). 4. The ZHER2:342-SR-H6 pool after IMAC purification is buffer changed to 10 mM sodium acetate, pH 4.6, using PD-10 desalting columns, and is then lyophilized and stored at 20  C. 3.2 General Solid-Phase Synthesis of the PNA-Based Hybridization Probes, HP1 and HP2

1. General amino acid coupling step: dissolve 10 equiv of PyBOP in 1–2 mL of DMF in a 15 mL polypropylene centrifuge tube. Add 10 equiv of the Fmoc-protected amino acid and 1–2 mL of DCM. Vortex the solution, and make sure everything is dissolved before adding 10 equiv of the amino acid base. Add the amino acid/activator solution to the resin. Let the coupling reaction go on for at least 40 min at RT with gentle agitation on an orbital shaker. 2. Coupling of AEEA linker or DOTA: dissolve 5 equiv of PyBOP in 1–2 mL of DMF in a 15 mL polypropylene centrifuge tube. Add 5 equiv of Fmoc-8-amino-3,6-dioxaoctanoic acid or DOTA-tri(tBu)-ester and 1–2 mL of DCM. Vortex the solution, and make sure everything is dissolved before adding 5 equiv of the amino acid base. Add the solution to the resin. Let the coupling reaction go on for at least 40 min at RT with gentle agitation on an orbital shaker. 3. General PNA monomer coupling step: dissolve 5 equiv of PyBOP in 1–2 mL of DMF. Add 5 equiv of the Fmoc-PNAOH monomer and 1–2 mL DCM, and vortex the solution until it is completely dissolved (see Note 3). 4. Add 5 equiv of the base for PNA couplings to the PyBOP/ monomer solution and pre-activate the PNA monomer for at least 1 min at room temperature before adding the solution to the resin. Let the coupling reaction go on for at least 40 min at RT with agitation. 5. Qualitative Kaiser test: take 10–15 lightly dried resin beads in a test tube, and add 2–3 drops each of reagent A and B. Mix by swirling and heat at 98  C for 2–4 min. Positive test ¼ blue/ purple beads, indicative of free amines on the beads. Negative test ¼ pale yellow beads.

3.3 Synthesis of HP1 (GGG-PNA)

1. Weigh 0.05 mmol (93 mg) of Rink amide ChemMatrix resin in a 10 mL fritted synthesis vessel, and allow the resin to swell in 2–3 mL of DCM under agitation for 30 min or overnight.

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2. Couple Fmoc-Glu(OtBu)-OH to the resin as described in Subheading 3.2, step 1. 3. After coupling, drain the solution by suction, and wash the resin for 30 s with 2 mL each of (a) EtOH, (b) NMP, (c) 5% DIEA in DCM, (d) NMP, and (e) DCM  3. 4. Monitor the completion of the reaction by performing a qualitative Kaiser test as described in Subheading 3.2, step 4. If the Kaiser test is positive, redo the monomer coupling step. If the test is negative, continue to the next step. 5. Add 2–3 mL capping solution to the peptide-resin, and incubate under agitation for 5 min  2. 6. Wash the peptide-resin with 2 mL of DCM  4. 7. Add 2–3 mL of the Fmoc deprotection solution to the resin, and let the solution react under agitation for 10 min  2. 8. Wash the resin with 2–3 mL of (a) DCM  3, (b) EtOH, (c) DMC  2, and (d) DCM  3. 9. Perform a qualitative Kaiser test. The test should be positive after Fmoc deprotection. 10. Couple the linker molecule AEEA (as Fmoc-8-amino-3,6dioxaoctanoic acid) to the peptide-resin as described in Subheading 3.2, step 2. Cap, wash, and deprotect the resin as described in steps 3–9. 11. Couple Fmoc-Lys(Mtt)-OH to the peptide-resin as described in Subheading 3.2, step 1. Cap and wash the resin as described in steps 3–6. 12. Remove the acid-labile Mtt group from the lysine residue by adding 3 mL of the Mtt deprotection solution to the resin. Leave to react for 2 min  10. 13. Wash the resin with 2 mL each of (a) 5% (v/v) DIEA in DCM  2 and (b) DCM  3. 14. Perform a qualitative Kaiser test. If the test is negative, redo steps 12 and 13. If the test is positive, continue to the next step. 15. Couple DOTA (as DOTA-tri(tBu)-ester) to the lysine side chain as described in Subheading 3.2, step 2. Cap, wash, and deprotect the resin as described in steps 3–9. 16. Couple Fmoc-Glu(OtBu)-OH to the resin as described in Subheading 3.2, step 1. Cap, wash, and deprotect the resin as described in steps 3–9. 17. Assemble the 15-mer PNA part (a-g-t-c-t-g-g-a-t-g-t-a-g-t-c). Couple each PNA monomer (Fmoc-PNA-A(Bhoc)-OH/ Fmoc-PNA-C(Bhoc)-OH/Fmoc-PNA-G(Bhoc)-OH or

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Fmoc-PNA-T-OH) sequentially as described in Subheading 3.2, step 3. Cap, wash, and deprotect the resin as described in steps 3–9 after each coupling step. 18. Complete the synthesis of HP1 by adding the N-terminal peptide part (G-G-G-S-S-) to the PNA-peptide chimera. Couple each amino acid monomer (Fmoc-Gly-OH or Fmoc-Ser (tBu)-OH) as described in Subheading 3.2, step 1. Cap, wash, and deprotect the resin as described in steps 3–9 after each coupling step. 3.4 Synthesis of HP2 (DOTA-PNA)

1. HP2 is synthesized on the same scale, on the same resin, and using the same coupling protocols as for HP1. 2. Couple the C-terminal peptide part (E-E-Y) to the resin. Each amino acid monomer (Fmoc-Tyr(tBu)-OH or Fmoc-Glu (OtBu)-OH) is coupled sequentially as described in Subheading 3.2, step 1. Cap, wash, and deprotect the resin as described in Subheading 3.3, steps 3–9, after each coupling step. 3. Assemble the PNA part of the molecule (g-a-c-t-a-c-a-t-c-c-ag-a-c-t), complementary to the PNA part of HP1. Couple each PNA monomer (Fmoc-PNA-A(Bhoc)-OH/Fmoc-PNA-C (Bhoc)-OH/Fmoc-PNA-G(Bhoc)-OH or Fmoc-PNA-TOH) sequentially as described in Subheading 3.2, step 3. Cap, wash, and deprotect the resin as described in Subheading 3.3, steps 3–9, after each coupling step. 4. Couple two serines (as Fmoc-Ser(tBu)-OH) sequentially as described in Subheading 3.2, step 1. Cap, wash, and deprotect the resin as described in Subheading 3.3, steps 3–9, after each coupling step. 5. Couple the linker molecule AEEA (as Fmoc-8-amino-3,6dioxaoctanoic acid) to the PNA-peptide chimera as described in Subheading 3.2, step 2. Cap, wash, and deprotect the resin as described in Subheading 3.3, steps 3–9, after each coupling step. 6. Complete the HP2 synthesis by adding DOTA (as DOTA-tri (tBu)-ester) to the N-terminal amine group as described in Subheading 3.2, step 2. Cap and wash the resin as described in Subheading 3.3, steps 3–6.

3.5 Cleavage of the PNA-Based Hybridization Probes

1. Allow the resin with the PNA-peptide chimera to air-dry at room temperature for at least 12 h in a 15 mL polypropylene centrifuge tube covered by aluminum foil. 2. Add 4 mL of the cleavage cocktail to the peptide-resin and incubate for 3 h under gentle agitation on an orbital shaker. 3. Carefully pipette the cleavage mixture into 40 mL of diethyl ether/Milli-Q water (50:50) in a 50 mL centrifuge tube. Close the lid.

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4. Shake the tube to mix the two phases, and open the lid slowly to ventilate. Wait for the phases to separate. 5. Decant off the ether phase and add 20 mL of new diethyl ether. Repeat the extraction twice. 6. Filter the water phase through a glass pipette with a bottom glass wool plug to remove the resin beads. 7. Freeze-dry the filtrate. 3.6 Sortase A-Mediated Affibody-PNA Ligation

1. Dilute the 10 sortase A ligation buffer to 1 in Milli-Q water. 2. Reconstitute the ZHER2:342-SR-H6 lyophilisate in 1 sortase A ligation buffer to a final concentration of 500 μM. 3. Reconstitute the HP1 lyophilisate in 10% DMSO in Milli-Q water to a final concentration of 500 μM. 4. Add 500 nmol of HP1 (1 mL of a 500 μM stock solution) to a 15 mL polypropylene centrifuge tube. 5. Add 100 μL 10 sortase A ligation buffer. 6. Add 1.25 μmol ZHER2:342-SR-H6 (2.5-fold molar excess over HP1; 2.5 mL of a 500 μM stock solution). 7. Adjust the volume to 5 mL with 1 sortase A ligation buffer, lowering the final DMSO concentration to 2%. 8. Add 2.5 μmol nickel(II) acetate (twofold molar excess over ZHER2:342-SR-H6; 40 μL of 10 mM stock solution) (see Note 4). 9. Check the pH of the solution and adjust to 7.5 with 50 mM NaOH if necessary. 10. Start the enzymatic reaction by adding sortase A3∗ to the reaction mixture at a final concentration of 5 μM (50 μL from a 500 μM sortase A3∗ stock). 11. Let the enzymatic reaction proceed at 37  C for 30 min on a rotating tube shaker. 12. Add 2 mL of IMAC resin to an empty gravity flow column and equilibrate the resin by letting 20 mL of IMAC binding buffer without imidazole pass-through. 13. Add the IMAC resin to the reaction mixture at the end of the 30-min reaction time, and leave at room temperature for 20 min on a rotating tube shaker. 14. Pour the resin/buffer slurry onto an empty gravity flow column. Make sure the end cap is securely fastened. 15. Remove the end cap, and collect the flow-through in 500 μL fractions in 1.5 mL microcentrifuge tubes (see Note 5). 16. Wash the resin by adding another 5 mL of IMAC binding buffer without imidazole, and continue to fractionate the flow-through.

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17. Measure the absorbance at 260 nm of all collected fractions, and pool those with active absorbance. 18. Change the buffer to 10 mM sodium acetate, pH 4.6, using PD-10 desalting columns. 19. Lyophilize the sample. 3.7 Purification of HP2

1. Reconstitute lyophilized HP2 (synthesis crude) in 10% CH3CN + 0.1% TFA in Milli-Q water, and pass the solution through a 0.45 μm PVDF syringe filter. 2. Load the sample on a semi-preparative Zorbax C18 column. 3. Elute with a linear gradient going from 10% to 25% B in A in 17.5 min (10% B in 2.5 min and 10–25% B in 2.5–17.5 min) and a flow rate of 3 mL/min. 4. Collect fractions absorbing at 260 nm; the retention time of HP2 is approximately 11 min. 5. Analyze fractions with matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectroscopy, and pool those that contain HP2. 6. Lyophilize the HP2 pool. 7. Reconstitute the lyophilisate in 10% DMSO in Milli-Q water. 8. Change the buffer to 10 mM sodium acetate, pH 4.6, using PD-10 desalting columns (see Note 6). 9. Divide HP2 into 50 μg aliquots in 1.5 mL microcentrifuge tubes, and lyophilize.

3.8 Purification of ZHER2:342-SR-HP1

1. Reconstitute lyophilized flow-through from the sortase Amediated ligation (Subheading 3.6, step 19) in 10% CH3CN + 0.1% TFA in Milli-Q water, and pass the solution through a 0.45 μm PVDF syringe filter. 2. Load the sample on a semi-preparative Zorbax C18 column. 3. Elute with an elution gradient going from 10% to 37% B in A in 32 min (10% B for 2.5 min, 10–20% B in 2.5–12.5 min, 20–30% B in 12.5–17.5 min, and 30–37% B in 17.5–32 min) and a flow rate of 3 mL/min. 4. Collect fractions absorbing at 260 nm; retention times of HP1 and ZHER2:342-SR-HP1 are approximately 8 and 26 min, respectively. 5. Analyze fractions with MALDI-TOF, and pool those that contain HP1 and ZHER2:342-SR-HP1 separately. 6. Lyophilize HP1 and ZHER2:342-SR-HP1. 7. Reconstitute the ZHER2:342-SR-HP1 lyophilisate in PBS. 8. Change the buffer to 10 mM sodium acetate, pH 4.6, using PD-10 desalting columns.

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9. Divide ZHER2:342-SR-HP1 into 50 μg aliquots in 1.5 mL microcentrifuge tubes and lyophilize. 10. Uncoupled lyophilized HP1 can be reconstituted in 10% DMSO in Milli-Q water and be reused for sortase A-mediated ligation. 3.9 Preparation of Buffers and Solution for Labelling

1. Prepare 0.2 M ammonium acetate buffer, pH 5.5 (for labelling with 111In or 57Co), 1 M ascorbic acid, pH 5.5 (for labelling with 177Lu), and/or 1.25 M sodium acetate buffer, pH 3.6 (for labelling with 68Ga), using a high-quality water and pure reagents. 2. Add Chelex 100 to prepared buffers (10 g/L of buffer), mix carefully, and let sit overnight. 3. Immediately before use, filter the buffer through a 0.4 μm filter into a disposable polypropylene vial. Use first 5 mL to rinse vials. 4. Prepare Na4EDTA solution in 0.625 M sodium acetate, pH 3.6.

3.10 Labelling of DOTA-PNA with 111In or 57Co

1. Calculate the volume of 111In or 57Co from stock solution required to provide approximately 15 MBq. 2. Add the volume of 111In or 57Co to an aliquot of DOTA-PNA conjugate (50 μg, 9.7 nmol) dissolved in 100 μL of 0.2 M ammonium acetate, pH 5.5. 3. Vortex the mixture carefully and incubate at 90  C for 30 min. 4. Remove the reaction vial from heating block and allow to cool for 2–3 min. 5. To evaluate purity of the radiolabelled PNA, take 1 μL sample, and place on ITLC strip (e.g., Tec-Control Chromatography 150-771 strip). Elute the strip with 0.2 M citric acid. 6. The radiolabelled PNA would stay at the application point, while free 111In/57Co would migrate with the solvent front. It is possible to quantitatively assess radiochemical yield and purity using PhosphorImager or TLC scanner. Alternatively a γ-spectrometer could be used. Simply cut the Tec-Control Chromatography 150-771 strip in the middle. After measurement of background (B), measure radioactivity of half A (with the application point) and half F (with the solvent front). Calculate the purity according to the following formula: %Yield ¼ ðA  BÞ∗100=ðA  BÞ þ ðF  BÞ ðsee Note 7Þ: 7. Dilute the radiolabelled PNA with 2% BSA in PBS solution, and store at 20  C if not used directly.

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3.11 Labelling of DOTA-PNA with 177Lu

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1. Calculate a volume of 177Lu stock solution required to provide 4.8 MBq/μg of DOTA-PNA. 2. Add the required volume of 177Lu to an aliquot of DOTA-PNA conjugate (50 μg, 9.7 nmol) in 100 μL of 1 M ascorbic acid, pH 5.5. 3. Vortex the mixture carefully and incubate at 95  C for 1 h. 4. Take 1 μL sample and place on Tec-Control Chromatography 150-771 strip. Elute the strip with 0.2 M citric acid. 5. Evaluate purity of 177Lu-radiolabelled DOTA-PNA as described in Subheading 3.10, steps 5 and 6. The radiolabelled PNA would stay at the application point, while free 177Lu would migrate with the solvent front (see Note 7). 6. Dilute the radiolabelled PNA with 2% BSA in PBS solution to minimize the effect of hydrolysis.

3.12 Labelling of DOTA-PNA with 68Ga

1. Pre-equilibrate a NAP-5 column with 1% BSA in PBS by passing at least 10 mL of the buffer through the column immediately before labelling. 2. Reconstitute the lyophilized DOTA-PNA (50 μg, 9.7 nmol) in 50 μL of 1.25 M sodium acetate buffer, pH 3.6. 3. Elute the generator with 0.1 M HCl collecting the eluate in 400 μL fractions. Measure activity of fraction and select one containing the maximum activity for labelling. 4. Add 350 μL of 68Ga-containing eluate (180–220 MBq) to the DOTA-PNA solution. 5. Vortex the mixture carefully and incubate at 95  C for 15 min. 6. Add Na4EDTA (4 mg, 10 μmol, 100 μL of 40 mg/mL 1.25 M sodium acetate, pH 3.6), vortex the mixture carefully, and incubate at 95  C for 5 min. 7. Load the reaction mixture on the column. Let it pass through the upper filter. Collect and discard the eluate. 8. Add 1000 μL of PBS to the column; collect the eluate. This fraction will contain 68Ga-DOTA-PNA. 9. Evaluate purity of 68Ga-DOTA-PNA as described in Subheading 3.10, steps 5 and 6. The radiolabelled PNA would stay at the application point, while free 68Ga would migrate with the solvent front.

3.13 Labelling of DOTA-Affibody-PNA with 68Ga

1. Pre-equilibrate a NAP-5 column with 1% BSA in PBS by passing at least 10 mL of the buffer through the column immediately before labelling. 2. Reconstitute the lyophilized DOTA-affibody-PNA (50 μg, 3.80 nmol) in 50 μL of 1.25 M sodium acetate buffer, pH 3.6.

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3. Elute the generator with 0.1 M HCl collecting the eluate in 400 μL fractions. Measure activity of fraction and select one containing the maximum activity for labelling. 4. Add 300 μL of 68Ga-containing eluate (165–185 MBq) to the DOTA-affibody-PNA solution. 5. Vortex the mixture carefully and incubate at 95  C for 15 min. 6. Add Na4EDTA (1.44 mg, 3.80 μmol, 72 μL of 20 mg/mL in 1.25 M sodium acetate, pH 3.6), vortex the mixture carefully, and incubate at 95  C for 5 min. 7. Load the reaction mixture on the column. Let it pass through the upper filter. Add 80 μL PBS and let it pass through the filter. Collect and discard the eluate. 8. Add 1000 μL of PBS to the column; collect the eluate. This fraction will contain 68Ga- DOTA-affibody-PNA. 9. Evaluate purity of 68Ga- DOTA-affibody-PNA as described in Subheading 3.10, steps 5 and 6. The radiolabelled PNA would stay at the application point, while free 68Ga would migrate with the solvent front. 3.14 Quality Control Using HPLC

1. To confirm the identity of a radiolabelled PNA, a radio-HPLC system can be used. For this purpose, dissolve the radiolabelled PNA in 5% of buffer B (diluted in deionized H2O). 2. Load the sample on a semi-preparative Zorbax C18 column. 3. Elute with an elution gradient going from 5% to 50% buffer B in A in 30 min and a flow rate of 1 mL/min. 4. The retention time of the radiolabelled PNA would be approximately 10.5 min.

3.15 Evaluation of Pretargeting In Vitro Specificity

1. Seed cells in 3 cm dishes (n ¼ 12) to have ca 1  106 cells/dish by the time of experiment. Take in account cell character (doubling time, receptor expression, time to stable attachment, etc.). 2. Add 1 mL of complete cell culture media to each dish, and leave the cells to incubate overnight at 37  C. 3. On the day of the experiment, calculate a volume of a primary targeting agent required to prepare a solution (solution 1) with concentration of 20  KD (where KD is an apparent dissociation constant of the primary targeting agent at equilibrium). 4. Calculate a volume of the radiolabelled complementary PNA required to prepare a solution (solution 2) with a concentration of 10 solution 1.

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5. Calculate a volume of the non-labelled blocking agent (agent binding to the same epitope as the primary agent) required to prepare a solution (solution 3) with concentration of 1000  KD. This will be used to block the binding of primary agent to targeted receptors. 6. Calculate a volume of the non-labelled complementary PNA required to prepare a solution (solution 4) with concentration of 2 solution 2. This will be used to block the interaction between the primary targeting agents and radiolabelled complementary PNA on the cell surface (i.e., PNA-PNA interaction). 7. All solutions are prepared using complete cell culture media with a 0.5 mL volume added per dish. 8. Divide the cell culture dishes into four groups A, B, C, and D, with three dishes each. 9. Remove the complete cell culture media (incubation media) from cell dishes of groups A, B, and C. 10. Wash cells once with 1 mL serum-free medium. 11. Add 0.5 mL of blocking solution (solution 3) to the cell culture dishes of group B. 12. Add 0.5 mL of complete cell culture media to cell culture dishes of groups A and C. 13. After a certain time (determined by how fast is the kon of the blocking agent), add an additional 0.5 mL of primary targeting agent solution (solution 1) to all dishes (A, B, and C). 14. Incubate the cell culture dishes at 4  C for 1 h. 15. After 1 h, remove incubation media from all cell culture dishes (A, B, C, and D). 16. Wash once with 1 mL serum-free medium. 17. To dishes of groups A, B, and D, add 0.5 mL of radiolabelled complementary PNA solution (solution 2). 18. To the dishes of group C, add 0.5 mL of cold non-labelled complementary PNA solution (solution 4). 19. Leave all cells to incubate for additional 60 min at 37  C. This is to allow for the hybridization (reaction) between PNA oligonucleotides (HP1 and HP2) to take place. 20. Remove cell culture dishes from incubator, and add 0.5 mL of radiolabelled complementary PNA solution (solution 2) to cells of group C. 21. To the cell culture dishes of groups A, B, and D, add 0.5 mL of complete cell culture media. 22. Allow cells to further incubate for 15 min at 37  C.

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23. During incubation time prepare the test tubes for separate collection of media and cell suspension from each dish. 24. Collect incubation media (1 mL) in the fraction tubes (media). 25. Wash cells with 1 mL serum-free medium. 26. Detach cells using 0.5 mL trypsin solution/dish at 37  C for 10 min. 27. Resuspend cells by adding 0.5 mL complete media, and collect cell suspension (1 mL) in the respective test tubes (cells). 28. Samples should γ-spectrometer.

be

measured

for

radioactivity

using

29. Calculate cell-associated radioactivity (as % of total added activity) in each dish.

4

Notes 1. Radionuclides emit ionizing radiation, which is potentially damaging for workers. During work, the radiation safety guidelines set by institutions and the national nuclear regulatory authorities must be followed strictly and meticulously. Protective equipment, personal dosimeters, and radiation survey monitors are required when handling any radioactive materials. 2. Sortase A3∗ can in our experience be stored at 80  C for at least 2 years without noticeable loss in activity upon thawing. 3. The PNA monomers, especially the C monomer, may be hard to dissolve in DMF/DCM, and the solution may need heating for 10 min at 85  C to become completely clear. 4. Addition of Ni2+-ions can enhance the ligation yield of sortase A-mediated ligations if the substrate protein has a C-terminal LPXTGGH motif [27]. In our experience nickel(II) acetate or nickel(II) sulfate salts can be used interchangeably as a source of Ni2+-ions. 5. The flow-through contains the affibody-PNA chimera, unreacted HP1, and by-products from the ligation reaction (hydrolyzed species of the affibody molecule with the His-tag cleaved off). 6. This buffer exchange step is important to make HP2 dissolve more easily after lyophilization. 7. The yield should be over 95%, if the procedure was performed using routines preventing metal contamination of the buffers and solutions.

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Fig. 3 Illustrative example of binding of radiolabelled PNA to cells in a binding specificity assay. In group (A) cells were incubated with primary targeting agent prior to the addition of radiolabelled PNA. In groups (B) and (C), cells were incubated with either an excess amount of unlabelled blocking agent prior to the addition of the primary agent or excess unlabelled PNA prior to the addition of radiolabelled PNA, respectively. Radiolabelled PNA was added directly to cells in group (D) without pretargeting (see also Notes 8–10)

8. If a significant difference between the means of A (red) and B (green) exists (Fig. 3), this means that binding of radiolabelled PNA is dependent on binding of primary targeting agent to cells expressing the target. 9. If a significant difference between the means of A (red) and C (blue) exists (Fig. 3), this means that binding of radiolabelled PNA to cells is mediated by PNA-PNA hybridization. 10. If a significant difference between the means of A (red) and D (black) exists (Fig. 3), this means that the binding of radiolabelled PNA to cells is very low without primary agent pretreatment. References 1. Larson SM, Carrasquillo JA, Cheung NK, Press OW (2015) Radioimmunotherapy of human tumours. Nat Rev Cancer 15:347–360 2. Patra M, Zarschler K, Pietzsch HJ, Stephan H, Gasser G (2017) New insights into the pretargeting approach to image and treat tumours. Chem Soc Rev 45:6415–6431 3. Bailly C, Bodet-Milin C, Rousseau C, FaivreChauvet A, Kraeber-Bode´re´ F, Barbet J (2017) Pretargeting for imaging and therapy in oncological nuclear medicine. EJNMMI Radiopharm Chem 2:6 4. Altai M, Membreno R, Cook B, Tolmachev V, Zeglis BM (2017) Pretargeted imaging and therapy. J Nucl Med 58:1553–1559 5. Schubert M, Bergmann R, Fo¨rster C, Sihver W, Vonhoff S, Klussmann S, Bethge L, Walther M, Schlesinger J, Pietzsch J, Steinbach J, Pietzsch

HJ (2017) Novel tumor pretargeting system based on complementary l-configured oligonucleotides. Bioconjug Chem 28:1176–1188 6. Liu G (2017) Use of morpholino oligomers for pretargeting. Methods Mol Biol 1565:161–179. https://doi.org/10.1007/ 978-1-4939-6817-6_14 7. Mardirossian G, Lei K, Rusckowski M, Chang F, Qu T, Egholm M, Hnatowich DJ (1997) In vivo hybridization of technetium99m-labeled peptide nucleic acid (PNA). J Nucl Med 38:907–913 8. Nielsen PE, Egholm M, Berg RH, Buchardt O (1991) Sequence-selective recognition of DNA by strand displacement with a thyminesubstituted polyamide. Science 254:1497–1500

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INDEX A Acrylamide..................................................................... 143 Acute toxicity .............................................. 242, 245, 249 Acyl-carrier protein (acpP) ........................................... 232 Acyl migration ................................................................... 4 Affibody ................................................................ 283–303 Agarose ................................................143–146, 244, 248 Aggregation ......................................................65, 71, 122 Alexa Fluor-680 (AF-680) ................................. 252, 256, 257, 259 Antibiotics ............................................... 4, 231–234, 292 Antibodies .................................................. 120, 123, 136, 203, 205, 207, 244, 247, 283–285 AntimiR ................................................................ 269–271 Antisense................................................................ 4, 7, 97, 101, 173, 175, 176, 201, 231–237, 242, 255 Apoptosis ..................................................... 204, 207, 208 Automated peptide synthesis....................................82, 83 Azide ..................................................................... 125, 135 Azido-lysine ................................................................... 125

B Bacteria ................................................................ 219, 222, 227, 231–237 Benzhydryloxycarbonyl (Bhoc)......................... 2–4, 6, 64 Benzoyladenine .........................................................42, 43 Benzoylcytosine.........................................................42, 43 Biofilms ................................................220, 221, 225, 227 BisPNA ................................................................. 262–265

C C18 ..................................................................81, 84, 101, 162, 165, 166, 289, 292 Capping ................................................................... 5, 7, 8, 50, 63, 67, 69, 70, 136, 162, 178, 181, 288, 294 Cell-penetrating peptides (CPPs) .......................... 76, 97, 100, 160, 173–175, 183, 253, 267, 269, 270 CF, see Cystic fibrosis (CF) CFTR, see Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Chloroquine (CQ) .............................................. 173, 175, 177, 180, 264, 265

Cholesterol .............................................................. 78, 80, 85, 87, 174, 175 Cholic acid................................................... 175, 178, 182 Chromatography ...............................................18, 19, 21, 23–32, 41, 42, 45, 56, 82, 290, 291, 298, 299 Cleavage............................................................. 2, 3, 5–10, 50–52, 54, 57, 58, 62, 64, 67, 68, 70–72, 80, 84, 136, 144, 146, 162, 164, 180, 181, 289, 294 Click chemistry........................................ 35–59, 188, 251 Combinatorial .....................................120, 122, 128, 137 Confocal fluorescence microscopy ............................... 167 Cooperativity ................................................................... 78 CPPs, see Cell-penetrating peptides (CPPs) CQ, see Chloroquine (CQ) Cryosection ................................................................... 247 Cy5....................................................................... 125, 126, 136, 188, 194, 195, 197 Cystic fibrosis (CF) ............................................. 200, 201, 210, 267–268 Cystic fibrosis transmembrane conductance regulator (CFTR) .................................................... 200, 201, 203, 205, 207, 210, 211, 267, 268

D DAPI, see 4’,6-Diamidino-2-phenylindole (DAPI) DCM, see Dichloromethane (DCM) Delivery............................................................76, 97, 100, 169, 173–184, 201, 208, 212, 253, 257, 258, 261–278, 283 Deprotection ............................................................2, 4–9, 48–51, 58, 66, 69–71, 80, 82, 83, 121, 133, 136, 137, 160, 164, 166, 169, 180, 181, 288, 294 4’,6-Diamidino-2-phenylindole (DAPI) ............................................. 167, 169, 247 Dichloromethane (DCM) .................................. 274, 276, 288, 289, 293, 301 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) ...................................................... 77, 78, 80, 85, 87, 90 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) .....................................78, 80, 85, 87, 90 DMD, see Duchenne muscular dystrophy (DMD) DMF, see N,N-dimethylformamide (DMF)

Peter E. Nielsen (ed.), Peptide Nucleic Acids: Methods and Protocols, Methods in Molecular Biology, vol. 2105, https://doi.org/10.1007/978-1-0716-0243-0, © Springer Science+Business Media, LLC, part of Springer Nature 2020

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PROTOCOLS

DOTA ..................................................285–287, 293, 294 Double-stranded RNA (dsRNA) ........................ 157–170 Duchenne muscular dystrophy (DMD) ............. 241, 242 Dystrophin....................................................241, 247–248

E Endosomal release......................................................... 174 Enterobacteriaceae ................................................ 223, 231 Epifluorescence ................................................... 218, 222, 226, 248, 257–258 Escherichia coli ............................................. 233, 237, 293 Exon skipping......................................242, 243, 247, 248

F FACS.............................................................................. 208 Fluoren-9-ylmethoxycarbonyl (Fmoc) ...............................................1–14, 50, 55, 58, 64, 121, 125, 133, 160, 164, 288, 294 Fluorescence ............................................................ 78, 80, 82, 89, 91–93, 123, 125–127, 167, 177, 190, 194, 195, 197, 217–228, 247, 251–259 Fluorescence in situ Hybridization (FISH)....................................................... 217–228 Fluorescence resonance energy transfer (FRET)...................................................... 187–197 Fluorescent ............................................................ 36, 159, 160, 164, 187, 251, 252 Fmoc, see Fluoren-9-ylmethoxycarbonyl (Fmoc) Fusogens ................................................65, 75, 76, 78, 79

G Gamma PNA .............................................................17–32 Gel......................................................................18, 19, 21, 24–28, 41, 42, 143–146, 148, 149, 152, 153, 203, 205, 248 Gene correction.................................................... 262–268 Gene editing .......................................... 18, 261–268, 271 β-globin ......................................................................... 263 β-globin gene (HBB) .................................. 263, 264, 268

H 2-(1H-7-azabenzotriazole-1-yl)-1,1,3,3tetramethyluronium hexafluorophosphate (HATU) ................................................... 2, 49, 57, 63, 67, 73, 127, 135, 161, 164 HBB, see β-globin gene (HBB) 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) ....................2, 4–9, 28, 82, 178, 181, 182 Heat maps............................................................. 123, 126 HeLa pLuc705 .............................................................. 177 HER2.................................................................... 285, 286

Hexafluoro-2-propanol (HFIP) ............................. 65, 70, 127, 133 High-performance liquid chromatography (HPLC).................................................... 2, 4–7, 9, 10, 13, 30, 32, 51, 65–67, 70, 72, 80, 81, 84–85, 98, 100–104, 132, 162–166, 169, 180, 182, 221, 249, 256, 259, 278, 290, 292, 300 High throughput screening (HTS) ................7, 101, 119 Homopurine......................................................... 158, 159 Hoogsteen ........................................................... 142, 143, 158, 159, 262, 264 HPLC, see High-performance liquid chromatography (HPLC) HTS, see High throughput screening (HTS) Hybridization ....................................................62, 64, 77, 120, 121, 123, 128, 132, 136, 144, 153, 188, 197, 205, 217, 219–222, 225–228, 265, 270, 284, 292, 294, 301, 303 1-Hydroxy-7-azabenzotriazole (HOAt) .................................................48, 49, 58, 63, 67, 73

I IM administration ................................................ 243, 245 Imaging....................................................... 248, 251–259, 276, 278, 285, 287 Invasion .................................................36, 144, 150, 264 In vitro transcription................................... 142, 146, 153 In vivo .................................................174, 201, 241–249, 251–259, 261–278, 284 IP administration .......................................................... 245 Isothermal titration calorimetry (ITC)................................................ 160, 163, 167 ITC, see Isothermal titration calorimetry (ITC) IV administration .......................................................... 245

K Kidneys ....................................................... 242, 252–255, 258, 269, 285, 286 Kill curves ............................................................. 236, 237 Kinase............................................................121–123, 125

L LC-MS ..................................................................... 6, 101, 132, 134, 165, 251 Library ................................................................. 120–123, 125, 126, 128, 132–136 Lipidated PNA ................................................................ 63 Lipid bilayers ................................................................... 76 Lipofectamine................................................................ 210 Liposome extrusion ........................................................ 82 Liposomes............................................ 62–65, 75–93, 201

PEPTIDE NUCLEIC ACIDS: METHODS

AND

PROTOCOLS Index 307

Liver ............................................................ 242, 253–255, 258, 259, 268, 269 Loading................................................... 7–10, 48, 57, 71, 133, 147, 152–154, 167, 188, 270, 276–278, 288

N,N-dimethylformamide (DMF)..............................2, 22, 36, 64, 79, 127, 161, 177, 288 NMP, see N-methyl-2-pyrrolidone NMR................................................................................ 31

M

P

Manual synthesis ..........................................4, 6–9, 58, 65 Matrix-Assisted Laser Desorption-Time of Flight (MALDI-TOF)............................................ 4, 5, 9, 10, 14, 58, 81, 85, 103, 180, 297 MBC, see Minimal bactericidal concentration (MBC) Mdx....................................................................... 241–249 Membranes ........................................................61, 75, 97, 162, 176, 196, 203, 221, 231, 262, 285 Microarray ........................................................... 120, 123, 128, 132, 136, 137 MicroRNAs (miRNAs) .............................. 158, 200–212, 269–271 Microscopy ............................................................ 78, 218, 219, 226, 228, 273 Microwave ........................................................... 1–14, 65, 68, 70, 71, 83, 160 Mimetics ............................................................. 61–73, 97 Minimal bactericidal concentration (MBC)...................................................... 234, 236 Minimal inhibitory concentration (MIC)........................................................ 231–237 MiniPEG....................................................................17–32 miR-145 ........................................................................ 200 miRNAs, see MicroRNAs (miRNAs) Mismatch ............................................................... 36, 128, 195, 223, 232, 236, 244 Monomers ........................................................... 1, 18, 35, 63, 82, 132, 160, 178, 259, 264, 288 4-Monomethyltrityl (Mtt)...................................... 3, 121, 125, 127, 132, 133, 289, 294 Mouse .................................................................. 205, 224, 241–249, 252, 254–259, 266, 268–270 mRNA.................................................................. 158, 159, 201, 203, 205, 207, 208, 210, 211, 232, 242, 243, 267, 269 Mtt, see 4-monomethyltrityl (Mtt)

Palmitoyl....................................................................62, 63 PCR...................................................................... 187, 202, 204, 205, 219, 248 PEG.............................................................. 121, 133, 134 Pentafluorophenyl ..........................................2, 43–44, 57 Pharmacokinetics ................................................ 242, 251, 252, 259, 285 Phosphorodiamidate morpholino (PMO)................... 232 Photochemical internalization (PCI)................. 173, 175, 180, 183 Photosensitizers...................................175–177, 180, 183 Plasmids ............................................................... 143, 144, 148, 149, 152, 267, 269, 292 PLGA, see Poly(lactic-co-glycolic acid) (PLGA) PMO, see Phosphorodiamidate morpholino (PMO) PNA encoded ...................................................... 120, 123, 125, 126, 128 PNA-peptide............................................................ 3, 4, 6, 61–73, 97–116, 184, 231–237, 242, 294, 295 PNA synthesis.............................................................3, 36, 47–50, 122, 135, 137, 160–164, 177, 180–181, 190, 256 PNA tags........................................................................ 128 Polyacrylamide ..................................................... 143, 152 Poly(lactic-co-glycolic acid) (PLGA) ........................................... 262, 266–272, 274–276, 278 Polymerase..................................141, 143, 150, 244, 248 Pretargeting .......................................................... 283–303 Promoter ..................................................... 143, 152, 210 Pseudoisocytosine ................................................ 142, 143 Pyrrolidinyl PNA.......................................................35–59

N Nanoparticles....................... 86, 188, 196, 197, 261–278 Near-Infrared........................................................ 251–259 N-hydroxybenzotriazole (HOBt) .............................2, 66, 82, 123, 127, 133, 134 N-methyl-2-pyrrolidone (NMP).............................2–6, 8, 9, 66, 67, 69, 72, 127, 133–135, 163, 288, 294 N,N-diisopropylethylamine (DIPEA)........................................................2, 4–6, 8, 9, 43, 45, 47–49, 58, 63, 67, 127, 135, 161, 164, 181

Q qPCR, see Quantitative PCR (qPCR) Quantitative PCR (qPCR)................................... 202, 204 Quantum dot ....................................................... 187–197

R Radionuclide........................................283, 285–287, 301 Real-time quantitative PCR (RT-qPCR) ............ 202, 204 Recognition ....................................................... 18, 61–65, 69–70, 76, 77, 144, 157–170, 200, 283, 284 Restriction enzymes ...................................................... 144 Reverse transcriptase PCR (RT-PCR) ............... 205, 210, 242, 243, 247–249 R-Loops ................................................................ 141–151 RNA polymerase (RNAP) .................. 141–143, 146–153

PEPTIDE NUCLEIC ACIDS: METHODS

308 Index

AND

PROTOCOLS

RP-C18............................................................... 65, 66, 70 rRNA .................................................................... 219–223 RT-qPCR, see Real-time quantitative PCR (RT-qPCR)

S Salmonella............................................................. 223, 233 SbmA ............................................................................. 233 SC administration ......................................................... 245 Screenings................................................................ 6, 120, 121, 125, 127, 136, 137 Serum................................................................... 174, 179, 183, 205, 251, 252, 292, 301, 302 16S ............................................................... 219, 222, 223 Size-exclusion chromatography ..................................... 82 SNAP-25 ...................................................................61, 62 Solid phase peptide synthesis (SPPS)............... 63, 68–71, 120, 121 Solid-phase ......................................................1, 9, 63–65, 77, 82, 97, 160, 287–289, 293 Solid-phase synthesis (SPS) ....................................2–4, 98 Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)...............61–73, 76, 78 Sortase A.............................................................. 285, 287, 289, 293, 296–298, 301 Split and mix................................................ 120, 125, 133 SPPS, see Solid phase peptide synthesis Sulforhodamine B (SRB) ........................................ 79, 80, 85, 88, 92 Synthesis .............................................................. 1–14, 18, 20–30, 35–59, 61–73, 75–93, 119–137, 160, 161, 163, 164, 169, 177, 178, 180–182, 184, 188, 189, 200, 208, 223, 271, 287–289, 293–295, 297

T Tags.............................................120, 128, 133, 284, 285 Tail-clamp PNA (tcPNA) ........................... 264, 266, 267

tcPNA, see Tail-clamp PNA (tcPNA) tert-butyloxycarbonyl (Boc) ................................... 1, 3, 4, 6–9, 26, 29, 30, 43, 56, 98, 160, 180, 181 TFA, see Trifluoroacetic acid (TFA) TFMSA .......................................................................... 181 β-thalassemia......................................................... 263, 268 Therapies ............................................................... 97, 201, 211, 242, 268, 283, 285–287 Tomography .................................................................. 255 Toxicity ......................................................... 97, 174, 183, 232, 233, 242, 245, 249, 262, 266 Transcription .......................................141–151, 210, 269 Transfection......................................................... 174, 175, 177–180, 182, 183, 212, 262, 266, 268 Transmembrane...................................63–66, 68, 71, 267 Trifluoroacetic acid (TFA) ...................................... 6, 7, 9, 32, 58, 67, 68, 80, 81, 84, 86, 101, 136, 162, 169, 181, 182, 189, 249, 256, 259, 288–290, 292, 297 Triple helix................................................... 158, 160, 167 Triplet ................................................................... 158, 159 Triplex..................................................142, 143, 157–170 Triton X-100 ........................................................... 80, 86, 91, 92, 222, 227 Tumors ................................................................. 283–287 23s................................................................ 219, 222, 223

W Watson-Crick................................ 62, 142, 143, 262, 264 Western blot ...............................203, 205, 207, 242, 247

X X-ray CT ........................................................................ 255

Z Zeta potential ...............................................272, 275–278