Sequence-Specific DNA Binders for the Therapy of Mitochondrial Diseases (Springer Theses) 9789811684357, 9789811684364, 9811684359

Table of contents :
Supervisor’s Foreword
Acknowledgments
Contents
1 Introduction
1.1 Mitochondrial Biology and Diseases
1.1.1 Mitochondrial Biology
1.1.2 Mitochondrial Diseases
1.2 Pyrrole-Imidazole Polyamides
1.2.1 Design of PIPs
1.2.2 Application of PIPs
References
2 Creation of a Synthetic Ligand for Mitochondrial DNA Sequence Recognition and Promoter-Specific Transcription Suppression
2.1 Introduction
2.2 Results
2.2.1 Design of MITO-PIPs
2.2.2 Promoter-Specific Transcription Control by MITO-PIP-LSP
2.2.3 Sequence-Selective DNA Binding of MITO-PIPs
2.2.4 Mitochondrial Accumulation of MITO-PIP-LSP
2.3 Discussion
2.4 Materials and Methods
2.4.1 General Information of Synthesis
2.4.2 Synthesis of PIP-LSP, MITO-PIP-LSP, and MITO-PIP-HSP1
2.4.3 Synthesis of MITO-PIP-TAMRA
2.4.4 Melting Temperature (Tm) Analysis
2.4.5 Cell Culture
2.4.6 Quantitative Reverse-Transcription PCR Analysis (RT-qPCR)
2.4.7 Live Cell Imaging
2.5 Characterization Data of Synthesized Compounds
References
3 Allele-Specific Replication Inhibition of Mitochondrial DNA by MITO-PIP Conjugated with Alkylation Reagent
3.1 Introduction
3.2 Results
3.2.1 Design and Synthesis of a Conjugate Recognizing Selective DNA Sequence
3.2.2 Validation of Sequence-Specific Adenine Alkylation by 8950A-Chb(Cl/OH)
3.2.3 8950A-Chb(Cl/OH) Shifts Heteroplasmy Level in Live Cells
3.3 Discussion
3.4 Materials and Methods
3.4.1 General Information of Synthesis
3.4.2 Solid-Phase Synthesis of MITO-PIPs and Chlorambucil Conjugation
3.4.3 Synthesis of 8950A-Chb(Cl/OH), Ctrl-Chb(Cl/OH) and 9037A-Chb(Cl/OH)
3.4.4 Synthesis of 8950A-Chb(OH/OH) and Ctrl-Chb(OH/OH)
3.4.5 Preparation of DMSO Solution of Each Compound
3.4.6 Melting Temperature (Tm) Analysis
3.4.7 Alkylation Assay by Capillary Electrophoresis
3.4.8 Compound Treatment and Quantitative PCR (qPCR) Analysis
3.5 Characterization Data of Synthesized Compounds
References
4 Enhanced Nuclear Accumulation of Pyrrole-Imidazole Polyamides by Incorporation of the Tri-Arginine Vector
4.1 Introduction
4.2 Results
4.2.1 Improved Nuclear Delivery of PIP–Tri-Arginine Conjugates
4.2.2 Sequence-Selective DNA Binding of PIP–Tri-Arginine Conjugates
4.2.3 Transcriptional Control of Endogenous Genes by PIP–Tri-Arginine Conjugates
4.3 Discussion
4.4 Materials and Methods
4.4.1 General Information of Synthesis
4.4.2 Synthesis of SOX2i-R3, SOX2i, Ctrl-R3, AP2i-R3, and AP2i
4.4.3 Synthesis of SOX2i-R3-TAMRA and SOX2i-TAMRA
4.4.4 Cell Culture
4.4.5 Live Cell Imaging
4.4.6 Flow Cytometry Analysis
4.4.7 Preparation of DMSO Solution of SOX2i-R3, SOX2i, Ctrl-R3, AP2i-R3, and AP2i
4.4.8 Electrophoretic Mobility Shift Assay
4.4.9 Quantitative Reverse-Transcription PCR (RT-qPCR) Analysis of 201B7 Cells
4.4.10 Quantitative Reverse-Transcription PCR (RT-qPCR) Analysis of SKBR-3 Cells
4.5 Characterization Data of Synthesized Compounds
References
Curriculum Vitae