Advancing development of synthetic gene regulators : with the power of high-throughput sequencing in chemical biology : doctoral thesis accepted by Kyoto University, Kyoto, Japan

This book focuses on an “outside the box” notion by utilizing the powerful applications of next-generation sequencing (NGS) technologies in the interface of chemistry and biology. In personalized medicine, developing small molecules targeting a specific genomic sequence is an attractive goal. N-methylpyrrole (P)–N-methylimidazole (I) polyamides (PIPs) are a class of small molecule that can bind to the DNA minor groove. First, a cost-effective NGS (ion torrent platform)-based Bind-n-Seq was developed to identify the binding specificity of PIP conjugates in a randomized DNA library. Their biological influences rely primarily on selective DNA binding affinity, so it is important to analyze their genome-wide binding preferences. However, it is demanding to enrich specifically the small-molecule-bound DNA without chemical cross-linking or covalent binding in chromatinized genomes. Herein is described a method that was developed using high-throughput sequencing to map the differential binding sites and relative enriched regions of non-cross-linked SAHA-PIPs throughout the complex human genome. SAHA-PIPs binding motifs were identified and the genome-level mapping of SAHA-PIPs-enriched regions provided evidence for the differential activation of the gene network. A method using high-throughput sequencing to map the binding sites and relative enriched regions of alkylating PIP throughout the human genome was also developed. The genome-level mapping of alkylating the PIP-enriched region and the binding sites on the human genome identifies significant genomic targets of breast cancer. It is anticipated that this pioneering low-cost, high through-put investigation at the sequence-specific level will be helpful in understanding the binding specificity of various DNA-binding small molecules, which in turn will be beneficial for the development of small-molecule-based drugs targeting a genome-level sequence. Supervisor’s Foreword 7 Part of this Thesis have been Published in the Following Journal Articles:Chandran A, Syed J, Li Y, Sato S, Bando T, Sugiyama H (2016) “Genome-Wide Assessment of the Binding Effects of Artificial Transcriptional Activators by High-Throughput Sequencing.” Chembiochem 17:1905–1910. doi: 10.1002/cbic.201600274.Chandran A, Syed J, Taylor RD, Kashiwazaki G, Sato S, Hashiya K, Bando T, Sugiyama H (2016) “Deciphering the genomic targets of alkylating polyamide conjugates using high-throughput sequencing.” Nucleic Acids Res 44:4014–4024. doi: 10.1093/nar/gkw283.Anandhakumar C, Kizaki S, Bando T, Pandian GN, Sugiyama H (2015) “Advancing small-molecule-based chemical biology with next-generation sequencing technologies.” ChemBioChem 16:20–38.doi: 10.1002/cbic.20140 2556.Anandhakumar C, Li Y, Kizaki S, Pandian GN, Hashiya K, Bando T, Sugiyama H (2014) “Next-generation sequencing studies guide the design of pyrrole-imidazole polyamides with improved binding specificity by the addition of β-alanine.” ChemBioChem 15:2647–2651. doi: 10.1002/cbic.201402497. 9 Acknowledgements 10 Contents 11 1 Overview of Next-Generation Sequencing Technologies and Its Application in Chemical Biology 14 Abstract 14 1.1 General Introduction: Next Generation Sequencing (NGS) Principles and Platforms 14 1.1.1 Sequencing by Synthesis (SBS) 16 1.1.1.1 Illumina 16 1.1.1.2 Roche 454 16 1.1.1.3 Ion Torrent 17 1.1.2 Single-Molecule Sequencing (SMS) 19 1.1.2.1 Helicos 19 1.1.2.2 Pacific Biosciences (PacBio) 19 1.1.3 Sequencing by Ligation (SBL) 20 1.1.3.1 Polonator and SOLiD (Support Oligonucleotide Ligation Detection) 21 1.2 Applications of NGS in Chemical Biology 23 1.2.1 Genome-Wide Localization of Non-B DNA Using Small Molecules 23 1.2.2 Decoding of DNA Base Modification in Single Molecule Level 25 1.2.2.1 Studies Targets Epigenetically Modified Bases 26 1.2.2.2 DNA Strand Breakage and Damaged Bases 29 1.2.3 Aptamer Selection Using Massively Parallel Sequencing 31 1.3 Development of N-Methylpyrrole (P)—N-Methylimidazole (I) Polyamides (PIP) and Its Conjugates 33 1.4 PIP Based Gene Regulation 35 1.5 Utilization of NGS in PIP Based Small Molecule Studies 36 1.5.1 Guiding the Design and Screening of Small Molecule Using NGS 36 1.5.2 Analysis of Gene Expression Induced by PIP Based Small Molecules 39 1.5.2.1 Transcriptome Studies 39 1.5.2.2 Protein-DNA Interaction Studies 41 1.6 Conclusion and Future Prospects 44 References 46 2 Next Generation Sequencing Studies Guide the Design of Pyrrole-Imidazole Polyamides with Improved Binding Specificity by the Addition of β-Alanine 55 Abstract 55 2.1 Introduction 55 2.2 Results and Discussion 58 2.2.1 PIP and PIP Conjugates Synthesis and Bind-n-Seq Analysis 58 2.2.2 PIP Redesign, ββ-PIP Synthesis and Bind-n-Seq Analysis 61 2.2.3 Binding Affinity Conformation by SPR 63 2.2.4 Discussion 64 2.3 Materials and Methods 67 2.3.1 General Materials and Synthesis 67 2.3.1.1 General Scheme for Synthesis of Biotin-Conjugated Polyamides 67 2.3.2 Oligomer Sequences Used in Bind-n-Seq Method 69 2.3.3 Bind-n-Seq 70 2.3.4 Surface Plasmon Resonance (SPR) 71 2.3.4.1 General Scheme for Synthesis of Polyamides Used for SPR Analysis 71 2.3.4.2 SPR Analysis 72 References 72 3 Genome-Wide Assessment of the Binding Effects of Artificial Transcriptional Activators by Utilizing the Power of High-Throughput Sequencing 74 Abstract 74 3.1 Introduction 74 3.2 Results and Discussion 76 3.2.1 Revealing the Unique Gene Set Activation Mechanism of PIP Conjugates in the Human Genome 80 3.2.2 Identification of the Preferential Binding Region of PIP Conjugates in the Tightly Packed Heterochromatin Region 81 3.2.3 Discussion 82 3.3 Materials and Methods 84 3.3.1 Nucleus Extraction and PIP Conjugate Incubation 84 3.3.2 MNase Digestion 84 3.3.3 Affinity Purification 85 3.3.4 High-Throughput Sequencing Library Construction and Sequencing 85 Appendix 87 References 88 4 Deciphering the Genomic Targets of Alkylating Polyamide Conjugates Using High-Throughput Sequencing 91 Abstract 91 4.1 Introduction 91 4.2 Results and Discussion 94 4.2.1 Bind-n-Seq with PIP-Indole-Seco-CBI Conjugate 1 94 4.2.2 Bind-n-Seq with PIP-Indole-Seco-CBI Conjugate 2 96 4.2.3 Bind-n-Seq with PIP-Conjugate 5 96 4.2.4 Identification of PIP-Indole-Seco-CBI Conjugate 2 High Affinity DNA-Alkylating Site in Chromatinized Human Genome 96 4.2.5 Identification of PIP-Indole-Seco-CBI Conjugate 2 Representative Enriched Sites in Chromatinized Human Genome 100 4.2.6 Genome-Wide Analysis of PIP-Indole-Seco-CBI Conjugate 2 Enriched Sites Distribution 101 4.2.7 Discussion 102 4.3 Materials and Methods 103 4.3.1 Synthesis of Biotin-Conjugated Alkylating Polyamide Conjugates 103 4.3.2 Bind-n-Seq Experiment and High-Throughput Sequencing 106 4.3.3 Affinity Purification-Based High-Throughput Sequencing of Human Genomic Regions Enriched with PIP-Indole-Seco-CBI Conjugate 2 107 4.3.4 Validating PIP-Indole-Seco-CBI Conjugate 2 Bound and Enriched Region in the Human Genome 109 Appendix 110 References 118 Curriculum Vitae 122 Front Matter ....Pages i-xv Overview of Next-Generation Sequencing Technologies and Its Application in Chemical Biology (Anandhakumar Chandran)....Pages 1-41 Next Generation Sequencing Studies Guide the Design of Pyrrole-Imidazole Polyamides with Improved Binding Specificity by the Addition of β-Alanine (Anandhakumar Chandran)....Pages 43-61 Genome-Wide Assessment of the Binding Effects of Artificial Transcriptional Activators by Utilizing the Power of High-Throughput Sequencing (Anandhakumar Chandran)....Pages 63-79 Deciphering the Genomic Targets of Alkylating Polyamide Conjugates Using High-Throughput Sequencing (Anandhakumar Chandran)....Pages 81-111 Back Matter ....Pages 113-114