DNA/RNA G-Quadruplexes: Structures, Transitions and Recognition

DNA/RNA G-四链体：结构、转换和识别

• 批准号: 7785988
• 负责人: DINSHAW J PATEL
• 金额: $ 43.25万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-12-01 至 2014-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7785988
• 关键词: 5' Untranslated Regions; Address; Adopted; Affinity; Antineoplastic Agents; Apoptosis Regulator; Architecture; Back; Base Pairing; Binding; Bioinformatics; Cell Proliferation; Cleaved cell; Complex; DNA; Data; Digestion; Dimensions; Drug Delivery Systems; Elements; Engineering; Enzymes; Exhibits; Foundations; Friend leukemia; G-Quartets; G-substrate; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Genome; Genome Stability; Goals; Guanine; Health; Higher Order Chromatin Structure; Human; Human Chromosomes; Human Genome; Image; In Vitro; Indium; Introns; Invaded; Investigation; Laboratories; Length; Ligand Binding; Ligands; Literature; Mediating; Methods; Modeling; Molecular Conformation; Monitor; Monovalent Cations; Mutation; Nucleic Acids; Oligonucleotides; Oncogenes; Oncogenic; Oxazoles; Peptides; Positioning Attribute; Promoter Regions; Property; Proteins; Proto-Oncogene Protein c-kit; Proto-Oncogenes; Protons; Publishing; RNA; RNA Stability; Regulation; Relative (related person); Reporting; Research; Role; Shapes; Site; Solutions; Structure; Telomerase; Telomerase Inhibitor; Telomerase inhibition; Telomere Maintenance; Testing; Therapeutic; Transcript; Transcription Factor AP-1; Transcription Initiation Site; Translations; Triad Acrylic Resin; Tumor Suppressor Genes; Vascular Endothelial Growth Factors; Vasopressins; analog; aptamer; base; c-myc Genes; cancer cell; conformer; design; drug candidate; drug development; immunogenic; in vivo; insight; interest; molecular recognition; neoplastic cell; novel; nuclease; peptide structure; prevent; promoter; public health relevance; recombinational repair; scaffold; small molecule; telomere; telomestatin; transcription factor

DESCRIPTION (provided by applicant): Guanine-rich tracts are observed in critical segments of eukaryotic genomes including telomeric, intronic and oncogenic promotor DNA regions, as well as within 5'-untranslated regions (UTRs) of oncogenic RNA transcripts. Such putative G-quadruplex-forming sequences are prevalent in proto-oncogenes (which promote cell proliferation) and essentially lacking in tumor-suppressor genes (which maintain genomic stability). Cellular proteins exist that bind, cleave, resolve, promote and disrupt G-quadruplex formation, with recent research providing increasing support for G-quadruplex formation in vivo. Our laboratory has ongoing projects aimed at NMR and x-ray structural characterization of G-quadruplex topologies formed by guanine-rich tracts in c-myc, c-kit, VEGF and c-RET oncogenic DNA promoters (Aim 1), in human telomeric and intronic DNA (Aim 2), and in N-ras, and related oncogenic RNA 5'-UTR segments (Aim 3). These structural studies should define the folding propensity and diversity of G-quadruplex topologies, as well as the energetics of interconversion between conformational states. The research will be extended to structural characterization of G-quadruplex-duplex junctions and to simplified models of telomeric t-loops, where the telomeric 3'-ovehang is protected through invasion into an adjacent duplex segment. Ligand-induced stabilization of telomeric G-quadruplex scaffolds in humans, resulting in the inhibition of telomerase activity, constitutes a promising strategy for anti-cancer drug development. We propose to structurally characterize complexes of ligands exhibiting unique selectivity towards telomeric, intronic, oncogenic promotor and 5'-UTR G-quadruplexes identified in our laboratory (Aim 4), thereby providing structural insights into the action of potent inhibitors of telomerase and oncogene regulation at the level of transcription (promoters) and translation (5'- UTR). Our initial efforts are focused on oxazole-containing macrocycles, analogs of telomestatin, the most potent inhibitor of telomerase, but will be expanded to promising small-molecule shape-sensitive G-quadruplex-interacting ligands. There are no structures reported in the literature of G-quadruplexes bound to peptides and proteins. To address this issue (Aim 5), we are currently undertaking the NMR-based structural characterization of the complex between L-vasopressin and an in vitro selected mirror-image 38-mer L-RNA aptamer (spiegelmer) that folds into a G-quadruplex scaffold. PUBLIC HEALTH RELEVANCE: Four-stranded nucleic acid scaffolds, designated G-quadruplexes are formed at guanine-rich tracts of oncogenic promoters, telomeres and intronic sequences, as well as 5'-untranslated regions of RNAs. Small molecule ligands can target and discriminate amongst the diverse sequence-dependent topologies of G-quadruplexes, thereby stabilizing their conformations and impacting on gene regulation at the transcriptional and translational levels. This application applies primarily NMR methods to determine the structures of G-quadruplex folds and their ligand complexes to decipher rules governing higher-order nucleic acid architecture and the principles dictating molecular recognition, with promising impact on human health, as reflected for instance, in the potential of stabilized G-quadruplexes generated at telomeric ends in inhibiting the activity of telomerase, an enzyme upregulated in tumor cells.

描述（由申请人提供）：在真核生物基因组的关键区段（包括端粒、内含子和致癌启动子DNA区域）以及致癌RNA转录物的5 '-非翻译区（UTR）内观察到富含鸟嘌呤的片段。这种推定的G-四链体形成序列在原癌基因（促进细胞增殖）中普遍存在，而在肿瘤抑制基因（维持基因组稳定性）中基本上缺乏。存在结合、切割、分解、促进和破坏G-四链体形成的细胞蛋白，最近的研究为体内G-四链体形成提供了越来越多的支持。我们的实验室正在进行的项目，旨在NMR和X射线结构表征的G-四链体拓扑结构形成的鸟嘌呤丰富的tracts在c-myc，c-kit，VEGF和c-RET致癌DNA启动子（目标1），在人类端粒和内含子DNA（目标2），并在N-ras，和相关的致癌RNA 5 '-UTR片段（目标3）。这些结构的研究应该定义的折叠倾向和多样性的G-四链体拓扑结构，以及构象状态之间的相互转换的能量学。这项研究将扩展到G-四链体-双链体连接的结构表征和端粒T环的简化模型，其中端粒3 '-末端通过侵入相邻的双链体片段而受到保护。配体诱导的端粒G-四链体支架在人类中的稳定化，导致端粒酶活性的抑制，构成了抗癌药物开发的有前途的策略。我们建议在结构上表征配体的复合物，这些配体对我们实验室中鉴定的端粒、内含子、致癌启动子和5 '-UTR G-四链体表现出独特的选择性（Aim 4），从而在转录（启动子）和翻译（5'-UTR）水平上提供对端粒酶和致癌基因调控的有效抑制剂的作用的结构见解。我们最初的努力集中在含恶唑的大环化合物，类似物的端粒酶抑制剂，最有效的端粒酶抑制剂，但将扩大到有前途的小分子形状敏感的G-四链体相互作用配体。文献中没有报道与肽和蛋白质结合的G-四链体的结构。为了解决这个问题（目标5），我们目前正在进行基于NMR的复合物之间的结构表征的L-加压素和在体外选择的镜像38聚体L-RNA适体（spiegelmer），折叠成一个G-四链体支架。 公共卫生相关性：四链核酸支架，称为G-四链体，形成于致癌启动子、端粒和内含子序列的富含鸟嘌呤的区域，以及RNA的5 '-非翻译区。小分子配体可以靶向和区分G-四链体的不同序列依赖性拓扑结构，从而稳定它们的构象并在转录和翻译水平上影响基因调控。本申请主要应用NMR方法来确定G-四链体折叠及其配体复合物的结构，以破译支配高阶核酸结构的规则和支配分子识别的原理，对人类健康具有有希望的影响，例如，如在抑制端粒酶活性中在端粒末端产生的稳定的G-四链体的潜力所反映的，端粒酶是一种在肿瘤细胞中上调的酶。