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

描述(申请人提供)：在真核生物基因组的关键片段，包括端粒、内含子和致癌基因启动子DNA区域，以及致癌RNA转录本的5‘-非翻译区(UTRs)中观察到富含鸟嘌呤的区域。这种假定的G-四链形成序列普遍存在于原癌基因(促进细胞增殖)中，而基本上缺乏肿瘤抑制基因(维持基因组稳定性)。细胞中存在结合、裂解、分解、促进和破坏G-四链形成的蛋白质，最近的研究为体内G-四链的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是针对c-myc、c-kit、VEGF和c-RET致癌DNA启动子(目标1)、人类端粒和内含子DNA(目标2)、N-ras以及相关的致癌RNA5‘-UTR片段(目标3)中富鸟嘌呤链形成的G-四链拓扑的核磁共振和X射线结构表征。这些结构研究应该定义G-四链拓扑的折叠倾向和多样性，以及构象状态之间相互转化的能量学。这项研究将扩展到G-四链-双链连接的结构特征和端粒t-环的简化模型，其中端粒3‘-卵悬挂通过入侵相邻的双链片段来保护。配体诱导稳定人类端粒G-四链支架，从而抑制端粒酶活性，构成了抗癌药物开发的一种有前途的策略。我们建议对我们实验室(AIM 4)中发现的对端粒、内含子、致癌启动子和5‘-UTRG-四链具有独特选择性的配体络合物进行结构表征，从而在转录(启动子)和翻译(5’-UTR)水平上提供对端粒酶和癌基因调节的有效抑制物的作用的结构洞察力。我们最初的努力集中在含有恶唑的大环，端粒酶最有效的抑制剂端粒酶的类似物，但将扩展到有希望的小分子形状敏感的G-四链相互作用的配体。目前尚无文献报道G-四链体与多肽和蛋白质结合的结构。为了解决这个问题(目标5)，我们目前正在对L加压素与体外选择的镜像38聚体L核糖核酸适配子(Spiegelmer)之间的复合体进行基于核磁共振的结构表征，该适配子折叠成G-四链支架。 与公共卫生相关：四链核酸支架，被称为G-四链体，形成于致癌启动子、端粒和内含子序列以及RNA的5‘-非翻译区的富含鸟嘌呤的区域。小分子配体可以靶向和区分G-四链体的不同序列依赖的拓扑结构，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。这项应用主要应用核磁共振方法来确定G-四链折叠及其配体复合体的结构，以破译控制高阶核酸结构的规则和指导分子识别的原理，对人类健康有希望的影响，例如，反映在端粒末端产生的稳定的G-四链抑制端粒酶活性的可能性，端粒酶是一种在肿瘤细胞中上调的酶。

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.