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

描述（由申请人提供）：在真核生物基因组的关键片段中观察到富鸟嘌呤束，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（UTRs）。这种假定的g -四重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）中基本缺乏。细胞中存在结合、切割、分解、促进和破坏g -四重体形成的蛋白质，最近的研究越来越多地支持g -四重体在体内的形成。我们实验室正在进行的项目旨在对c-myc、c-kit、VEGF和c-RET致癌DNA启动子（Aim 1）、人类端粒和内含子DNA （Aim 2）、N-ras和相关致癌RNA 5'-UTR片段（Aim 3）中富含鸟嘌呤的束形成的g -四重体拓扑结构进行核磁共振和x射线结构表征。这些结构研究应该定义g -四复体拓扑结构的折叠倾向和多样性，以及构象状态之间相互转换的能量学。该研究将扩展到g -四工-双工连接的结构表征和简化的端粒t环模型，其中端粒3'-悬垂通过侵入相邻的双工段而受到保护。配体诱导的端粒g -四重体支架的稳定，导致端粒酶活性的抑制，构成了抗癌药物开发的一个有前途的策略。我们建议从结构上表征我们实验室鉴定的对端粒、内含子、致癌启动子和5'-UTR g -四联体具有独特选择性的配体复合物（Aim 4），从而在转录（启动子）和翻译（5'- UTR）水平上对端粒酶和癌基因调控的有效抑制剂的作用提供结构上的见解。我们最初的努力集中在含恶唑的大环，端粒酶最有效的抑制剂端粒抑素的类似物，但将扩大到有前途的小分子形状敏感的g -四联体相互作用配体。文献中还没有报道g -四联体与多肽和蛋白质结合的结构。为了解决这个问题（Aim 5），我们目前正在对l -加压素和体外选择的镜像38-mer L-RNA适体（spiegelmer）之间的复合物进行基于核磁共振的结构表征，该复合物可折叠成g -四重支架。

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.