Developing a genomic toolkit to identify RNAs within non-canonical DNA structures

项目总结 这项研究计划的目标是开发新的技术，以识别龙的区域 参与R-环和RNA三链形成的非编码RNA。长的非编码RNA(LncRNA)可以 定位于染色质并调节重要的核过程，如转录、重组和修复。 LncRNAs通过与特定蛋白质的结合或直接相互作用而被靶向染色质 染色质中的DNA。LncRNAs直接接触DNA和负责的RNA区域的程度 因为这些联系在很大程度上是未知的。我们将通过开发新的基因组学来弥合这一知识差距 这些技术将产生高分辨率的区域地图，显示所有核RNA中通过 R-环和三链结构。RNA-DNA在染色质上的相互作用是通过形成两个不同的 结构：1)RNA及其互补DNA之间通过Watson-Crick碱基配对形成的R-环 链，以及2)RNA-形成三联体，因为RNA占据双螺旋的主槽并形成 Hoogsteen或反向Hoogsteen氢键与Watson-Crick DNA链中的嘌呤形成氢键。抗体- 存在依赖和独立的策略来识别R-环结构的全基因组分布。 然而，这些方法中的大多数都是对R环的DNA成分进行测序。而来自 DNA透视提供了整个基因组中R-环形成的位置的观点，它无法回答R-环是否在基因组中形成 在特定基因组区域形成的环作为转录的副产物或由于调节基因的存在而形成 非编码RNA。此外，现有的依赖抗体的方法恢复R-RNA组分- 循环缺乏敏感性、特异性和分辨率。了解R环的RNA成分将使我们能够 区分调节性R-环和共转录R-环，发现新的 基因组调控中基于R-环的机制。对识别RNA三联体的新工具的需求更加迫切。 虽然已经开发了预测三联体形成的计算方法，但基因组方法检测 体内不存在内源性三联体。检测R环和RNA三联体中的RNA的新方法将 增强我们对这些结构的理解，并确定它们在基因调控中的功能程度。这些 可以使用工具来研究编码和非编码RNA在与染色质和 可以帮助定义通过RNA相互作用进行基因调控的新原则。在这里，我们建议开发新的 不依赖抗体的策略，利用表位转录组学来丰富和鉴定病毒的RNA组分 R环和RNA三联体。

PROJECT SUMMARY The goal of this research proposal is to develop new technologies for the identification of regions within long non-coding RNAs that engage in R-loop and RNA-triplex formation. Long non-coding RNAs (lncRNAs) can localize to chromatin and regulate important nuclear processes such as transcription, recombination, and repair. LncRNAs are targeted to chromatin through their association with specific proteins or they can interact directly with the DNA in chromatin. The extent to which lncRNAs directly contact DNA and the RNA regions responsible for these connections are largely unknown. We will bridge this gap in knowledge by developing new genomics technologies that will yield high-resolution maps of regions within all nuclear RNAs that interact with DNA through R-loop and triplex structures. RNA-DNA interactions on chromatin occur through the formation of two distinct structures: 1) R-loops that form through Watson-Crick base pairing between RNA and its complementary DNA strand, and 2) RNA-triplexes that form because an RNA occupies the major groove of the double helix and forms Hoogsteen or reverse Hoogsteen hydrogen bonds with the purines in the Watson-Crick DNA strands. Antibody- dependent and -independent strategies exist to identify the genome-wide distribution of R-loop structures. However, most of these methods sequence the DNA component of R-loops. While information from the perspective of DNA provides a view of where R-loops form across the genome, it cannot answer whether R- loops at specific genomic regions form as a by-product of transcription or because of the presence of regulatory non-coding RNAs. Moreover, the existing antibody-dependent approach to recover the RNA component of R- loops lacks sensitivity, specificity, and resolution. Knowledge of the RNA component of R-loops will allow us to distinguish regulatory R-loops, which may involve lncRNAs, from co-transcriptional R-loops and discover new R-loop based mechanisms in genome regulation. The need for new tools to identify RNA-triplexes is more urgent. While computational approaches to predict triplex formation have been developed, genomic methods to detect endogenous triplexes in vivo do not exist. New methods to detect RNAs within R-loops and RNA-triplexes will enhance our understanding of these structures and identify the extent of their function in gene regulation. These tools can be applied to examine how coding and non-coding RNAs differ in their interactions with chromatin and can help define new principles for gene regulation through RNA interactions. Here, we propose to develop new antibody-independent strategies that employ epitranscriptomics to enrich for and identify the RNA component of R-loops and RNA-triplexes.