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

项目摘要 这项研究提案的目标是开发新技术，用于识别长时间内的区域。 参与R环和RNA三链体形成的非编码RNA。长链非编码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。 LncRNA通过与特定蛋白质的结合靶向染色质，或者它们可以直接相互作用 与染色质中的DNA结合。lncRNA直接接触DNA的程度和负责的RNA区域 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鸿沟 这些技术将产生所有核RNA中与DNA相互作用的区域的高分辨率地图， R环和三链体结构。染色质上的RNA-DNA相互作用通过形成两种不同的 结构：1）通过RNA和其互补DNA之间的沃森-克里克碱基配对形成的R环 2）RNA-三链体，因为RNA占据双螺旋大沟并形成 与沃森-克里克DNA链中的嘌呤形成Hoogsteen或反向Hoogsteen氢键。抗体- 存在依赖性和非依赖性策略来鉴定R环结构的全基因组分布。 然而，这些方法中的大多数测序R环的DNA组分。虽然来自 DNA的观点提供了一个R环在基因组中形成的观点，它不能回答R环是否 在特定基因组区域的环作为转录的副产物或由于调节因子的存在而形成。 非编码RNA。此外，现有的回收R-RNA组分的抗体依赖性方法， 循环缺乏灵敏度、特异性和分辨率。了解R环的RNA成分将使我们能够 将可能涉及lncRNA的调控R环与共转录R环区分开来，并发现新的 基因组调控中基于R环的机制。对识别RNA三链体的新工具的需求更为迫切。 虽然已经开发了预测三链体形成的计算方法，但检测三链体形成的基因组方法仍然存在。 体内不存在内源性三链体。检测R环和RNA三链体中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 工具可以应用于检查编码和非编码RNA在与染色质的相互作用中如何不同， 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 采用表位转录组学来富集和鉴定 R环和RNA三链体。

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.