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

开发基因组工具包来识别非规范 DNA 结构中的 RNA

• 批准号: 10506451
• 负责人: Kavitha Sarma
• 金额: $ 27.35万
• 依托单位: WISTAR INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-22 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10506451
• 关键词: Antibodies; Base Pairing; Behavior; Binding; Biochemical; Biological; Chemistry; Chromatin; Chromatin Structure; Code; Complementary DNA; Cytosine; DNA; DNA Sequence; DNA Structure; Detection; Development; Disease; Enzymes; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Genome; Genomic Segment; Genomics; Goals; Hydrogen Bonding; Knowledge; Libraries; Location; Major Groove; Maps; Methodology; Methods; Modification; Nuclear; Nuclear RNA; Pathogenicity; Preparation; Process; Proteins; Purines; RNA; RNA methylation; Regulation; Regulator Genes; Research Proposals; Resolution; Ribonuclease H; Role; Sensitivity and Specificity; Specific qualifier value; Structure; Technology; Untranslated RNA; Work; base; bisulfite; epitranscriptomics; genome-wide; in vivo; insight; new technology; nuclease; nucleic acid structure; programs; recombinational repair; targeted nucleases; tool; triplex DNA

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.

项目概要 该研究计划的目标是开发新技术来识别长期内的区域 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，例如转录、重组和修复。 LncRNA 通过与特定蛋白质结合来靶向染色质，或者它们可以直接相互作用 与染色质中的 DNA 一起。 lncRNA 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 技术将产生所有核 RNA 内与 DNA 相互作用的区域的高分辨率图谱 R 环和三链体结构。 RNA-DNA 在染色质上的相互作用是通过形成两种不同的 结构：1) 通过 RNA 与其互补 DNA 之间的 Watson-Crick 碱基配对形成的 R 环 链，以及 2) 由于 RNA 占据双螺旋的主沟并形成而形成的 RNA 三链体 Hoogsteen 或反向 Hoogsteen 氢键与 Watson-Crick DNA 链中的嘌呤形成。抗体- 存在依赖和独立策略来识别 R 环结构的全基因组分布。 然而，大多数这些方法都是对 R 环的 DNA 成分进行测序。虽然信息来自 DNA 的视角提供了 R 环在基因组中形成的位置的视图，但它无法回答 R 是否 特定基因组区域的环作为转录的副产物或由于调节的存在而形成 非编码RNA。此外，现有的依赖抗体的方法来回收 R- 的 RNA 成分 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 区分可能涉及 lncRNA 的调节 R 环与共转录 R 环，并发现新的 基于 R 环的基因组调控机制。对识别 RNA 三链体的新工具的需求更加迫切。 虽然已经开发出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 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 工具可用于检查编码和非编码 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理。在此，我们建议开发新的 不依赖于抗体的策略，利用表观转录组学来富集和鉴定RNA成分 R 环和 RNA 三链体。