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.

项目摘要 该研究建议的目的是开发新技术，以识别长期内的区域 参与R环和RNA三重形成的非编码RNA。长的非编码RNA（LNCRNA）可以 本地化为染色质并调节重要的核过程，例如转录，重组和修复。 LNCRNA通过与特定蛋白的缔合靶向染色质，或者它们可以直接相互作用 与染色质中的DNA。 LNCRNA直接接触DNA和负责RNA区域的程度 因为这些连接在很大程度上未知。我们将通过开发新的基因组学来弥合知识的差距 将在与DNA相互作用的所有核RNA中产生高分辨率区域图的技术 R环和三重结构。染色质上的RNA-DNA相互作用是通过形成两个不同的 结构：1）通过RNA及其互补DNA之间的Watson-Crick碱基对形成的R环 链，以及2）形成的RNA三倍体，因为RNA占据了双螺旋的主要凹槽并形成 Hoogsteen或反向Hoogsteen氢与Watson-Crick DNA链中的嘌呤键合。抗体- 存在依赖和非依赖性的策略，以识别R环结构的全基因组分布。 但是，这些方法中的大多数将R-loops的DNA成分序列。而来自 DNA的透视图提供了整个基因组中R环形成的位置的观点，它无法回答是否R- 特定基因组区域的循环形式为转录的副产品或由于存在调节 非编码RNA。此外，现有的抗体依赖性方法是恢复R-的RNA成分 循环缺乏灵敏度，特异性和分辨率。了解R-loops的RNA成分将使我们能够 区分可能涉及LNCRNA的调节R环与共同转录R环并发现新的 基因组调节中基于R环的机制。需要新工具来识别RNA三重工具的需求更为紧急。 尽管已经开发了预测三重形成的计算方法，但基因组方法检测 体内不存在内源性三倍体。检测R环和RNA三倍体内RNA的新方法将 增强我们对这些结构的理解，并确定其在基因调节中的功能程度。这些 可以应用工具来检查编码和非编码RNA与染色质和染色质和 可以通过RNA相互作用来帮助定义基因调节的新原理。在这里，我们建议开发新的 采用表十一次的抗体策略来丰富并确定的RNA成分 R环和RNA三倍体。