Transcriptome Analysis with RNA-Reactive Probes

使用 RNA 反应探针进行转录组分析

基本信息

批准号：
10793323
负责人：
ERIC T. KOOL
金额：
$ 4.58万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-05-01 至 2027-04-30
项目状态：
未结题

项目摘要

Summary of parent project (R35 GM145357) Work in the last decade from many labs has underlined the critical importance of RNA-mediated cellular pathways, and clear connections of specific RNAs to human health. RNAs are increasingly viewed both as appealing therapeutic targets, and as therapeutic agents themselves. We hypothesize that obtaining a deeper and broader understanding about how ligands interact with the many RNA species of the cell will provide important new insights into RNA networks and functions, provide new understanding of how current drugs cause cellular toxicity, and lend novel insights into improving RNA therapies. We are convinced that the analysis of RNA interactions transcriptome-wide is essential to future biomedicine. Unfortunately, methods for assessing RNA interactions directly in the cell lag well behind those for protein and proteome analysis. This project will consolidate our RNA work into a broad program that will develop a new set of RNA-reactive reagents and methods, and will apply them to provide specific, quantitative information about ligand interactions with the transcriptome. We will develop first-in-class methods for functionalizing native RNAs at specific sites, and novel strategies for controlling RNAs with red light. Combining our reactive acyl tools and methods with next-gen sequencing, we will pinpoint and quantify ligand binding sites in the whole transcriptome. These methodologies, together termed Reactivity-Based RNA Profiling (RBRP), will be applied to analyzing off-target RNA binding by known small-molecule drugs with clinically limiting toxicity, to profiling RNA interactions of endogenous secondary metabolites, and to the analysis of how modified bases in next-generation mRNA vaccines and therapeutics affect their structures and interactions in the cell. This work is significant because it seeks answers to system-wide clinically-relevant questions regarding RNA interactions. Further, it develops the 2’-OH group as a nearly universal handle for manipulation, conjugation, and study of RNAs, introducing enabling molecular technologies that will broadly benefit researchers in the fields of RNA biology and contribute to improving future RNA therapies.

家长项目摘要（R35 GM145357）在过去十年中，许多实验室的工作强调了RNA介导的重要性细胞途径，以及特定RNA与人类健康的明确联系。 RNA是越来越多地将两者视为出现治疗靶标，又是治疗剂自己。我们假设，对如何获得更深入，更广泛的了解配体与细胞的许多RNA物种相互作用，将为您提供重要的新见解 RNA网络和功能，对当前药物如何引起细胞提供了新的了解毒性，并为改善RNA疗法提供新颖的见解。我们坚信 RNA相互作用整个转录组的分析对于未来的生物医学是必不可少的。不幸的是，直接在细胞滞后的细胞滞后中评估RNA相互作用的方法用于蛋白质和蛋白质分析。该项目将使我们的RNA工作合并为一个广泛的计划，该计划将开发一套新的 RNA反应性试剂和方法，并将它们应用于特定的定量有关配体与转录组相互作用的信息。我们将开发一流的在特定站点上功能化本机RNA的方法，以及控制的新型策略带红光的RNA。将我们的反应性酰基工具和方法与下一代测序相结合，我们将在整个转录组中查明并量化配体结合位点。这些方法将基于反应性的RNA分析（RBRP）一起应用于分析具有临床上限制毒性的已知小分子药物的脱靶RNA结合，分析内源性二级代谢产物的RNA相互作用，并分析下一代mRNA疫苗和治疗中的修改碱会影响其结构，细胞中的相互作用。这项工作很重要，因为它寻求整个系统范围的答案有关RNA相互作用的临床上的问题。此外，它将2'-oh群体发展为 RNA的操纵，结合和研究几乎通用的手柄，引入了启用将在RNA领域广泛受益的分子技术生物学并有助于改善未来的RNA疗法。