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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.

母项目总结（R35 GM 145357）在过去的十年里，许多实验室的工作强调了RNA介导的细胞通路，以及特定RNA与人类健康的明确联系。rna是越来越多地被视为有吸引力的治疗靶点和治疗剂，自己我们假设，获得更深入和更广泛的了解，配体与细胞的许多RNA种类相互作用将提供重要的新见解， RNA网络和功能，为当前药物如何引起细胞凋亡提供了新的理解。毒性，并为改善RNA疗法提供新的见解。我们深信全转录组范围的RNA相互作用分析对未来的生物医学是必不可少的。不幸的是，直接在细胞中评估RNA相互作用的方法远远落后于这些方法。用于蛋白质和蛋白质组分析。这个项目将把我们的RNA工作整合成一个广泛的计划， RNA反应试剂和方法，并将应用它们提供特异性，定量关于配体与转录组相互作用的信息。我们将开发一流的在特定位点功能化天然RNA的方法，红色发光的RNA。将我们的反应酰基工具和方法与下一代测序相结合，我们将精确定位并量化整个转录组中的配体结合位点。这些方法，统称为基于反应性的RNA分析（RBRP），将应用于分析具有临床限制毒性的已知小分子药物的脱靶RNA结合，分析内源性次级代谢物的RNA相互作用，并分析如何下一代mRNA疫苗和治疗剂中的修饰碱基影响它们的结构，细胞内的相互作用。这项工作意义重大，因为它寻求解决全系统的问题，关于RNA相互作用的临床相关问题。此外，它还产生了2 '-OH基团，一个几乎通用的手柄，用于RNA的操作、接合和研究，使分子技术能够使RNA领域的研究人员广泛受益，生物学和有助于改善未来的RNA疗法。