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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.

父项目摘要（R35 GM145357）许多实验室过去十年的工作强调了 RNA 介导的至关重要性细胞通路以及特定 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药物他们自己。我们假设获得更深入和更广泛的理解配体与细胞中许多RNA种类的相互作用将为以下方面提供重要的新见解 RNA网络和功能，提供了对当前药物如何引起细胞损伤的新认识毒性，并为改进 RNA 疗法提供新的见解。我们深信，转录组范围内 RNA 相互作用的分析对于未来的生物医学至关重要。不幸的是，直接评估细胞内 RNA 相互作用的方法远远落后于那些方法。用于蛋白质和蛋白质组分析。该项目将把我们的 RNA 工作整合为一个广泛的计划，该计划将开发一套新的 RNA反应试剂和方法，并将应用它们来提供特异性、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位点功能化天然 RNA 的方法，以及控制的新策略红光下的 RNA。将我们的反应性酰基工具和方法与下一代测序相结合，我们将查明并量化整个转录组中的配体结合位点。这些统称为基于反应性的 RNA 分析 (RBRP) 的方法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，分析内源次级代谢物的 RNA 相互作用，并分析如何下一代 mRNA 疫苗和疗法中的修饰碱基会影响其结构和细胞内的相互作用。这项工作意义重大，因为它寻求全系统范围的答案有关 RNA 相互作用的临床相关问题。此外，它将 2'-OH 基团发展为用于操作、接合和研究 RNA 的几乎通用的手柄，引入使分子技术广泛惠及 RNA 领域的研究人员生物学并为改善未来的 RNA 疗法做出贡献。