Transcriptome Analysis with RNA-Reactive Probes

父项目摘要(R35 GM145357) 在过去的十年中，许多实验室的研究强调了RNA介导的关键重要性 细胞途径，以及特定RNA与人类健康的明确联系。RNA是 越来越多的人认为既是吸引人的治疗目标，也是治疗药物 他们自己。我们假设，对如何获得更深更广的理解 配体与细胞的许多RNA物种相互作用将提供重要的新见解 RNA网络和功能，提供了对当前药物如何导致细胞 毒性，并为改进RNA疗法提供了新的见解。我们确信， 对转录范围内的RNA相互作用的分析对未来的生物医学是必不可少的。 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 用于蛋白质和蛋白质组分析。 该项目将把我们的RNA工作整合到一个广泛的计划中，该计划将开发一套新的 RNA反应性试剂和方法，并将其应用于提供特异、定量 关于配体与转录组相互作用的信息。我们将发展一流 在特定位置使本地RNA功能化的方法以及用于控制的新策略 红灯下的RNA。将我们的反应性酰基工具和方法与下一代测序相结合， 我们将精确定位并量化整个转录组中的配体结合部位。这些 方法学，统称为基于反应的RNA分析(RBRP)，将应用于 分析已知的临床毒性有限的小分子药物与靶外RNA的结合， 分析内源性次生代谢物的RNA相互作用，并分析 下一代RNA疫苗和治疗药物中的修饰碱基影响其结构和 细胞内的相互作用。这项工作意义重大，因为它寻求系统范围内 关于RNA相互作用的临床相关问题。此外，它将2‘-OH基团发展为 一个几乎通用的处理、接合和研究RNA的句柄，介绍 使分子技术能够广泛惠及RNA领域的研究人员 生物学，并有助于改进未来的RNA疗法。

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.