Transcriptome Analysis with RNA-Reactive Probes

在过去的十年里，许多实验室的研究已经强调了RNA介导的细胞的关键重要性 途径，以及特定RNA与人类健康的明确联系。RNA越来越多地被视为 吸引人的治疗靶点，以及作为治疗剂本身。我们假设获得一个 对配体如何与细胞的许多RNA物种相互作用的更深入和更广泛的理解将 对RNA网络和功能提供重要的新见解，提供对当前 药物会引起细胞毒性，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， 直接评估细胞中的RNA相互作用远远落后于蛋白质和蛋白质组分析。 这个实验室最近的工作已经建立了许多新的分子工具来分析 与生物学和临床相关的RNA。我们开发了第一个用于功能化的高产率反应策略 RNA2‘-OH基团，建立了用途广泛的酰基咪唑试剂。我们设计了细胞渗透性和 广泛使用的结构作图试剂NAI和NAI-N3--现已商业化--及其应用 使用RNA Seq，我们在哺乳动物细胞中绘制了16000个mRNA的折叠结构。我们开发了快速和 利用荧光标记、生物素、疏水基团使RNA功能化的简单化学方法， 交联剂和笼状基团。此外，我们设计了广泛或具体标记rna的策略。 网站。与最近的酶标记RNA方法不同，我们的方法不需要工程结构或 序列，因此可以快速和容易地与任何来源或长度的原生RNA一起使用。 拟议的项目将把我们的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反应试剂和方法，并将应用它们来提供关于 配基与转录组的相互作用。我们将开发一流的本机函数化方法 在特定位置的RNA，以及用红灯控制RNA的新策略。结合我们的反应性酰基 工具和方法通过下一代测序，我们将在整体上精确定位和量化配体结合位点 转录组。这些方法，一起被称为基于反应的RNA分析(RBRP)，将是 用于分析已知的具有临床限制性毒性的小分子药物与靶外RNA的结合，以 分析内源性次生代谢物的RNA相互作用，以及如何修饰碱基的分析 在下一代信使核糖核酸中，疫苗和疗法会影响它们在细胞内的结构和相互作用。这 这项工作意义重大，因为它寻求有关RNA的全系统临床相关问题的答案 互动。此外，它将2‘-OH基团发展为几乎通用的手柄，用于操纵、接合、 和对RNA的研究，引入使能分子技术，将使研究人员在 这项研究将促进RNA生物学领域的研究，并有助于改进未来的RNA疗法。

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. Recent work from this laboratory has established numerous new molecular tools for analysis of biologically and clinically relevant RNAs. We developed the first high-yield reaction strategy for functionalizing RNA 2'-OH groups, establishing broad utility of acylimidazole reagents. We designed the cell-permeable and broadly used structure-mapping reagents NAI and NAI-N3 - now commercially available - and applying them with RNA Seq, we mapped folded structures of 16000 mRNAs in mammalian cells. We developed rapid and simple chemical approaches for functionalizing RNA with fluorescent labels, biotin, hydrophobic groups, crosslinkers, and caging groups. Further, we designed strategies for labeling RNA either broadly or at specific sites. Unlike recent enzymatic approaches for RNA labeling, our methods require no engineered structure or sequence, and thus can be employed rapidly and easily with native RNAs of any origin or length. The proposed 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.