Transcriptome Analysis with RNA-Reactive Probes

使用 RNA 反应探针进行转录组分析

• 批准号: 10602470
• 负责人: ERIC T. KOOL
• 金额: $ 62.34万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 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
• 来源: https://reporter.nih.gov/project-details/10602470
• 关键词: Address; Affect; Binding Sites; Biology; Biotin; Cells; Chemicals; Clinical; Complex; Crosslinker; Drug Interactions; Engineering; Future; Health; Human; Hydrophobicity; Imidazole; Label; Laboratories; Length; Ligand Binding; Ligands; Light; Mammalian Cell; Maps; Mediating; Messenger RNA; Metabolite Interaction; Methodology; Methods; Modification; Molecular; Pathway interactions; Permeability; Pharmaceutical Preparations; Proteins; Proteome; RNA; RNA Binding; RNA analysis; RNA vaccine; Reaction; Reagent; Research; Research Personnel; Site; Structure; System; Technology; Therapeutic; Therapeutic Agents; Toxic effect; Work; base; clinically relevant; design; improved; insight; next generation; next generation sequencing; novel; novel strategies; programs; secondary metabolite; small molecule; therapeutic RNA; therapeutic target; tool; transcriptome; transcriptome sequencing

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.

在过去的十年里，许多实验室的工作强调了RNA介导的细胞凋亡的至关重要性。 通路，以及特定RNA与人类健康的明确联系。RNA越来越被视为 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种类相互作用的更深入和更广泛的理解将 为RNA网络和功能提供了重要的新见解，为当前的 药物引起细胞毒性，并为改进RNA疗法提供了新的见解。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相互作用远远落后于蛋白质和蛋白质组分析。 该实验室最近的工作建立了许多新的分子工具，用于分析 生物学和临床相关的RNA。我们开发了第一个高产率的反应策略， RNA 2 '-OH基团，建立了酰基咪唑试剂的广泛用途。我们设计了可渗透细胞的 广泛使用的结构绘图试剂NAI和NAI-N3 -现在可商购获得-并将它们应用于 利用RNA Seq，我们绘制了哺乳动物细胞中16000个mRNA的折叠结构。我们发展迅速， 用荧光标记、生物素、疏水基团 交联剂和笼状基团。此外，我们设计了广泛或特定标记RNA的策略 网站.与最近用于RNA标记的酶促方法不同，我们的方法不需要工程化的结构， 序列，因此可以快速和容易地与任何来源或长度的天然RNA一起使用。 拟议中的项目将把我们的RNA工作整合成一个广泛的计划，该计划将开发一套新的 RNA反应试剂和方法，并将应用它们提供具体的，定量的信息， 配体与转录组的相互作用。我们将开发一流的方法来功能化本机 在特定位点的RNA，以及用红光控制RNA的新策略。结合我们的活性酰基 工具和方法与下一代测序，我们将查明和量化配体结合位点的整体 转录组这些方法统称为基于反应性的RNA分析（RBRP）， 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产物的RNA相互作用，并分析如何修饰碱基 在下一代mRNA疫苗和治疗中，基因突变会影响它们在细胞中的结构和相互作用。这 这项工作很重要，因为它寻求有关RNA的全系统临床相关问题的答案 交互.此外，它还开发了2 '-OH基团，作为一种几乎通用的手柄，用于操纵，缀合， 和RNA的研究，引入使分子技术，将广泛受益于研究人员在 RNA生物学领域，并有助于改善未来的RNA疗法。