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.

许多实验室过去十年的工作强调了 RNA 介导的细胞的至关重要性 途径以及特定 RNA 与人类健康的明确联系。 RNA 越来越被视为 有吸引力的治疗靶点，以及作为治疗剂本身。我们假设获得 更深入、更广泛地了解配体如何与细胞的多种 RNA 相互作用 提供对 RNA 网络和功能的重要新见解，提供对当前如何进行的新理解 药物会引起细胞毒性，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，方法 直接评估细胞中 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来分析 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 RNA 2'-OH 基团，建立了酰基咪唑试剂的广泛用途。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（现已上市）及其应用 通过 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， 使用荧光标记、生物素、疏水基团对 RNA 进行功能化的简单化学方法 交联剂和笼化基团。此外，我们设计了广泛或特定标记 RNA 的策略 网站。与最近的 RNA 标记酶法不同，我们的方法不需要工程结构或 序列，因此可以快速、轻松地与任何来源或长度的天然 RNA 一起使用。 拟议的项目将把我们的 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有关RNA反应的具体、定量信息 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 特定位点的 RNA，以及用红光控制 RNA 的新策略。结合我们的反应性酰基 借助下一代测序的工具和方法，我们将在整个过程中精确定位和量化配体结合位点 转录组。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， 分析内源次级代谢物的 RNA 相互作用，并分析碱基修饰方式 下一代 mRNA 疫苗和治疗方法会影响它们在细胞中的结构和相互作用。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，它还开发了 2'-OH 基团作为操作、缀合、 和 RNA 的研究，引入使研究人员广泛受益的分子技术 RNA 生物学领域，并为改善未来的 RNA 疗法做出贡献。