Probing the Transcriptome with Multifunctional Acylation Chemistry

用多功能酰化化学探索转录组

• 批准号: 9494223
• 负责人: ERIC T. KOOL
• 金额: $ 31.65万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-07-01 至 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9494223
• 关键词: Acylation; Azides; Biological; Biological Process; Biology; CRISPR/Cas technology; Cells; Chemicals; Chemistry; Complex; DNA; Data; Development; Disease; Family; Fluorescent Probes; Gene Activation; Gene Expression; Genes; Health; Human; Immobilization; Label; Light; Link; Measurement; Measures; Messenger RNA; Methods; Molecular; Molecular Probes; Nucleotides; One-Step dentin bonding system; Optics; Organism; Pathology; Peripheral; Permeability; Phenotype; Proteins; RNA; RNA Folding; RNA analysis; RNA-Protein Interaction; Reagent; Research; Research Personnel; Resolution; Role; Sampling; Science; Signal Transduction; Structure; Technology; Time; Untranslated RNA; Work; base; deacylation; design; experimental study; improved; in vivo; innovation; mRNA Expression; novel; physical separation; programs; public health relevance; scaffold; tool; transcriptome; unpublished works

Recent studies from many labs have uncovered great complexity in cellular RNA biology, and critically important connections of RNA to human health. It is becoming increasingly apparent that RNA biology, like protein biology, is not merely peripheral, but rather central to cellular phenotypes and pathologies. Unfortunately, methods for study of RNA, such as tools for functionalization, labeling and control, lag well behind those used widely for proteins. Preliminary experiments have established the promise of a suite of novel molecular strategies for study of RNAs, based on multifunctional acylating agents that react at the 2'-OH group. This started with the development of the first cell-permeable acylating agents, based on a nicotinyl scaffold, that react with accessible 2'-OH groups in RNAs. These reagents allow unprecedented measurement of RNA structure and protein-RNA interactions in vivo at nucleotide resolution. In unpublished work, studies have shown that an azide functional handle can be employed on these acylating scaffolds to enable mild, bioorthogonal reversal of the acylation by Staudinger reduction. Excitingly, experiments show that this acylation/deacylation strategy can be used to block and initiate hybridization of RNA. Moreover, the data establishes that a label can be incorporated into such an acylating agent, enabling one-step, reversible fluorescent labeling of native RNA. These preliminary experiments suggest a suite of new acylating reagents as tools to isolate, immobilize, label, and analyze RNAs, and a range of molecular strategies to control their biological activities with chemical or optical signals. During the term of this project, the development of reversible protecting reagents for stabilizing and capturing RNAs from biological samples are proposed. Reagents for covalent delivery and release of RNAs into cells are also described. Further, new fluorescent acylating agents and methods will be developed and employed to measure protein-RNA interactions. Finally, a novel range of unprecedented chemical caging and release strategies will be developed for controlling biological function of RNAs in living systems, enabling initiation of mRNA expression, RNA folding, and gene editing in time and space. This work is significant because it will develop enabling molecular technologies that will greatly enhance the study of RNA biology and biomedicine. This new premise of multifunctional acylation will lead to universal and easy-to-use reagents that will markedly improve the isolation, analysis, delivery, and control of RNAs for researchers worldwide. Unlike previous methods, these reagents will function with large and native RNAs, and are simple enough that non-chemists can apply them. The research program is innovative because it develops a suite of new molecular probes and novel molecular strategies, making use of the concept of reversible labeling and functionalization of RNA via new selective bond-forming and –breaking strategies.

许多实验室最近的研究揭示了细胞 RNA 生物学的巨大复杂性，并且至关重要 RNA与人类健康的重要联系。越来越明显的是，RNA 生物学，如 蛋白质生物学不仅是细胞表型和病理学的外围，而且是中心。 不幸的是，RNA 研究方法，例如功能化、标记和控制工具，严重滞后 落后于那些广泛用于蛋白质的物质。 初步实验已经确定了一套用于研究的新颖分子策略的前景 RNA，基于在 2'-OH 基团上反应的多功能酰化剂。这始于 开发出第一个基于烟酰支架的细胞渗透性酰化剂，它与 RNA 中可接近的 2'-OH 基团。这些试剂可以前所未有地测量 RNA 结构和 核苷酸分辨率下体内蛋白质-RNA 相互作用。在未发表的工作中，研究表明 叠氮化物功能手柄可用于这些酰化支架上，以实现温和的生物正交逆转 通过施陶丁格还原进行酰化。令人兴奋的是，实验表明这种酰化/脱酰化策略 可用于阻断和启动RNA杂交。此外，数据表明标签可以 掺入这种酰化剂中，可以对天然 RNA 进行一步、可逆的荧光标记。 这些初步实验提出了一套新的酰化试剂作为分离、固定、 标记和分析 RNA，以及一系列分子策略来控制其生物活性 化学或光学信号。本项目期间，可逆保护试剂的开发 提出了用于稳定和捕获生物样品中的RNA的方法。用于共价传递的试剂和 还描述了 RNA 释放到细胞中的情况。此外，新的荧光酰化剂和方法将被开发出来。 开发并用于测量蛋白质-RNA 相互作用。最后，一系列前所未有的新颖 将开发化学笼养和释放策略来控制活体中RNA的生物学功能 系统，能够在时间和空间上启动 mRNA 表达、RNA 折叠和基因编辑。 这项工作意义重大，因为它将开发使能分子技术，从而大大增强 RNA生物学和生物医学的研究。多功能酰化的这一新前提将导致普遍的 和易于使用的试剂将显着改善 RNA 的分离、分析、递送和控制 全世界的研究人员。与以前的方法不同，这些试剂将与大的天然 RNA 一起发挥作用， 并且非常简单，非化学家也可以应用它们。该研究计划具有创新性，因为它 利用以下概念开发了一套新的分子探针和新颖的分子策略 通过新的选择性成键和断裂策略对 RNA 进行可逆标记和功能化。