Probing the Transcriptome with Multifunctional Acylation Chemistry

用多功能酰化化学探索转录组

• 批准号: 9926279
• 负责人: ERIC T. KOOL
• 金额: $ 31.87万
• 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/9926279
• 关键词: 3-Dimensional; 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; Optics; Organism; Pathology; Peripheral; Permeability; Phenotype; Proteins; RNA; RNA Folding; RNA analysis; RNA delivery; RNA-Protein Interaction; Reagent; Research; Research Personnel; Resolution; Role; Sampling; Science; Signal Transduction; Structural Protein; 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mRNA Expression; novel; physical separation; programs; public health relevance; scaffold; tool; transcriptome; unpublished works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 In unpublished work, studies have shown that an azide functional handle can be employed on these acylating scaffolds to enable mild, bioorthogonal reversal of the acylation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.

最近来自许多实验室的研究揭示了细胞RNA生物学的巨大复杂性，而且至关重要的是 核糖核酸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，不仅是外围的，而且是细胞表型和病理学的中心。 不幸的是，研究RNA的方法，如功能化、标记和控制工具，都滞后得很好 落后于那些被广泛用于蛋白质的物质。 初步实验已经确定了一套新的分子策略研究的前景 RNAs，基于在2‘-OH基上反应的多功能酰化剂。这始于 基于尼古丁支架的第一种细胞渗透型酰化剂的开发 RNA中可访问的2‘-OH基团。这些试剂可以前所未有地测量RNA的结构和 核苷酸分辨率下蛋白质-RNA在体内的相互作用。在未发表的研究中，研究表明， 可以在这些酰化支架上使用叠氮功能手柄，以实现温和的、生物正交的反转 以Staudinger还原的酰化反应。令人兴奋的是，实验表明，这种酰化/去酰化策略 可用于阻断和启动RNA的杂交。此外，数据建立了标签可以 加入到这样的酰化剂中，实现了天然RNA的一步可逆荧光标记。 这些初步实验表明，一套新的酰化试剂可以作为分离、固定化、 标记和分析RNA，以及一系列控制其生物活性的分子策略 化学或光学信号。在本项目期间，可逆保护剂的发展 提出了一种从生物样本中稳定和捕获RNA的方法。共价递送试剂和 还描述了将RNA释放到细胞中的过程。此外，新的荧光酰化试剂和方法将是 开发并用于测量蛋白质-RNA相互作用。最后，一系列史无前例的 将开发化学笼化和释放策略来控制RNA在生物中的生物功能 系统，能够启动mRNA表达、RNA折叠和在时间和空间上的基因编辑。 这项工作意义重大，因为它将开发使能分子技术，这将大大提高 核糖核酸生物学和生物医学的研究。这一多功能酰化的新前提将导致普遍 以及易于使用的试剂，将显著改善RNA的分离、分析、传递和控制 世界各地的研究人员。与以前的方法不同，这些试剂将与大的和天然的RNA一起发挥作用， 而且足够简单，非化学家也可以应用。这项研究计划具有创新性，因为它 开发一套新的分子探针和新的分子策略，利用 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。