Probing the Transcriptome with Multifunctional Acylation Chemistry

最近许多实验室的研究揭示了细胞RNA生物学的巨大复杂性， RNA与人类健康的重要联系。越来越明显的是，RNA生物学，如 蛋白质生物学不仅仅是细胞表型和病理学的外围，而是中心。 不幸的是，RNA的研究方法，如功能化，标记和控制工具，都很落后 被广泛应用于蛋白质。 初步的实验已经确立了一套新的分子策略的研究承诺， RNA，基于在2 '-OH基团反应的多功能酰化剂。这一切始于 开发了第一种基于烟碱基支架的可渗透细胞的酰化剂， RNA中的可接近的2 '-OH基团。这些试剂允许前所未有地测量RNA结构， 蛋白质-RNA相互作用在体内的核苷酸分辨率。在未发表的工作中，研究表明， 叠氮化物功能柄可用于这些酰化支架上以实现温和生物正交逆转 通过施陶丁格还原的酰化。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的杂交。此外，数据表明，标签可以 掺入到这样的酰化剂中，使得能够一步可逆地荧光标记天然RNA。 这些初步实验表明，一套新的酰化试剂作为工具，分离， 标记和分析RNA，以及一系列分子策略来控制它们的生物活性， 化学或光学信号。在本项目期间，可逆保护剂的开发 用于从生物样品中稳定和捕获RNA。用于共价递送的试剂和 还描述了RNA向细胞中的释放。此外，将开发新的荧光酰化剂和方法。 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 化学笼和释放策略将被开发用于控制RNA在生活中的生物学功能， 系统，能够启动mRNA表达，RNA折叠和基因编辑的时间和空间。 这项工作意义重大，因为它将开发使能分子技术， RNA生物学和生物医学的研究。这种多功能酰化的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，交付和控制， 全世界的研究人员。与以前的方法不同，这些试剂将与大的和天然的RNA一起起作用， 而且非常简单，非化学家也能应用。该研究项目具有创新性，因为它 开发了一套新的分子探针和新的分子策略，利用的概念， 通过新的选择性键形成和断裂策略可逆标记和功能化RNA。

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 In unpublished work, studies have shown that an azide functional handle can be employed on these acylating scaffolds to enable mild, bioorthogonal reversal of the acylation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.