Covalent Profiling of RNA Targets and Off-targets

许多实验室最近的工作强调了RNA控制的细胞通路的至关重要性， 特定RNA与人类健康的临床相关联系。因此，发展小- 与传统的蛋白质靶点相比，RNA的分子配体现在提供了一种有前途的方法， 未来的疾病目标。除了这种设计方法之外，似乎目前许多蛋白质- 靶向药物可以很好地与RNA脱靶结合并在那里引起非预期的生物学效应。因此 RNA-小分子相互作用的转录组范围的分析是至关重要的。不幸的是， 直接在细胞中分析RNA-配体相互作用远远落后于蛋白质-配体结合分析。 该实验室的初步实验已经建立了多种新的分子工具，用于分析 生物学和临床相关的RNA。已经开发了新的基于RNA Seq的方法， 直接识别RNA碱基修饰。这个新项目的核心是最近的发展， 第一种细胞可渗透的RNA酰化剂，其基于烟碱基支架，与可接近的2 ′-OH反应 组转录组范围内。这使得前所未有的测量RNA结构和蛋白质-RNA 在核苷酸分辨率下的体内相互作用。在最近的工作中，新的酰化试剂，可以多酰化 已经开发了暂时灭活RNA的体外RNA。然后可以将这些组移除 以化学或光化学方式暂时或局部控制RNA活性。总的来说，这部新作品 提出了一套新的分子试剂，工具和测序方法，可以直接用于细胞 在一个实验中分析整个转录组的配体-RNA相互作用。 拟议的项目将开发一套新的功能化RNA反应试剂，包括酰化 和烷基化物质，它们可以进入细胞并提供有关配体结合的特异性定量信息 在转录组中。结合下一代测序，该方法将精确定位特定的结合位点。 成绩单这些方法，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药物的脱靶结合。此外，新的基于反应性的 包括反应性药物样片段的方法将用于鉴定癌症相关RNA的配体。 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，在转录组范围内直接分析药物相互作用， cell.此外，它将概述蛋白质靶向药物脱靶结合的现象有多严重， RNA。该研究计划是创新的，因为它开发了一套新的分子探针和新的 分子策略，利用RNA反应性。它将开发前所未有的数据， 临床上有用但有毒的药物。它还将开发新的细胞渗透性配体，用于在细胞内上调的RNA。 癌症，它可以广泛用作癌症研究的分子工具。

Recent work from many labs has underlined the critical importance of RNA-controlled cellular pathways, and clinically relevant connections of specific RNAs to human health. As a result, the development of small- molecule ligands for RNAs, in contrast to traditional protein targets, is now offering a promising approach for future targeting of diseases. In addition to this designed approach, it seems likely that many current protein- targeted drugs may well bind to RNAs off-target and cause unintended biological effects there. Thus, the analysis of RNA-small molecule interactions transcriptome-wide is critical. Unfortunately, methods for analyzing RNA-ligand interactions directly in the cell lag far behind those for protein-ligand binding analysis. Preliminary experiments from this laboratory have established multiple new molecular tools for analysis of biologically and clinically relevant RNAs. New RNA Seq-based approaches have been developed for identifying RNA base modifications directly. Central to this new project was the recent development of the first cell-permeable RNA acylating agents, based on a nicotinyl scaffold, that react with accessible 2'-OH groups transcriptome-wide. This allows unprecedented measurement of RNA structure and protein-RNA interactions in vivo at nucleotide resolution. In very recent work, new acylating reagents that can polyacylate RNAs in vitro, temporarily inactivating the RNA have been developed. These groups can then be removed chemically or photochemically to control RNA activity temporally or locally. Overall, this recent new work suggests a suite of new molecular reagents, tools and sequencing methods that can be used directly in cells to analyze ligand-RNA interactions for the whole transcriptome in one experiment. The proposed project will develop a new set of functionalized RNA-reactive reagents, including acylating and alkylating species, that can enter cells and provide specific, quantitative information about ligand binding in the transcriptome. Combined with next-gen sequencing, the methods will pinpoint binding sites in specific transcripts. These methodologies, together termed Reactivity-Based RNA Profiling (RBRP), will be applied to analyzing off-target binding by known drugs with clinically limiting toxicity. Further, new reactivity-based approaches - involving reactive druglike fragments – will be used to identify ligands for cancer-related RNAs. This work is significant because it will develop enabling molecular technologies that will greatly enhance the study of RNA biology and biomedicine, analyzing drug interactions transcriptome-wide and directly in the cell. In addition, it will outline how serious is the phenomenon of protein-targeted drugs binding off target to RNAs. The research program is innovative because it develops a suite of new molecular probes and novel molecular strategies, making use of RNA reactivity. It will develop unprecedented data regarding existing clinically useful but toxic drugs. It will also develop novel cell-permeable ligands for RNAs upregulated in cancer, which can be broadly useful as molecular tools for cancer research.