Covalent Profiling of RNA Targets and Off-targets

RNA 靶标和脱靶的共价分析

基本信息

批准号：
10294248
负责人：
ERIC T. KOOL
金额：
$ 37.6万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-01-10 至 2022-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10294248
关键词：
Acylation Affect Alkylation Antibiotics Binding Binding Proteins Binding Sites Biological Biology Biopsy Specimen CUG repeat Cells Chemicals Clinical Data Development Disease Dose Drug Interactions Drug Targeting Future Genomics Health Human In Vitro Ligand Binding Ligands Link Malignant Neoplasms Measurement Measures Methodology Methods Modification Molecular Molecular Probes Myotonic Dystrophy Nucleotides Pathway interactions Permeability Pharmaceutical Preparations Proteins RNA RNA Probes RNA analysis RNA chemical synthesis RNA-Protein Interaction Reagent Research Resolution Specificity Structure Technology Toxic effect Transcript Untranslated RNA Work analytical method anticancer research base clinically relevant design experimental study improved in vivo innovation kinase inhibitor laboratory experiment next generation sequencing novel off-target site programs scaffold screening small molecule tool transcriptome transcriptome sequencing unpublished works

项目摘要

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.

来自许多实验室的最新工作强调了RNA控制的细胞途径的重要性，特定RNA与人类健康的临床相关联系。结果，小型的发展与传统蛋白质靶标相反，RNA的分子配体现在为有前途的方法提供了一种未来的疾病靶向。除了这种设计的方法外，似乎许多当前的蛋白质似乎都可能靶向药物很可能与靶向脱靶的RNA结合，并在那里引起意外的生物学效应。那， RNA-MALL分子相互作用在整个转录组中的分析至关重要。不幸的是，方法直接分析RNA - 配体相互作用的细胞滞后，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基于烟素支架，首先可与可访问的2'-OH反应的第一个细胞渗透RNA酰化剂组全转录组。这允许对RNA结构和蛋白-RNA的前所未有的测量核苷酸分辨率的体内相互作用。在最近的工作中，可以多酰胺的新酰基化试剂 RNA在体外，暂时灭活RNA。然后可以删除这些组在化学或光化学上以临时或局部控制RNA活性。总体而言，这项最新的新作品建议一套可以直接在细胞中使用的新分子试剂，工具和测序方法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组新的功能化RNA反应试剂，包括酰化和藻类物种，可以进入细胞并提供有关配体结合的特定定量信息在转录组中。结合下一代测序，这些方法将在特定的特定中查明结合位点成绩单。这些方法共同将基于反应性的RNA分析（RBRP）一起应用于分析具有临床限制毒性的已知药物的靶向靶向结合。此外，基于新的反应性方法 - 涉及反应性药物片段 - 将用于鉴定与癌症相关的RNA的配体。这项工作很重要，因为它将开发能够大大增强的分子技术 RNA生物学和生物医学的研究，分析了整个转录组的药物相互作用，直接在细胞。此外，它将概述蛋白质靶向药物的现象有多严重。 RNA。该研究计划具有创新性，因为它发展了一系列新的分子问题和新颖的问题分子策略，利用RNA反应性。它将制定有关现有的前所未有的数据临床上有用但有毒药物。它还将开发出可更新的RNA的新型细胞渗透配体癌症，可以作为癌症研究的分子工具广泛用来。