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sequencing novel off-target site programs scaffold screening small molecule tool transcriptome transcriptome sequencing unpublished works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.

许多实验室最近的工作强调了 RNA 控制的细胞途径的至关重要性，并且因此，特定 RNA 与人类健康的临床相关联系的发展。与传统的蛋白质靶标相比，RNA 的分子配体现在为除了这种设计方法之外，目前的许多蛋白质似乎也可能是未来的目标。靶向药物很可能与脱靶的 RNA 结合，并在那里引起意想不到的生物效应。不幸的是，对转录组范围内的 RNA-小分子相互作用进行分析至关重要。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来分析生物学和临床相关的 RNA 已经开发出来。直接识别RNA碱基修饰是这个新项目的核心是最近的发展。第一种可渗透细胞的 RNA 酰化剂，基于烟酰支架，可与可接近的 2'-OH 发生反应这使得对 RNA 结构和蛋白质-RNA 的前所未有的测量成为可能。在最近的工作中，新的酰化试剂可以多酰化。已经开发出体外RNA，使RNA暂时失活，然后可以去除这些基团。总体而言，这项最近的新工作是通过化学或光化学来暂时或局部控制 RNA 活性。提出了一套可直接在细胞中使用的新分子试剂、工具和测序方法在一项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，包括酰化试剂和烷基化物质，可以进入细胞并提供有关配体结合的特定定量信息与下一代测序相结合，这些方法将精确定位特定的结合位点。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。涉及反应性药物样片段的方法将用于识别癌症相关 RNA 的配体。这项工作意义重大，因为它将开发使能分子技术，从而大大增强 RNA生物学和生物医学的研究，分析转录组范围内的药物相互作用并直接在此外，还将概述蛋白质靶向药物脱靶结合的现象有多严重。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颖的RNA。分子策略，利用 RNA 反应性，它将开发有关现有的前所未有的数据。它还将开发新的细胞渗透性配体，用于在细胞中上调的RNA。癌症，可作为癌症研究的分子工具广泛使用。