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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sequencing novel off-target site programs scaffold screening small molecule tool transcriptome transcriptome sequencing unpublished works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控制的细胞通路的关键重要性，以及特定RNA与人类健康的临床相关联系。因此，小型企业的发展- 与传统的蛋白质靶标不同，核糖核酸的分子配体现在提供了一种很有希望的方法未来针对疾病的目标。除了这种设计的方法，似乎许多目前的蛋白质- 靶向药物很可能与脱靶的RNA结合，并在那里造成意想不到的生物效应。因此，在转录范围内分析RNA-小分子相互作用是至关重要的。不幸的是，直接分析细胞内RNA与配体的相互作用远远落后于蛋白质与配体结合的分析。这个实验室的初步实验已经建立了多种新的分子工具来分析与生物学和临床相关的RNA。基于RNA序列的新方法已经被开发用于直接鉴定RNA碱基修饰。这个新项目的核心是最近的发展第一种细胞通透性的RNA酰化剂，基于尼古丁基支架，与可访问的2‘-OH反应整个转录组。这使得前所未有的对RNA结构和蛋白质-RNA的测量成为可能核苷酸分辨率下的体内相互作用。在最近的工作中，新的酰化试剂可以聚酰化 RNA在体外，暂时失活的RNA已经开发出来。然后可以删除这些组通过化学或光化学手段暂时或局部地控制RNA的活性。总体而言，最近的这项新工作提出了一套可直接用于细胞的新分子试剂、工具和测序方法在一次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，包括酰化和烷化物种，它们可以进入细胞并提供关于配体结合的特定、定量信息在转录组中。结合下一代测序，这些方法将精确定位特定的结合位点成绩单。这些方法一起被称为基于反应的RNA分析(RBRP)，将被应用于分析临床毒性有限的已知药物的靶外结合。此外，新的基于反应性的涉及反应性类药物片段的方法将被用于识别癌症相关RNA的配体。这项工作意义重大，因为它将开发使能分子技术，这将大大提高 RNA生物学和生物医学的研究，分析药物相互作用在转录范围内和直接在手机。此外，它还将概述蛋白质靶向药物与靶向结合的现象有多严重 RNA。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分子策略，利用RNA的反应性。它将开发关于现有数据的前所未有的数据临床上有用但有毒的药物。它还将为上调的RNA开发新的细胞渗透性配体癌症，可广泛用作癌症研究的分子工具。