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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sequencing novel off-target site programs scaffold screening small molecule tool transcriptome transcriptome sequencing unpublished works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都强调了rna控制的细胞通路的重要性