Oligonucleotide-directed in situ proximity biotinylation: a unified method for mapping RNA-interacting proteomes, transcriptomes and genomic loci within intact cells.

寡核苷酸引导的原位邻近生物素化：一种绘制完整细胞内 RNA 相互作用蛋白质组、转录组和基因组位点的统一方法。

• 批准号: 10620766
• 负责人: David Michael Shechner
• 金额: $ 33.82万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10620766
• 关键词: Address; Adopted; Affinity; Antisense Oligonucleotides; Architecture; Binding; Biochemical; Biological; Biological Models; Biology; Biotinylation; Cell Culture Techniques; Cell Line; Cell Nucleolus; Cell physiology; Cells; Cellular biology; Chemicals; Clinical; Code; Communication; Complex; DNA; DNA sequencing; Data; Development; Disease; Embryonic Development; Engineering; Enzymes; Exhibits; Fractionation; Gene Expression; Genomics; Goals; Human Pathology; Image; In Situ; Individual; Infection; Interphase Cell; Kinetics; Laboratories; Length; Malignant Neoplasms; Mammalian Cell; Maps; Membrane; Messenger RNA; Metabolic; Methods; Modeling; Molecular; Monitor; Neurodegenerative Disorders; Noise; Nuclear; Nucleic Acid Probes; Oligonucleotides; Organelles; Organism; Play; Proteins; Proteome; Proteomics; Protocols documentation; RNA; RNA Probes; RNA analysis; RNA-Protein Interaction; Resolution; Resources; Role; Sampling; Specificity; Streptavidin; Structure; Techniques; Technology; Testing; Untranslated RNA; Viral Pathogenesis; X Inactivation; biological adaptation to stress; cell fixing; cell type; clinically relevant; design; experimental study; field study; genetic manipulation; genomic locus; human disease; molecular assembly/self assembly; neurotransmission; novel; portability; programs; rRNA Precursor; recruit; response; scaffold; single molecule; therapeutic RNA; therapeutic target; tool; transcriptome; transcriptome sequencing

PROJECT SUMMARY Throughout biology, RNA molecules form complex and dynamic networks of molecular interactions that are essential to their function, but which remain challenging to investigate. These networks of RNA-interacting proteins, RNAs, and genomic loci regulate nearly all aspects of mRNA function, enable noncoding RNAs that regulate gene expression at various levels, and scaffold molecular assemblies that control cellular gene- expression, metabolic, and stress-response programs. Dysregulation of RNA-interactions has been causally implicated in numerous human pathologies, suggesting that these interactions may represent a significant class of untapped therapeutic targets. Yet, despite the central importance of RNA to basic biology and human disease, methods for elucidating the factors that interact with any given RNA remain limited. Current state-of- the-art approaches—which use biotinylated antisense oligonucleotides to pull down target RNAs from crude cell lysates—are noisy, suffer from low target RNA specificity, and lack biological context. Emerging strategies that use transgenically expressed enzymes to affinity-tag RNA-interactors in situ require complicated cell-line engineering that limits their applicability across cell types and target RNAs. Therefore, there is a pressing need for straightforward and generalizable tools that can elucidate intra-cellular RNA-interactions at high resolution, without cumbersome biochemical fractionation or cell-line engineering. To meet this challenge, and in response to RFA PAR 19-253, this proposal will develop Oligonucleotide-Directed Biotinylation (ODB). This novel technique combines high-resolution single-molecule RNA-FISH and in situ proximity-biotinylation to map RNA interaction networks within their native cellular context. In pilot experiments, ODB exhibited exceptionally precise targeting of individual RNAs in situ, and enabled proteomic analysis of RNA-scaffolded structures that are difficult to isolate biochemically. We have also recently demonstrated that proximity-biotinylation approaches like ODB can be used to probe nucleic acids as well as proteins. Given these promising proof-of- principle results, we propose developing ODB into a unified, “multi-‘omic” method for identifying the proteins, RNAs, and/or genomic loci that interact with a broad range of target RNAs. In Aim 1, we will optimize the core steps of the ODB workflow, developing robust protocols for deploying ODB to a target RNA at high spatial precision, and for isolating RNA-interacting proteins, RNAs, and genomic loci from an ODB experiment. We will develop general-use strategies for applying ODB in an array of different mammalian cell lines and RNA targets. In Aim 2, we will “field test” ODB on a dynamic, developmentally-regulated nuclear-architectural RNA that has been difficult to characterize by conventional approaches. These experiments will develop a versatile and straightforward technology for interrogating RNA interactions in situ, and which is easily adoptable by most laboratories. Given the pervasive roles played by RNA throughout biology, this transformative method will pave the way for paradigm-shifting discoveries in cell biology, and reveal novel RNA-based therapeutic targets.

项目总结