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.

项目摘要 在整个生物学中，RNA分子形成复杂的动态分子相互作用网络， 这对它们的功能至关重要，但仍难以调查。这些RNA相互作用的网络 蛋白质、RNA和基因组位点几乎调控mRNA功能的所有方面，使非编码RNA能够 在不同水平上调节基因表达，并为控制细胞基因的分子组装提供支架， 表达、代谢和应激反应程序。RNA相互作用的失调是导致 与许多人类病理学有关，这表明这些相互作用可能代表了一种重要的 一类尚未开发的治疗靶点。然而，尽管RNA对基础生物学和人类 然而，由于疾病，用于阐明与任何给定RNA相互作用的因子的方法仍然有限。当前状态 最先进的方法--使用生物素化的反义寡核苷酸从粗RNA中拉下靶RNA， 细胞裂解物是嘈杂的，具有低靶RNA特异性，并且缺乏生物学背景。新兴战略 使用转基因表达的酶原位亲和标记RNA相互作用物需要复杂的细胞系 这限制了它们在细胞类型和靶RNA中的适用性。因此，迫切需要 对于能够以高分辨率阐明细胞内RNA相互作用的简单和可推广的工具， 而无需繁琐的生化分离或细胞系工程。为了迎接这一挑战， 响应RFA PAR 19-253，该提案将开发寡核苷酸指导的生物素化（ODB）。这 一种新的技术结合了高分辨率的单分子RNA-FISH和原位邻近生物素化， RNA相互作用网络在其天然细胞环境中。在中试实验中，ODB表现出异常的 精确定位单个RNA，并对RNA支架结构进行蛋白质组学分析， 很难用生化方法分离出来我们最近也证明了邻近生物素化 类似ODB的方法可用于探测核酸以及蛋白质。鉴于这些有希望的证据- 原则的结果，我们建议发展ODB成为一个统一的，“多”组学”的方法来识别蛋白质， RNA和/或与广泛的靶RNA相互作用的基因组基因座。在目标1中，我们将优化核心 ODB工作流程的步骤，开发用于以高空间分辨率将ODB部署到靶RNA的稳健方案， 精确度，以及用于从ODB实验中分离RNA相互作用蛋白质、RNA和基因组位点。我们将 开发将ODB应用于不同哺乳动物细胞系和RNA靶标阵列的通用策略。 在目标2中，我们将在动态的、发育调节的核结构RNA上“实地测试”ODB， 很难用传统的方法来描述。这些实验将开发出一种多功能的， 直接的技术，用于询问RNA相互作用的原位，这是很容易采用的大多数 laboratories.考虑到RNA在整个生物学中所扮演的普遍角色，这种变革性的方法将为 这是细胞生物学中范式转变发现的方式，并揭示了新的基于RNA的治疗靶点。