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: 10029882
• 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/10029882
• 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; Complex; DNA; DNA sequencing; Data; Development; Disease; Embryonic Development; Engineering; Enzymes; Exhibits; Fractionation; Gene Expression; Genomics; Goals; Human Pathology; Hybrids; Image; In Situ; Individual; Infection; Interphase Cell; Kinetics; Laboratories; Length; Malignant Neoplasms; Mammalian Cell; Maps; Messenger RNA; Metabolic stress; 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; Resolution; Resources; Role; Sampling; Specificity; Streptavidin; Structure; Techniques; Technology; Testing; Transgenic Organisms; Untranslated RNA; Viral Pathogenesis; X Inactivation; base; biological adaptation to stress; 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 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.

项目概要 在整个生物学中，RNA 分子形成复杂且动态的分子相互作用网络 对于它们的功能至关重要，但研究起来仍然具有挑战性。这些RNA相互作用网络 蛋白质、RNA 和基因组位点调节 mRNA 功能的几乎所有方面，使非编​​码 RNA 能够 在不同水平上调节基因表达，并支架控制细胞基因的分子组装体 表达、代谢和应激反应程序。 RNA相互作用的失调是因果关系 与许多人类病理有关，表明这些相互作用可能代表了重要的 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至关重要 疾病，阐明与任何给定 RNA 相互作用的因素的方法仍然有限。目前的状态- 最先进的方法——使用生物素化的反义寡核苷酸从粗产物中提取目标RNA 细胞裂解物——噪音大、靶标 RNA 特异性低且缺乏生物学背景。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 工程限制了它们在细胞类型和目标 RNA 上的适用性。因此，迫切需要 寻找能够以高分辨率阐明细胞内 RNA 相互作用的简单且通用的工具， 无需繁琐的生化分离或细胞系工程。为了应对这一挑战，并在 为了响应 RFA PAR 19-253，该提案将开发寡核苷酸定向生物素化 (ODB)。这 新技术结合了高分辨率单分子 RNA-FISH 和原位邻近生物素化来绘制图谱 RNA 相互作用网络在其天然细胞环境中。在试点实验中，ODB 表现出色 原位精确靶向单个 RNA，并对 RNA 支架结构进行蛋白质组学分析 很难通过生化方法分离。我们最近还证明了邻近生物素化 ODB 等方法可用于探测核酸和蛋白质。鉴于这些有希望的证据- 原则结果，我们建议将 ODB 开发为一种统一的“多组学”方法来识别蛋白质， RNA 和/或与多种靶 RNA 相互作用的基因组位点。在目标1中，我们将优化核心 ODB 工作流程的步骤，开发用于将 ODB 部署到高空间目标 RNA 的稳健协议 精度，并用于从 ODB 实验中分离 RNA 相互作用蛋白、RNA 和基因组位点。我们将 开发在一系列不同哺乳动物细胞系和 RNA 靶标中应用 ODB 的通用策略。 在目标 2 中，我们将在动态的、发育调控的核结构 RNA 上“现场测试”ODB，该 RNA 具有 很难用传统的方法来表征。这些实验将开发出一种多功能且 原位检测 RNA 相互作用的简单技术，并且很容易被大多数人采用 实验室。鉴于 RNA 在整个生物学中发挥的普遍作用，这种变革性方法将为 细胞生物学范式转变的发现之路，并揭示新的基于 RNA 的治疗靶点。