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.

项目摘要 在整个生物学中，RNA分子形成了分子相互作用的复杂和动态网络， 对它们的功能至关重要，但仍然挑战进行调查。这些RNA相互作用网络 蛋白质，RNA和基因组局部几乎调节mRNA功能的所有方面，使非编​​码RNA能够 调节各种水平的基因表达，以及控制细胞基因的支架分子组件 表达，代谢和应力反应程序。 RNA相互作用的失调一直是因果关系 在许多人类病理中实施，表明这些相互作用可能代表着重要的 一类未开发的治疗靶标。然而，努力RNA对基本生物学和人类的核心重要性 疾病，阐明与任何给定RNA相互作用的因素的方法仍然有限。当前的最新 ART方法 - 使用生物素化的反义寡核苷酸从粗核苷酸降低目标RNA 细胞裂解物 - 噪声，靶RNA特异性低，缺乏生物学环境。新兴策略 使用翻译表达的酶与亲和力标签RNA相互作用的原位需要复杂的细胞线 工程限制其跨单元类型和目标RNA的适用性。因此，有紧迫的需求 对于可以在高分辨率下阐明细胞内RNA相互作用的直接且可推广的工具， 没有繁琐的生化分馏或细胞系工程。满足这一挑战，并在 对RFA PAR 19-253的反应，该提案将发展为寡核苷酸指导的生物素化（ODB）。 新型技术组合高分辨率的单分子RNA-FISH和原位接近 - 生物素化以映射 RNA相互作用网络在其天然细胞环境中。在飞行员实验中，ODB异常暴露 精确靶向单个RNA的原位，并实现了RNA相互影响的结构的蛋白质组学分析 我们最近还证明了接近生物素化 诸如ODB之类的方法可用于探测核酸和蛋白质。考虑到这些证明 原理结果，我们建议将ODB开发为一种识别蛋白质的统一的“多”方法， 与广泛的靶RNA相互作用的RNA和/或基因组基因组基因局基因局基因局基因局。在AIM 1中，我们将优化核心 ODB工作流程的步骤，开发可靠的协议，以将ODB部署到高空间的目标RNA 精度，用于从ODB实验中分离RNA相互作用的蛋白，RNA和基因组基因组基因局。我们将 制定一般使用策略以在不同的哺乳动物细胞系和RNA靶标中应用ODB。 在AIM 2中，我们将“现场测试” ODB对具有动态的，开发的核架构RNA 它们很难通过常规方法来表征。这些实验将开发出多功能和 直接询问RNA相互作用的直接技术，并且大多数都很容易适应 实验室。鉴于RNA在整个生物学中扮演的普遍角色，这种变革方法将铺路 细胞生物学中范式转移发现的方式，并揭示了基于RNA的新型治疗靶标。