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

项目总结 在整个生物学中，RNA分子形成复杂而动态的分子相互作用网络，这些网络 对它们的功能至关重要，但研究起来仍然具有挑战性。这些RNA相互作用的网络 蛋白质、RNAs和基因组基因座几乎调控着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，并构建控制细胞基因的分子组件。 表达、新陈代谢和压力反应程序。RNA相互作用的失调是原因之一 与许多人类病理有关，这表明这些相互作用可能代表着一个重要的 一类未开发的治疗靶点。然而，尽管RNA对基础生物学和人类的核心重要性 对于疾病，阐明与任何特定RNA相互作用的因素的方法仍然有限。当前的状态- ART方法--使用生物素化的反义寡核苷酸从原油中提取靶RNA 细胞裂解物--噪音大，靶标RNA特异性低，缺乏生物学背景。新兴战略 利用转基因表达的酶在原位亲和标记RNA相互作用子需要复杂的细胞系 限制其在细胞类型和目标RNA之间的适用性的工程。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， 没有繁琐的生化分级或细胞系工程。迎接这一挑战，并在 响应RFA PAR 19-253，这一建议将发展寡核苷酸导向的生物素化(ODB)。这 高分辨率单分子RNA-FISH和原位邻近生物素化相结合的新技术 RNA相互作用网络在它们的自然细胞环境中。在试点实验中，ODB表现得格外出色 在原位精确定位单个RNA，并能够对RNA支架结构进行蛋白质组学分析 很难用生化方法分离。我们最近还证明了邻近生物素化作用 像ODB这样的方法既可以用来探测蛋白质，也可以用来探测核酸。鉴于这些有希望的证据- 主要结果，我们建议将ODB发展成为一种统一的、“多组”的蛋白质鉴定方法， 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，开发用于将ODB部署到高空间目标RNA的健壮协议 精确度，并用于从ODB实验中分离RNA相互作用的蛋白质、RNA和基因组位点。我们会 开发在一系列不同的哺乳动物细胞系和RNA靶标中应用ODB的通用策略。 在目标2中，我们将在动态的、受发育调节的核结构RNA上进行现场测试，该RNA具有 很难用传统的方法来描述。这些实验将开发出一种多功能和 用于原位询问RNA相互作用的简单技术，并且容易被大多数人采用 实验室。鉴于RNA在整个生物学中扮演的普遍角色，这种变革性的方法将为 为细胞生物学中的范式转换发现铺平道路，并揭示基于RNA的新治疗靶点。

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.