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。
基本信息
- 批准号:10620766
- 负责人:
- 金额:$ 33.82万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 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:
- 关键词:AddressAdoptedAffinityAntisense OligonucleotidesArchitectureBindingBiochemicalBiologicalBiological ModelsBiologyBiotinylationCell Culture TechniquesCell LineCell NucleolusCell physiologyCellsCellular biologyChemicalsClinicalCodeCommunicationComplexDNADNA sequencingDataDevelopmentDiseaseEmbryonic DevelopmentEngineeringEnzymesExhibitsFractionationGene ExpressionGenomicsGoalsHuman PathologyImageIn SituIndividualInfectionInterphase CellKineticsLaboratoriesLengthMalignant NeoplasmsMammalian CellMapsMembraneMessenger RNAMetabolicMethodsModelingMolecularMonitorNeurodegenerative DisordersNoiseNuclearNucleic Acid ProbesOligonucleotidesOrganellesOrganismPlayProteinsProteomeProteomicsProtocols documentationRNARNA ProbesRNA analysisRNA-Protein InteractionResolutionResourcesRoleSamplingSpecificityStreptavidinStructureTechniquesTechnologyTestingUntranslated RNAViral PathogenesisX Inactivationbiological adaptation to stresscell fixingcell typeclinically relevantdesignexperimental studyfield studygenetic manipulationgenomic locushuman diseasemolecular assembly/self assemblyneurotransmissionnovelportabilityprogramsrRNA Precursorrecruitresponsescaffoldsingle moleculetherapeutic RNAtherapeutic targettooltranscriptometranscriptome sequencing
项目摘要
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
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。
项目成果
期刊论文数量(2)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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David Michael Shechner其他文献
David Michael Shechner的其他文献
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{{ truncateString('David Michael Shechner', 18)}}的其他基金
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 - 财政年份:2020
- 资助金额:
$ 33.82万 - 项目类别:
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:
10404121 - 财政年份:2020
- 资助金额:
$ 33.82万 - 项目类别:
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:
10247689 - 财政年份:2020
- 资助金额:
$ 33.82万 - 项目类别:
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