A Chemical Biology Approach to Tagging RNAs in Live Cells

标记活细胞中 RNA 的化学生物学方法

• 批准号: 8146809
• 负责人: Ming Chen Hammond
• 金额: $ 230.25万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-30 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8146809
• 关键词: Affinity; Affinity Chromatography; Basic Science; Binding; Biology; Cell physiology; Cells; Cellular biology; Chemicals; Defect; Dendrites; Fluorescence; Fragile X Syndrome; Human; Inherited; Label; Life; Ligands; Mental Retardation; Messenger RNA; Methods; Neuraxis; Organism; Permeability; Play; Proteins; RNA; Role; Spatial Distribution; System; Techniques; Testing; Yeasts; abstracting; cellular imaging; chemical function; fluorescence imaging; innovation; neuron development; paralogous gene; protein complex; public health relevance; small molecule; tool

DESCRIPTION (Provided by the applicant) Abstract: RNA localization is an important mechanism for regulating the spatial distribution of proteins inside the cell. Defects in mRNA localization have been shown in different organisms to disrupt the formation of a normal body plan and to inhibit the function of the central nervous system. In humans, the loss of a single mRNA that is localized to dendrites leads to Fragile X syndrome, which is the most common inherited form of mental retardation. There is mounting evidence that RNA localization plays a large role in the spatial cell biology of many organisms, but studies are often limited to static snapshots of this intrinsically dynamic cellular process. Existing strategies for fluorescent labeling of RNAs have several drawbacks when applied to live cells, a major limitation being high background fluorescence. Thus, there remains a critical need for technical advances in the field of live cell imaging of RNAs. In this proposal, we present an innovative strategy for tagging RNAs with fluorescent small molecule probes. This method should have several advantages for fluorescence imaging applications, including specific targeting, low background, and interchangeability of the small molecule probes. We have developed a synthetic strategy to access two classes of fluorescent ligand probes. The cell permeability and binding affinity of these new compounds to the corresponding RNA tags will be tested. We will then apply this chemical tagging system to study the localization of mRNAs encoding protein paralogs in yeast. We also will develop the system for use in other organisms through collaborative projects. Finally, we will explore other functions for the chemical handle appended on the tagged RNA, including affinity purification and selective inactivation of the RNA and its associated protein partners. This new fluorescent labeling strategy, which involves chemically tagging RNAs in live cells, is expected to provide a powerful tool for basic research into RNA localization. Public Health Relevance: The asymmetric distribution of RNAs inside cells is required for the function of specialized cells like neurons and for the development of a normal body plan. In order to understand how and why certain RNAs are localized, we propose to develop a technique to chemically tag RNAs for live cell imaging. Furthermore, the utility of our tagging strategy for other applications, including isolating RNA-protein complexes and inactivating RNAs, will be investigated.

描述(由申请人提供) 摘要：RNA定位是调节细胞内蛋白质空间分布的重要机制。在不同的生物体中，已经发现了信使核糖核酸定位缺陷，破坏了正常身体计划的形成，并抑制了中枢神经系统的功能。在人类中，定位于树突的单个mRNA的丢失会导致脆性X综合征，这是最常见的遗传性智力低下形式。越来越多的证据表明，RNA定位在许多生物体的空间细胞生物学中发挥着重要作用，但研究往往局限于这一内在动态细胞过程的静态快照。现有的RNA荧光标记策略在应用于活细胞时有几个缺陷，一个主要的限制是高背景荧光。因此，在RNA的活细胞成像领域仍然有迫切的技术进步的需要。在这项提案中，我们提出了一种用荧光小分子探针标记RNA的创新策略。这种方法在荧光成像应用中应该具有几个优点，包括特异性靶向性、低本底和小分子探针的互换性。我们开发了一种合成策略来访问两类荧光配体探针。将测试这些新化合物对相应RNA标签的细胞渗透性和结合亲和力。然后，我们将应用这个化学标记系统来研究编码蛋白质类似物的mRNAs在酵母中的定位。我们还将通过合作项目开发该系统，用于其他生物体。最后，我们将探索附加在标记RNA上的化学句柄的其他功能，包括亲和纯化和选择性失活RNA及其相关蛋白质伙伴。这一新的荧光标记策略涉及对活细胞中的RNA进行化学标记，有望为RNA定位的基础研究提供一个强大的工具。 与公共卫生相关：RNA在细胞内的不对称分布对于神经元等特殊细胞的功能和正常身体计划的发展是必需的。为了了解某些RNA是如何以及为什么被定位的，我们建议开发一种用于活细胞成像的化学标记RNA的技术。此外，我们还将研究我们的标记策略在其他应用中的应用，包括分离RNA-蛋白质复合体和灭活RNA。