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（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。在不同生物体中已显示MRNA定位缺陷，以破坏正常身体计划的形成并抑制中枢神经系统的功能。在人类中，局部在树突上的单个mRNA的丧失导致易碎的X综合征，这是最常见的遗传智力低下形式。 有越来越多的证据表明，RNA定位在许多生物的空间细胞生物学中起着重要作用，但是研究通常仅限于这种本质动态的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具有几个缺点，主要的局限性是高背景荧光。因此，在RNA的实时细胞成像领域中，对技术进步仍然存在着迫切的需求。 在此提案中，我们提出了一种创新的策略，用于用荧光小分子探针标记RNA。该方法对于荧光成像应用应该具有几个优点，包括特定的靶向，低背景和小分子探针的互换性。我们已经开发了一种合成策略来访问两类荧光配体探针。这些新化合物对相应的RNA标签的细胞渗透性和结合亲和力将进行测试。然后，我们将应用这种化学标记系统来研究编码蛋白质旁系同源物的mRNA定位。我们还将通过协作项目开发在其他生物体中使用的系统。最后，我们将探索附加在标记的RNA上的化学手柄的其他功能，包括亲和力纯化和RNA及其相关蛋白质伴侣的选择性失活。这种新的荧光标签策略涉及在活细胞中化学标记的RNA，预计将为RNA定位提供基础研究的强大工具。 公共卫生相关性：细胞内RNA的不对称分布是神经元（如神经元）的功能和正常身体计划的发展所必需的。为了了解某些RNA如何定位，我们建议开发一种化学标记RNA进行活细胞成像的技术。此外，将研究我们针对其他应用的标签策略的实用性，包括隔离RNA - 蛋白质复合物和失活的RNA。