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） 翻译后摘要：RNA定位是调节细胞内蛋白质的空间分布的重要机制。在不同的生物体中，mRNA定位的缺陷已经显示出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，位于树突的单个mRNA的丢失导致脆性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起着重要作用，但研究往往限于这种内在动态细胞过程的静态快照。当应用于活细胞时，用于RNA的荧光标记的现有策略具有若干缺点，主要限制是高背景荧光。因此，仍然迫切需要RNA的活细胞成像领域的技术进步。 在这个建议中，我们提出了一个创新的策略标记RNA与荧光小分子探针。这种方法对于荧光成像应用应该具有几个优点，包括特异性靶向、低背景和小分子探针的可重复性。我们已经开发了一种合成策略来获得两类荧光配体探针。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，我们将应用这种化学标记系统来研究酵母中编码蛋白质旁系同源物的mRNA的定位。我们还将通过合作项目开发用于其他生物体的系统。最后，我们将探索附加在标记RNA上的化学手柄的其他功能，包括亲和纯化和RNA及其相关蛋白质伴侣的选择性失活。这种新的荧光标记策略，涉及在活细胞中化学标记RNA，有望为RNA定位的基础研究提供有力的工具。 公共卫生相关性：细胞内RNA的不对称分布是神经元等特化细胞功能和正常身体发育所必需的。为了了解某些RNA是如何以及为什么被定位的，我们建议开发一种技术来化学标记RNA用于活细胞成像。此外，我们的标签策略的其他应用程序，包括分离RNA-蛋白质复合物和灭活RNA的效用，将进行调查。