Fluorescent labeling of cellular mRNA using self-alkylating ribozymes

使用自烷基化核酶对细胞 mRNA 进行荧光标记

• 批准号: 9175416
• 负责人: Jennifer Margaret Heemstra
• 金额: $ 28.7万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-01 至 2017-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9175416
• 关键词: Actins; Address; Alkylation; Alzheimer' s Disease; Binding; Binding Proteins; Biological; Biological Process; Catalytic RNA; Cell physiology; Cells; Development; Differentiation and Growth; Disease; Disease Progression; Drosophila genus; Dyes; Elements; Excision; Fluorescein; Fluorescence; Fragile X Syndrome; Generations; Goals; Huntington Disease; Image; Imagery; Immunoprecipitation; Intracellular Transport; Kinetics; Label; Life; Malignant Neoplasms; Membrane; Messenger RNA; Methods; Monitor; Movement; Nature; Oligonucleotides; Pattern; Physiologic pulse; Play; Property; Proteins; RNA; RNA Sequences; RNA Transport; RNA-Binding Proteins; RNA-Protein Interaction; Reaction; Reporting; Research; Series; Signal Transduction; Site; Stress; Structure; Technology; Therapeutic; Time; Transcript; Untranslated Regions; Work; abstracting; analog; aptamer; base; drug development; fluorophore; improved; insight; interest; malignant neurologic neoplasms; molecular recognition; nervous system disorder; new therapeutic target; novel; reaction rate; research study; response; small molecule; tool

Project Summary/Abstract Asymmetrical localization and specific protein binding patterns of mRNA play a key role in many cellular pro- cesses, and aberrant mRNA localization has been observed in cancer and several neurological diseases. Gain- ing a deeper understanding of mRNA localization patterns and the corresponding mechanisms of mRNA transport would provide valuable information regarding disease progression and potential therapeutic ap- proaches. However, the RNA labeling methods currently available suffer from various challenges and limitations, creating an ongoing demand for improved methods for labeling and imaging of specific mRNA sequences in living cells. Harnessing the power of molecular recognition between RNA and small molecules, we have devel- oped ribozyme sequences that are capable of self-alkylation with an electrophilic fluorescein analogue, and can be fused to an mRNA of interest and expressed in cells. These ribozymes are anticipated to serve the dual purpose of enabling fluorescence-based visualization of mRNA and providing a handle for immunoprecipitation of proteins bound to specific RNA sequences. Compared with other RNA labeling technologies, the proposed ribozyme-based approach offers the benefits of smaller fusion size, potential for use with a broad palette of small-molecule fluorophores, and reduction of background signal by removal of excess fluorophore. Additionally, the proposed ribozyme-based approach enables new applications that are not possible with other RNA labeling technologies, such as pulse-chase labeling and transcript-specific immunoprecipitation. The specific aims of this project are to: (1) utilize the ribozymes to isolate and identify transcript-specific RNA-binding proteins; (2) fluorescently label and visualize mRNAs in living cells and monitor time-resolved mRNA dynamics; (3) utilize our RNA labeling and immunoprecipitation methods to gain insight into the mechanism and specific localization pat- terns of non-canonical ER-localized mRNAs. This research is anticipated to provide powerful tools for studying the localization and transport mechanisms of mRNA in living cells, and will put these tools to immediate use to answer biological questions regarding ER-localized RNAs. This is in turn expected to further our understanding of fundamental cellular processes and offer new insights into the mechanisms and treatment of disease.

项目摘要/摘要 mRNA的不对称定位和特定的蛋白质结合模式在许多细胞促值中起着关键作用 在癌症和几种神经系统疾病中观察到了CESSES和异常的mRNA定位。获得- 对mRNA定位模式和mRNA的相应机制有了更深入的了解 运输将提供有关疾病进展和潜在治疗性的有价值的信息 促进。但是，当前可用的RNA标签方法遇到了各种挑战和局限性， 创造了对改进方法的持续需求，以将特定mRNA序列的标记和成像成像 活细胞。利用RNA和小分子之间的分子识别的能力，我们具有DEVEL- 能够用亲电荧光素类似物自我烷基化的OPED核酶序列，并且可以 将其融合到感兴趣的mRNA中并在细胞中表达。这些核酶预计将服务于双重 实现mRNA的基于荧光的可视化并提供免疫沉淀的手柄的目的 与特定RNA序列结合的蛋白质。与其他RNA标记技术相比 基于核酶的方法提供了较小的融合尺寸的好处，可与广泛的调色板一起使用 小分子荧光团，以及通过去除过量的荧光团来减少背景信号。此外， 提出的基于核酶的方法可实现其他RNA标记不可能的新应用 技术，例如脉冲马的标记和转录本特异性免疫沉淀。具体目的 该项目是：（1）利用核酶分离和鉴定转录本特异性的RNA结合蛋白； （2） 荧光标记和可视化活细胞中的mRNA并监测时间分辨的mRNA动力学； （3）利用我们的 RNA标记和免疫沉淀方法可深入了解机制和特定定位 非典型ER定位的mRNA的元素。预计这项研究将为研究提供强大的工具 MRNA在活细胞中的定位和运输机制，并将这些工具立即使用 回答有关ER定位的RNA的生物学问题。反过来，这又可以进一步理解 基本的细胞过程，并为疾病的机制和治疗提供新的见解。