Fluorescent mRNA Labeling Using Self-Alkylating Ribozymes

项目概要/摘要 mRNA的不对称定位和特异性蛋白结合模式在许多细胞前体中起着关键作用， 在癌症和几种神经系统疾病中观察到了异常的mRNA定位。增益- 更深入地了解mRNA的定位模式和mRNA的相应机制， 转运将提供关于疾病进展和潜在治疗应用的有价值的信息， 接近然而，目前可用的RNA标记方法受到各种挑战和限制， 产生了对用于标记和成像特定mRNA序列的改进方法的持续需求， 活细胞利用RNA和小分子之间的分子识别能力，我们已经开发了 能够与亲电荧光素类似物自烷基化的寡核苷酸核酶序列， 与感兴趣的mRNA融合并在细胞中表达。这些核酶预计将服务于双 目的是实现mRNA的基于荧光的可视化并提供用于免疫沉淀的手柄 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更小的融合尺寸的好处， 小分子荧光团，以及通过除去过量荧光团来降低背景信号。此外，本发明还 所提出的基于核酶的方法使得其他RNA标记不可能实现的新应用成为可能 技术，如脉冲追踪标记和转录特异性免疫沉淀。的具体目标 本项目的主要目标是：（1）利用核酶分离和鉴定转录本特异性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，以了解机制和特异性定位模式， 非典型ER定位mRNA的表达。这项研究有望提供强有力的工具，研究 mRNA在活细胞中的定位和运输机制，并将这些工具立即用于 回答有关ER定位RNA的生物学问题。这反过来有望进一步加深我们对 基本的细胞过程，并提供新的见解的机制和治疗疾病。

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.