Fluorescent mRNA Labeling Using Self-Alkylating Ribozymes

项目摘要/摘要 MRNA的不对称定位和特定的蛋白结合模式在许多细胞周期中起着关键作用。 在癌症和一些神经系统疾病中已经观察到了异常的mRNA定位。收益- 加深对信使核糖核酸定位模式及相应机制的认识 运输将提供关于疾病进展和潜在治疗应用的有价值的信息。 行尸走肉。然而，目前可用的RNA标记方法受到各种挑战和限制， 产生了对改进的方法的持续需求，用于标记和成像特定的 活细胞。利用RNA和小分子之间的分子识别能力，我们开发了- OpED核酶序列，能够与亲电荧光素类似物自烷基化，并且可以 与目的基因融合，并在细胞中表达。这些核酶有望服务于这两个 实现基于荧光的mRNA可视化并为免疫沉淀提供句柄的目的 结合特定RNA序列的蛋白质。与其他RNA标记技术相比，提出的 基于核酶的方法提供了更小的融合大小的好处，潜在地与广泛的调色板一起使用 小分子荧光团，以及通过去除多余的荧光团来减少背景信号。另外， 所提出的基于核酶的方法实现了其他rna标记所不能实现的新应用。 例如脉冲追逐标记和转录本特异性免疫沉淀等技术。的具体目标 本项目的目的是：(1)利用核酶分离和鉴定转录特异性RNA结合蛋白；(2) 荧光标记和显示活细胞中的mRNAs，并监测时间分辨的mRNAs动态；(3)利用我们的 RNA标记和免疫沉淀方法，以深入了解其作用机制和特异性定位方法。 非规范内质网定位的mRNAs的Terns。本研究可望为今后的研究提供有力的工具 活细胞中信使核糖核酸的定位和运输机制，并将立即使用这些工具来 回答有关内质网定位的RNA的生物学问题。这反过来有望进一步加深我们的理解 并为疾病的机制和治疗提供了新的见解。

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.