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利用Pumilio同源结构域突变体对内源RNA进行活细胞成像:原理与应用

Live Cell Imaging of Endogenous RNAs Using Pumilio Homology Domain Mutants: Principles and Applications.

作者信息

Yoshimura Hideaki

机构信息

Department of Chemistry, School of Science, The University of Tokyo , 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.

出版信息

Biochemistry. 2018 Jan 16;57(2):200-208. doi: 10.1021/acs.biochem.7b00983. Epub 2017 Dec 5.

Abstract

Recently, dynamic changes in the location of RNA in space and time in living cells have become a target of interest in biology because of their essential roles in controlling physiological phenomena. To visualize RNA, methods for the fluorescent labeling of RNA in living cells have been developed. For RNA labeling, oligonucleotide-based RNA probes have mainly been used because of their high selectivity for target RNAs. By contrast, protein-based RNA probes have not been used widely because of their lack of design flexibility, although they have various potential advantages compared with nucleotide-based probes, such as controllability of intracellular localization, high detectability, and ease of introduction into cells and transgenic organisms in a cell type and tissue specific manner by genetic engineering techniques. This Perspective focuses on a possible approach to the development of protein-based RNA probes using Pumilio homology domain (PUM-HD) mutants. The PUM-HD is a domain of an RNA binding protein that allows custom-made modifications to recognize a given eight-base RNA sequence. PUM-HD-based RNA probes have been applied to visualize various RNAs in living cells. Here, the techniques and RNA imaging results obtained using the PUM-HD are introduced.

摘要

最近,活细胞中RNA在空间和时间上的动态变化因其在控制生理现象中的重要作用而成为生物学研究的一个热点。为了可视化RNA,人们已经开发出了在活细胞中对RNA进行荧光标记的方法。对于RNA标记,基于寡核苷酸的RNA探针因其对靶RNA具有高选择性而被广泛使用。相比之下,基于蛋白质的RNA探针虽然与基于核苷酸的探针相比具有多种潜在优势,如细胞内定位的可控性、高检测性以及能够通过基因工程技术以细胞类型和组织特异性方式轻松导入细胞和转基因生物,但由于缺乏设计灵活性,尚未得到广泛应用。本观点聚焦于一种利用Pumilio同源结构域(PUM-HD)突变体开发基于蛋白质的RNA探针的可能方法。PUM-HD是一种RNA结合蛋白的结构域,它允许进行定制修饰以识别给定的八个碱基的RNA序列。基于PUM-HD的RNA探针已被应用于活细胞中各种RNA的可视化。在此,介绍了使用PUM-HD获得的技术和RNA成像结果。

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