RNA Imaging with Dimeric Broccoli in Live Bacterial and Mammalian Cells.
RNA Imaging with Dimeric Broccoli in Live Bacterial and Mammalian Cells.
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DOI：
10.1002/9780470559277.ch150174
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发表时间：
2016-03-16
影响因子：
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通讯作者：
Jaffrey SR
中科院分区：
文献类型：
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作者：
Filonov GS;Jaffrey SR
RNA的空间动态在细胞生理学中起着至关重要的作用，因此监测活细胞中RNA定位的能力可以为重要的生物学问题提供深入见解。本文重点介绍使用一种“绿色荧光蛋白（GFP）的RNA模拟物”对RNA进行成像。这种方法依赖于一种名为二聚体西兰花（dimeric Broccoli）的RNA适体，它与DFHBI结合并开启其荧光，DFHBI是一种模拟GFP中荧光团的小分子。二聚体西兰花被标记到异源表达的RNA上，在与DFHBI结合后，二聚体西兰花的荧光信号报告转录本在细胞中的定位。本实验方案描述了在体外和细胞内验证二聚体西兰花标记的转录本荧光的过程、通过流式细胞术分析确定细胞内总体荧光水平，以及在细菌和哺乳动物细胞中进行荧光成像。总体而言，当前的实验方案对于寻求对高丰度RNA进行成像的研究人员应该是有用的，例如对细菌中由T7启动子转录的RNA或哺乳动物细胞中由依赖Pol III的启动子转录的RNA进行成像。
RNA spatial dynamics play a crucial role in cell physiology and thus the ability to monitor RNA localization in live cells can provide insight into important biological problems. This article focuses on imaging RNAs using an “RNA mimic of GFP”. This approach relies on a RNA aptamer, called dimeric Broccoli, which binds to and switches on the fluorescence of DFHBI, a small molecule mimicking the fluorophore in GFP. Dimeric Broccoli is tagged to heterologously expressed RNAs and upon DFHBI binding the fluorescent signal of dimeric Broccoli reports the transcript’s localization in cells. This protocol describes the process of validating the fluorescence of dimeric Broccoli-labeled transcripts in vitro and in cells, flow cytometry analysis to determine overall fluorescence levels in cells, and fluorescence imaging in bacterial and mammalian cells. Overall, the current protocol should be useful for researchers seeking to image high abundance RNAs, such as transcribed off the T7 promoter in bacteria or off Pol III-dependent promoters in mammalian cells.