RNA visualization in live bacterial cells using fluorescent protein complementation
RNA visualization in live bacterial cells using fluorescent protein complementation
复制标题
DOI：
10.1038/nmeth1023
复制
发表时间：
2007-05-01
期刊：
影响因子：
48
通讯作者：
Broude, Natalia E.
中科院分区：
文献类型：
--
作者：
Valencia-Burton, Maria;McCullough, Ron M.;Broude, Natalia E.
我们描述了一种用于检测和定位活细胞中RNA转录本的技术。该方法是基于荧光蛋白互补调节的分裂RNA结合蛋白与其相应的RNA适体的相互作用。在我们的设计中，RNA结合蛋白是真核起始因子4A（eIF4A）。将eIF 4A切割成两个片段，并且每个片段与增强型绿色荧光蛋白（EGFP）的分裂片段融合。在含有eIF4A相互作用RNA适体的转录本存在下，两种蛋白融合物的共表达导致大肠杆菌细胞中EGFP荧光的恢复。我们还将这种技术应用于适体标记的mRNA和5S核糖体RNA（rRNA）的可视化。我们观察到不同的空间和时间的变化，在单细胞内的荧光，反映了转录的性质。
We describe a technique for the detection and localization of RNA transcripts in living cells. The method is based on fluorescent-protein complementation regulated by the interaction of a split RNA-binding protein with its corresponding RNA aptamer. In our design, the RNA-binding protein is the eukaryotic initiation factor 4A (eIF4A). eIF4A is dissected into two fragments, and each fragment is fused to split fragments of the enhanced green fluorescent protein (EGFP). Coexpression of the two protein fusions in the presence of a transcript containing eIF4A-interacting RNA aptamer resulted in the restoration of EGFP fluorescence in Escherichia coli cells. We also applied this technique to the visualization of an aptamer-tagged mRNA and 5S ribosomal RNA (rRNA). We observed distinct spatial and temporal changes in fluorescence within single cells, reflecting the nature of the transcript.