RNAs mediate many different processes that are central to cellular function. The ability to quantify or image RNAs in live cells is very useful in elucidating such functions of RNA. RNA aptamer–fluorogen systems have been increasingly used in labeling RNAs in live cells. Here, we use the malachite green aptamer (MGA), an RNA aptamer that can specifically bind to malachite green (MG) dye and induces it to emit far-red fluorescence signals. Previous studies on MGA showed a potential for the use of MGA for genetically tagging other RNA molecules in live cells. However, these studies also exhibited low fluorescence signals and high background noise. Here we constructed and tested RNA scaffolds containing multiple tandem repeats of MGA as a strategy to increase the brightness of the MGA aptamer-fluorogen system as well as to make the system fluoresce when tagging various RNA molecules, in live cells. We demonstrate that our MGA scaffolds can induce fluorescence signals by up to ∼20-fold compared to the basal level as a genetic tag for other RNA molecules. We also show that our scaffolds function reliably as genetically encoded fluorescent tags for mRNAs of fluorescent proteins and other RNA aptamers.

中文翻译：

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使用孔雀石绿适体支架作为荧光探针标记活细胞中的RNA

RNA介导许多对细胞功能至关重要的过程。在活细胞中对RNA进行定量或成像的能力对于阐明RNA的这种功能非常有用。RNA适体–氟系统已越来越多地用于标记活细胞中的RNA。在这里，我们使用孔雀石绿适体（MGA），这是一种RNA适体，可以特异性结合孔雀石绿（MG）染料并诱导其发出远红色荧光信号。先前对MGA的研究表明，使用MGA进行遗传标记活细胞中其他RNA分子的潜力。但是，这些研究还显示出低荧光信号和高背景噪声。在这里，我们构建并测试了包含多个MGA串联重复序列的RNA支架，以此作为增加MGA适配体-荧光素系统亮度并在活细胞中标记各种RNA分子时使系统发出荧光的策略。我们证明了我们的MGA支架与其他RNA分子的遗传标签相比，可诱导荧光信号的亲和力高达基础水平的20倍左右。我们还表明，我们的支架功能可靠地作为遗传编码的荧光标签，用于荧光蛋白和其他RNA适体的mRNA。