Light-up and FRET aptamer reporters; evaluating their applications for imaging transcription in eukaryotic cells.
Light-up and FRET aptamer reporters; evaluating their applications for imaging transcription in eukaryotic cells.
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DOI：
10.1016/j.ymeth.2015.12.009
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发表时间：
2016-04-01
期刊：
影响因子：
--
通讯作者：
Nilsen-Hamilton M
中科院分区：
文献类型：
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作者：
Ilgu M;Ray J;Bendickson L;Wang T;Geraskin IM;Kraus GA;Nilsen-Hamilton M
RNA转录的调节是细胞功能的核心。基因表达的变化驱动分化和细胞对损伤等事件的反应。RNA运输也可以对蛋白质表达及其定位产生很大影响。因此，在真实的时间和活细胞中成像RNA转录和运输的能力是一个很难实现的有价值的目标。当被适体结合时引起其配体荧光增加的“点亮”适体的可用性已经显示出用于报告体内RNA产生和定位的前景。在这里，我们已经调查了两个发光的适体（孔雀石绿色适体和菠菜适体）的适用性作为报告的RNA表达在体内使用两种真核细胞类型，酵母和哺乳动物。我们的分析集中在适体配体，它们对背景噪声的贡献，以及串联适体串对信号强度和配体亲和力的影响。尽管背景荧光在体外非常低，但对于细胞成像并不总是如此。我们的研究结果表明，在使用发光适体作为RNA成像报告时需要谨慎。特别是，应由对样品身份不知情的操作员收集和分析图像。每个实验中必须包括配体与细胞在不存在适体表达的情况下的适当对照条件。该对照条件确定了配体与适体的特异性相互作用，而不是与未知细胞元件的非特异性相互作用，是观察到的荧光信号的原因。由于适体配体与细胞组分的非特异性相互作用而产生的高背景信号可以通过使用IMAGE标签（细胞内多适体遗传标签）来最小化，IMAGE标签通过FRET发出信号并且是用于成像转录的有前景的RNA报告物。
The regulation of RNA transcription is central to cellular function. Changes in gene expression drive differentiation and cellular responses to events such as injury. RNA trafficking can also have a large impact on protein expression and its localization. Thus, the ability to image RNA transcription and trafficking in real time and in living cells is a worthwhile goal that has been difficult to achieve. The availability of “light-up” aptamers that cause an increase in fluorescence of their ligands when bound by the aptamer have shown promise for reporting on RNA production and localization in vivo. Here we have investigated two light-up aptamers (the malachite green aptamer and the Spinach aptamers) for their suitability as reporters of RNA expression in vivo using two eukaryotic cell types, yeast and mammalian. Our analysis focused on the aptamer ligands, their contributions to background noise, and the impact of tandem aptamer strings on signal strength and ligand affinity. Whereas the background fluorescence is very low in vitro, this is not always true for cell imaging. Our results suggest the need for caution in using light-up aptamers as reporters for imaging RNA. In particular, images should be collected and analyzed by operators blinded to the sample identities. The appropriate control condition of ligand with the cells in the absence of aptamer expression must be included in each experiment. This control condition establishes that the specific interaction of ligand with aptamer, rather than nonspecific interactions with unknown cell elements, is responsible for the observed fluorescent signals. High background signals due to nonspecific interactions of aptamer ligands with cell components can be minimized by using IMAGEtags (Intracellular Multiaptamer GEnetic tags), which signal by FRET and are promising RNA reporters for imaging transcription.