Department of Chemistry and Henry Eyring Center for Cell & Genome Science, University of Utah.
Department of Chemistry and Henry Eyring Center for Cell & Genome Science, University of Utah;
J Vis Exp. 2022 Aug 9(186). doi: 10.3791/64367.
Fluorogenic RNA aptamers have been applied in live cells to tag and visualize RNAs, report on gene expression, and activate fluorescent biosensors that detect levels of metabolites and signaling molecules. In order to study dynamic changes in each of these systems, it is desirable to obtain real-time measurements, but the accuracy of the measurements depends on the kinetics of the fluorogenic reaction being faster than the sampling frequency. Here, we describe methods to determine the in vitro and cellular turn-on kinetics for fluorogenic RNA aptamers using a plate reader equipped with a sample injector and a flow cytometer, respectively. We show that the in vitro kinetics for the fluorescence activation of the Spinach2 and Broccoli aptamers can be modeled as two-phase association reactions and have differing fast phase rate constants of 0.56 s and 0.35 s, respectively. In addition, we show that the cellular kinetics for the fluorescence activation of Spinach2 in Escherichia coli, which is further limited by dye diffusion into the Gram-negative bacteria, is still sufficiently rapid to enable accurate sampling frequency on the minute timescale. These methods to analyze fluorescence activation kinetics are applicable to other fluorogenic RNA aptamers that have been developed.
荧光 RNA 适体已被应用于活细胞，用于标记和可视化 RNA、报告基因表达，并激活荧光生物传感器，以检测代谢物和信号分子的水平。为了研究这些系统中的每一个的动态变化，获得实时测量是很理想的，但测量的准确性取决于荧光反应的动力学是否快于采样频率。在这里，我们描述了使用配备有样品注射器的平板读数仪和流式细胞仪分别确定荧光 RNA 适体的体外和细胞内开启动力学的方法。我们表明，Spinach2 和 Broccoli 适体的荧光激活的体外动力学可以被模拟为两相缔合反应，并且具有不同的快速相速率常数，分别为 0.56 s 和 0.35 s。此外，我们表明，Spinach2 在大肠杆菌中的荧光激活的细胞动力学仍然足够快，足以在分钟尺度上实现准确的采样频率，其进一步受到染料扩散到革兰氏阴性细菌的限制。这些用于分析荧光激活动力学的方法适用于已经开发的其他荧光 RNA 适体。
