Determination of In Vitro and Cellular Turn-on Kinetics for Fluorogenic RNA Aptamers.

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-1 and 0.35 s-1, 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.

Hysteresis in poly-2'-deoxycytidine i-motif folding is impacted by the method of analysis as well as loop and stem lengths.

In DNA, i-motif (iM) folds occur under slightly acidic conditions when sequences rich in 2'-deoxycytidine (dC) nucleotides adopt consecutive dC self base pairs. The pH stability of an iM is defined by the midpoint in the pH transition (pHT ) between the folded and unfolded states. Two different experiments to determine pHT values via circular dichroism (CD) spectroscopy were performed on poly-dC iMs of length 15, 19, or 23 nucleotides. These experiments demonstrate two points: (1) pHT values were dependent on the titration experiment performed, and (2) pH-induced denaturing or annealing processes produced isothermal hysteresis in the pHT values. These results in tandem with model iMs with judicious mutations of dC to thymidine to favor particular folds found the hysteresis was maximal for the shorter poly-dC iMs and those with an even number of base pairs, while the hysteresis was minimal for longer poly-dC iMs and those with an odd number of base pairs. Experiments to follow the iM folding via thermal changes identified thermal hysteresis between the denaturing and annealing cycles. Similar trends were found to those observed in the CD experiments. The results demonstrate that the method of iM analysis can impact the pHT parameter measured, and hysteresis was observed in the pHT and Tm values.