Incorporation of a FRET Pair into a Riboswitch RNA to Measure Mg²⁺ Concentration and RNA Conformational Change in Cell

Riboswitches are natural biosensors that can regulate gene expression by sensing small molecules. Knowledge of the structural dynamics of riboswitches is crucial to elucidate their regulatory mechanism and develop RNA biosensors. In this work, we incorporated the fluorophore, Cy3, and its quencher, TQ3, into a full-length adenine riboswitch RNA and its isolated aptamer domain to monitor the dynamics of the RNAs in vitro and in cell. The adenine riboswitch was sensitive to Mg²⁺ concentrations and could be used as a biosensor to measure cellular Mg²⁺ concentrations. Additionally, the TQ3/Cy3-labeled adenine riboswitch yielded a Mg²⁺ concentration that was similar to that measured using a commercial assay kit. Furthermore, the fluorescence response to the adenine of the TQ3/Cy3-labeled riboswitch RNA was applied to determine the proportions of multiple RNA conformational changes in cells. The strategy developed in this work can be used to probe the dynamics of other RNAs in cells and may facilitate the developments of RNA biosensors, drugs and engineering.