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

Yanyan Xue,Yu Liu

doi:10.3390/ijms23031493

Yanyan Xue, Yu Liu

Open Access

https://doi.org/10.3390/ijms23031493

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Abstract

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.

Highlights

Riboswitch RNA to Measure Mg2+Riboswitches are non-coding RNAs located in the 50 -untranslated region of mRNAs and are composed of an aptamer domain and an expression platform
We found that the dynamics of the kissing loop (KL) in both RNAs were af‐
Site‐Specific of Tide Quencher 3 (TQ3)/Cy3‐apt and TQ3/Cy3‐fl incorporated into the kissing loop (KL) of the full-length adenine riboswitch RNA and its

Summary

Introduction

Riboswitch RNA to Measure Mg2+Riboswitches are non-coding RNAs located in the 50 -untranslated region of mRNAs and are composed of an aptamer domain and an expression platform. The aptamer domain can differentiate its target ligand from countless metabolites in cells, trigger structural switches, and regulate the transcription or translation of the genes downstream of the riboswitches [1,2]. The protein-free regulatory function of riboswitch RNAs relies on their structural flexibility during their binding with specific ligands, and highlights the potential for natural RNA biosensors to detect cellular metabolites [14]. Spinach, mango, and broccoli aptamers have been coupled to riboswitches to monitor the folding of riboswitch RNA, and have been used as fluorescent light-up aptamers (FLAPs) to detect metabolites in cells [15,16,17,18]. The non-negligible size of an aptamer may disturb the target RNA structure and function [16,20,21]

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