Rapid RNA–ligand interaction analysis through high-information content conformational and stability landscapes

The structure and biological properties of RNAs are a function of changing cellular conditions, but comprehensive, simultaneous investigation of the effect of multiple interacting environmental variables is not easily achieved. We have developed an efficient, high-throughput method to characterize RNA structure and thermodynamic stability as a function of multiplexed solution conditions using Förster resonance energy transfer (FRET). In a single FRET experiment using conventional quantitative PCR instrumentation, 19,400 conditions of MgCl2, ligand and temperature are analysed to generate detailed empirical conformational and stability landscapes of the cyclic diguanylate (c-di-GMP) riboswitch. The method allows rapid comparison of RNA structure modulation by cognate and non-cognate ligands. Landscape analysis reveals that kanamycin B stabilizes a non-native, idiosyncratic conformation of the riboswitch that inhibits c-di-GMP binding. This demonstrates that allosteric control of folding, rather than direct competition with cognate effectors, is a viable approach for pharmacologically targeting riboswitches and other structured RNA molecules.