title: Rapid RNA-ligand interaction analysis through high-information content conformational and stability landscapes.
abstract: 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.
authors.affiliation: National Heart, Lung and Blood Institute, NIH, 50 South Dr, Bethesda, Maryland 20892, USA.
authors.forename: Nathan J
authors.initials: NJ
authors.lastname: Baird
authors.affiliation: National Center for Advancing Translational Sciences, NIH, 9800 Medical Center Dr, Rockville, Maryland, 20850, USA.
authors.forename: James
authors.initials: J
authors.lastname: Inglese
authors.affiliation: National Heart, Lung and Blood Institute, NIH, 50 South Dr, Bethesda, Maryland 20892, USA.
authors.forename: Adrian R
authors.initials: AR
authors.lastname: Ferré-D'Amaré
mesh_terms: D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000737:chemistry / Q000235:genetics; D031541:Fluorescence Resonance Energy Transfer; D057166:High-Throughput Screening Assays; D008024:Ligands; D009690:Nucleic Acid Conformation; D020871:RNA Stability; D012329:RNA, Bacterial / Q000737:chemistry* / Q000235:genetics; D058928:Riboswitch; D014734:Vibrio cholerae / Q000737:chemistry / Q000235:genetics*
chemical_list: D008024:Ligands; D012329:RNA, Bacterial; D058928:Riboswitch; C062025:bis(3',5')-cyclic diguanylic acid; D006152:Cyclic GMP