Journal
NATURE COMMUNICATIONS
Volume 6, Issue -, Pages -Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/ncomms9898
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Funding
- National Heart, Lung and Blood Institute, NIH
- National Center for Advancing Translational Sciences, NIH
- Director's Challenge Innovation Award, NIH
- K22 NHLBI Career Transition Award [K22-HL121113A]
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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 Forster 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.
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