Baird Nathan J, Inglese James, Ferré-D'Amaré Adrian R
National Heart, Lung and Blood Institute, NIH, 50 South Dr, Bethesda, Maryland 20892, USA.
National Center for Advancing Translational Sciences, NIH, 9800 Medical Center Dr, Rockville, Maryland, 20850, USA.
Nat Commun. 2015 Dec 7;6:8898. doi: 10.1038/ncomms9898.
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.
RNA的结构和生物学特性是细胞条件变化的函数，但要全面、同时研究多个相互作用的环境变量的影响并非易事。我们开发了一种高效、高通量的方法，利用Förster共振能量转移（FRET）来表征RNA结构和热力学稳定性与多重溶液条件的函数关系。在使用传统定量PCR仪器的单次FRET实验中，分析了19400种MgCl2、配体和温度条件，以生成环二鸟苷酸（c-di-GMP）核糖开关详细的经验性构象和稳定性图谱。该方法能够快速比较同源和非同源配体对RNA结构的调节作用。图谱分析表明，卡那霉素B稳定了核糖开关的一种非天然、特异的构象，这种构象会抑制c-di-GMP的结合。这表明，折叠的变构控制而非与同源效应物的直接竞争，是一种可行的药理靶向核糖开关和其他结构化RNA分子的方法。
