Rapid RNA-ligand interaction analysis through high-information content conformational and stability landscapes
Rapid RNA-ligand interaction analysis through high-information content conformational and stability landscapes
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DOI：
10.1038/ncomms9898
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发表时间：
2015-12-01
影响因子：
16.6
通讯作者：
Ferre-D'Amare, Adrian R.
中科院分区：
文献类型：
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作者：
Baird, Nathan J.;Inglese, James;Ferre-D'Amare, Adrian R.
RNA的结构和生物学特性是细胞条件变化的函数，但要全面、同时研究多种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、高通量的方法来表征RNA的结构和热力学稳定性，它是使用Forster共振能量转移(FRET)来表征多重溶液条件的函数。在使用常规定量PCR仪器的单一FRET实验中，分析了19,400个氯化镁、配体和温度的条件，以生成环状二鸟苷(c-di-GMP)核糖开关的详细经验构象和稳定性图景。该方法可以快速比较同源和非同源配体对RNA结构的调节。景观分析表明，卡那霉素B稳定了核糖开关的非天然、特殊构象，从而抑制了c-di-GMP结合。这表明，变构控制折叠，而不是与同源效应器直接竞争，是药理学靶向核糖开关和其他结构RNA分子的可行方法。
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.