Laboratory of Chemical Physics, National Institute of Diabetes, Digestive and Kidney Diseases , National Institutes of Health , Bethesda , Maryland 20892 , United States.
J Am Chem Soc. 2018 Jun 6;140(22):6978-6983. doi: 10.1021/jacs.8b03298. Epub 2018 May 25.
NMR approaches using nucleotide-specific deuterium labeling schemes have enabled structural studies of biologically relevant RNAs of increasing size and complexity. Although local structure is well-determined using these methods, definition of global structural features, including relative orientations of independent helices, remains a challenge. Residual dipolar couplings, a potential source of orientation information, have not been obtainable for large RNAs due to poor sensitivity resulting from rapid heteronuclear signal decay. Here we report a novel multiple quantum NMR method for RDC determination that employs flip angle variation rather than a coupling evolution period. The accuracy of the method and its utility for establishing interhelical orientations are demonstrated for a 36-nucleotide RNA, for which comparative data could be obtained. Applied to a 78 kDa Rev response element from the HIV-1 virus, which has an effective rotational correlation time of ca. 160 ns, the method yields sensitivity gains of an order of magnitude or greater over existing approaches. Solution-state access to structural organization in RNAs of at least 230 nucleotides is now possible.
NMR 方法使用核苷酸特异性氘标记方案，已经能够对越来越大、越来越复杂的具有生物学相关性的 RNA 进行结构研究。尽管这些方法可以很好地确定局部结构，但定义全局结构特征，包括独立螺旋的相对取向，仍然是一个挑战。由于异核信号衰减迅速导致灵敏度差，因此对于大 RNA，尚未获得残磁偶合（一种潜在的取向信息源）。在这里，我们报告了一种新的 RDC 测定的多量子 NMR 方法，该方法采用翻转角变化而不是偶合演化周期。对于 36 个核苷酸的 RNA，我们证明了该方法的准确性及其用于确定螺旋间取向的适用性，因为可以获得比较数据。将该方法应用于 HIV-1 病毒的 Rev 反应元件（有效旋转相关时间约为 160 ns），与现有方法相比，该方法的灵敏度提高了一个数量级或更多。现在可以在至少 230 个核苷酸的 RNA 中获得溶液状态的结构组织。
