Institute for Organic Chemistry and Chemical Biology, Johann Wolfgang Goethe-University Frankfurt, Max-von-Laue-Strasse 7, 60438 Frankfurt am Main, Germany.
J Am Chem Soc. 2010 Aug 4;132(30):10318-29. doi: 10.1021/ja910015n.
The quantitative Gamma-(HCP) experiment, a novel heteronuclear NMR pulse sequence for the determination of the RNA backbone angles alpha(O3'(i-1)-P(i)-O5'(i)-C5'(i)) and zeta(C3'(i)-O3'(i)-P(i+1)-O5'(i+1)) in (13)C-labeled RNA, is introduced. The experiment relies on the interaction between the CH bond vector dipole and the (31)P chemical shift anisotropy (CSA), which affects the relaxation of the (13)C,(31)P double- and zero-quantum coherence and thus the intensity of the detectable magnetization. With the new pulse sequence, five different cross-correlated relaxation rates along the phosphodiester backbone can be measured in a quantitative manner, allowing projection-angle and torsion-angle restraints for the two backbone angles alpha and zeta to be extracted. Two versions of the pulse sequence optimized for the CH and CH(2) groups are introduced and demonstrated for a 14-mer cUUCGg tetraloop RNA model system and for a 27-mer RNA with a previously unknown structure. The restraints were incorporated into the calculation of a very high resolution structure of the RNA model system (Nozinovic, S.; et al. Nucleic Acids Res. 2010, 38, 683). Comparison with the X-ray structure of the cUUCGg tetraloop confirmed the high quality of the data, suggesting that the method can significantly improve the quality of RNA structure determination.
定量 Gamma-(HCP) 实验是一种新的异核 NMR 脉冲序列，用于测定 (13)C 标记 RNA 中的 RNA 骨架角 alpha(O3'(i-1)-P(i)-O5'(i)-C5'(i)) 和 zeta(C3'(i)-O3'(i)-P(i+1)-O5'(i+1))。该实验依赖于 CH 键向量偶极子与 (31)P 化学位移各向异性 (CSA) 之间的相互作用，这会影响 (13)C、(31)P 双量子和零量子相干的弛豫，从而影响可检测磁化强度。通过新的脉冲序列，可以定量测量沿磷酸二酯骨架的五个不同的交叉相关弛豫率，从而可以提取两个骨架角 alpha 和 zeta 的投影角和扭转角约束。引入了两种针对 CH 和 CH(2) 基团进行优化的脉冲序列，并在一个 14 -mer cUUCGg 四环 RNA 模型系统和一个具有未知结构的 27-mer RNA 上进行了演示。这些约束被纳入 RNA 模型系统的非常高分辨率结构计算中（Nozinovic，S.；等人。核酸研究。2010, 38, 683）。与 cUUCGg 四环的 X 射线结构进行比较证实了数据的高质量，表明该方法可以显著提高 RNA 结构测定的质量。
