Direct 13C-detected NMR experiments for mapping and characterization of hydrogen bonds in RNA
Direct 13C-detected NMR experiments for mapping and characterization of hydrogen bonds in RNA
复制标题
DOI：
10.1007/s10858-016-0021-5
复制
发表时间：
2016-03-01
影响因子：
2.7
通讯作者：
Schwalbe, Harald
中科院分区：
文献类型：
--
作者：
Fuertig, Boris;Schnieders, Robbin;Schwalbe, Harald
在RNA二级结构测定中，确定核苷酸是否是碱基配对是必不可少的。核苷酸的碱基配对由氢键介导。氢键的NMR表征依赖于与可交换质子的NMR共振相关的实验，并且可以最好地用于RNA的结构化部分，其中不稳定的氢原子被保护免于溶剂交换。然而，RNA中功能重要的区域经常显示出增加的动力学紊乱，这通常导致可交换质子的NMR信号被加宽超过H-1检测。在这里，我们开发了C-13直接检测实验来观察RNA中的所有核苷酸，无论它们是否参与氢键。利用由于交换过程导致的标量耦合的自解耦，可以确定每个单独核苷酸的氢键供体的氢键行为。此外，HNN-COSY实验用于C-13直接检测的适应允许供体-受体对的相关性和氢键受体核苷酸的定位。因此，建议的C-13直接检测实验提供的信息不服从常规质子检测方法的分子位点。这些信息使得通过NMR确定RNA二级结构更加准确，并有助于验证基于生物信息学的二级结构预测。
In RNA secondary structure determination, it is essential to determine whether a nucleotide is base-paired and not. Base-pairing of nucleotides is mediated by hydrogen bonds. The NMR characterization of hydrogen bonds relies on experiments correlating the NMR resonances of exchangeable protons and can be best performed for structured parts of the RNA, where labile hydrogen atoms are protected from solvent exchange. Functionally important regions in RNA, however, frequently reveal increased dynamic disorder which often leads to NMR signals of exchangeable protons that are broadened beyond H-1 detection. Here, we develop C-13 direct detected experiments to observe all nucleotides in RNA irrespective of whether they are involved in hydrogen bonds or not. Exploiting the self-decoupling of scalar couplings due to the exchange process, the hydrogen bonding behavior of the hydrogen bond donor of each individual nucleotide can be determined. Furthermore, the adaption of HNN-COSY experiments for C-13 direct detection allows correlations of donor-acceptor pairs and the localization of hydrogen-bond acceptor nucleotides. The proposed C-13 direct detected experiments therefore provide information about molecular sites not amenable by conventional proton-detected methods. Such information makes the RNA secondary structure determination by NMR more accurate and helps to validate secondary structure predictions based on bioinformatics.