Direct 13C-detected NMR experiments for mapping and characterization of hydrogen bonds in RNA
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 1H detection. Here, we develop 13C 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 13C direct detection allows correlations of donor–acceptor pairs and the localization of hydrogen-bond acceptor nucleotides. The proposed 13C 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.
Keywords13C direct detection RNA Exchange Hydrogen bonds Base pairs
This work was funded by the German funding agency (DFG) in Collaborative Research Center 902: Molecular principles of RNA-based regulation and in Graduate College: CLIC. Financial support by the Access to Research Infrastructures activity in the 6th Framework Programme of the EC (Contract # RII3-026145, EU-NMR) for conducting the research is gratefully acknowledged. Harald Schwalbe is member of the DFG-funded Cluster of Excellence: macromolecular complexes (EXC115). Christina Helmling is supported by the Fonds of the Chemical Industry. Work at BMRZ is supported by the state of Hesse.
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