NMR experiments for the rapid identification of P=O···Hâ??X type hydrogen bonds in nucleic acids

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NMR experiments for the rapid identification of P=O···Hâ??X type hydrogen bonds in nucleic acids

Abstract

Hydrogen bonds involving the backbone phosphate groups occur with high frequency in functional RNA molecules. They are often found in well-characterized tertiary structural motifs presenting powerful probes for the rapid identification of these motifs for structure elucidation purposes. We have shown recently that stable hydrogen bonds to the phosphate backbone can in principle be detected by relatively simple NMR-experiments, providing the identity of both the donor hydrogen and the acceptor phosphorous within the same experiment (Duchardt-Ferner et al., Angew Chem Int Ed Engl 50:7927â??7930, 2011). However, for imino and hydroxyl hydrogen bond donor groups rapidly exchanging with the solvent as well as amino groups broadened by conformational exchange experimental sensitivity is severely hampered by extensive line broadening. Here, we present improved methods for the rapid identification of hydrogen bonds to phosphate groups in nucleic acids by NMR. The introduction of the SOFAST technique into 1H,31P-correlation experiments as well as a BEST-HNP experiment exploiting 3hJN,P rather than 2hJH,P coupling constants enables the rapid and sensitive identification of these hydrogen bonds in RNA. The experiments are applicable for larger RNAs (up to ~â??100-nt), for donor groups influenced by conformational exchange processes such as amino groups and for hydrogen bonds with rather labile hydrogens such as 2â?²-OH groups as well as for moderate sample concentrations. Interestingly, the size of the through-hydrogen bond scalar coupling constants depends not only on the type of the donor group but also on the structural context. The largest coupling constants were measured for hydrogen bonds involving the imino groups of protonated cytosine nucleotides as donors.



Source: Journal of Biomolecular NMR