Study of nucleic acid structure by novel NMR methods

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Upon alignment of oligonucleotides in a magnetic field, the downfield, TROSY-component of the 13C-1H doublet changes its resonance frequency as a result of residual dipolar 13C-1H dipolar coupling (RDC) and residual 13C chemical shift anisotropy (RCSA), and the sum of these two second rank tensors is referred to as the pseudo-CSA. The experimentally measured difference in the resonance frequency of the 13C TROSY component in the aligned and isotropic samples is referred to as residual pseudo-CSA (RPCSA), and it can be used directly as a restraint during structure calculation. Because measurement of the RPCSA involves detection of the narrow TROSY 13C doublet component, it is applicable to systems with larger rotational correlation times than RDC measurement. The method is demonstrated for structure refinement of the helical region of a 24-nt stem-loop segment or ribosomal helix 35, uniformly enriched in 13C and 15N, with RPCSA values measured at 5 and 25 C. Substantial cross-validated improvements in structural accuracy are obtained upon incorporation of RPCSA restraints.