Enhanced spectral resolution in RNA HCP spectra for measurement of (3)J(C2'P) and (3)J(C4'P) couplings and (31)P chemical shift changes upon weak alignment.

The 'out-and-back' 3D HCP experiment, using gradient- and sensitivity-enhanced detection, provides a convenient method for assignment of the (31)P NMR spectra and accurate measurement of the (31)P chemical shifts of ribonucleic acids. The (13)C resolution in such spectra can be doubled, at the cost of a 50% reduction in sensitivity, by combining (13)C evolution during the (13)C-[(31)P] de- and rephasing periods. The multiple connectivities observable for a given (31)P, including correlations to the intranucleotide C5'H(2) and C4'H groups, and the C2'H, C3'H and C4'H groups of the preceding nucleotide, permit independent measurements of the (31)P shift. The (13)C spectrum of these groups is typically crowded for an RNA molecule in isotropic solution and overlap becomes more problematic in media used to achieve partial alignment. However, many of these correlations are resolvable in the combined-evolution HCP spectrum. The difference in (31)P chemical shift between isotropic solution and a medium containing liquid crystalline Pf1 provides information on the orientation of phosphate groups. The intensities measured in the 3D HCP spectrum, obtained for an isotropic sample, yield values for the (3)J(C2'P) and (3)J(C4'P) couplings, thereby providing important restraints for the backbone torsion angles epsilon and beta. The experiments are illustrated for a uniformly (13)C-enriched, 24-residue stem-loop RNA sequence, and results for the helical stem region show close agreement between observed Deltadelta((31)P) values and those predicted for a model A-form RNA helix when using a uniform (31)P CSA tensor. This confirms that Deltadelta((31)P) values can be used directly as restraints in refining nucleic acid structures.