Resolution-optimized NMR measurement of (1)D(CH), (1)D(CC) and (2)D(CH) residual dipolar couplings in nucleic acid bases.

New methods are described for accurate measurement of multiple residual dipolar couplings in nucleic acid bases. The methods use TROSY-type pulse sequences for optimizing resolution and sensitivity, and rely on the E.COSY principle to measure the relatively small two-bond (2)D(CH) couplings at high precision.

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.

Measurement of five dipolar couplings from a single 3D NMR multiplet applied to the study of RNA dynamics.

A new three-dimensional NMR experiment is described that yields five scalar or dipolar couplings from a single cross-peak between three spins. The method is based on the E.COSY principle and is demonstrated for the H1'-C1'-C2' fragment of ribose sugars in a uniformly 13C-enriched 24-nucleotide RNA stem-loop structure, for which a complete set of couplings was obtained for all nonmodified nucleotides.

1H-1H dipolar couplings provide a unique probe of RNA backbone structure.

A NMR method is described that permits simultaneous measurement of the geminal 2JH1H2 + 2DH1H2 splitting and the sum of the 1JCH1 + 1DCH1 + 1JCH2 + 1DCH2 couplings for methylene groups, where 2DH1H2 and 1DCH are residual dipolar couplings, occurring when molecules are weakly oriented relative to the magnetic field. By suppressing either the upfield or downfield half of the 1H-1H geminal doublet, the experiment yields improved resolution relative to regular two-dimensional 1H-13C correlation spectra, making it applicable to systems of considerable complexity. The method is demonstrated for measurement of all 2DH5'H5'' couplings in a 24-nucleotide 13C-enriched RNA stem loop structure, weakly aligned in liquid crystalline Pf1.