Measurement of carbon-phosphorus J coupling constants in RNA using spin-echo difference constant-time HCCH-COSY

We report a novel NMR technique for the measurement of carbon-phosphorus coupling constants in RNA oligomers. This method, spin-echo difference constant-time HCCH-COSY, takes advantage of the well-dispersed H1' and C1' resonances to analyze couplings involving the more poorly dispersed ribose carbon and phosphorus resonances. The technique was applied to analysis of the (3)J(C2'P) coupling constants related to backbone epsilon torsion angles in a 30-nucleotide lead-dependent ribozyme. (3)J(C2'P) coupling constants were obtained for similar to 90% of the residues in this RNA, which is over twice as many as could be obtained with previous methods. (C) 1998 Academic Press.