Determination of three-bond 1H3′-31P couplings in nucleic acids and protein nucleic acid complexes by quantitative J correlation spectroscopy

A new sensitive two-dimensional quantitative J correlation experiment is described for measuring 3J(H3'-P) couplings in nucleic acids and protein-nucleic acid complexes. The method is based on measuring the change in intensity of the H-1-H-1 cross peaks in a constant-time H-1-H-1 COSY experiment which occurs in the presence and absence of 3J(H3'-P) dephasing during the constant-time evolution period. For protein-nucleic acid complexes where the protein is C-13-labeled but the nucleic acid is not,C-12-filtering is readily achieved by the application of a series of C-13 purge pulses during the constant time evolution period without any loss of signal-to-noise of the nucleic acid cross peaks. The method is demonstrated for the Dickerson DNA dodecamer and a 19 kDa complex of the transcription factor SRY with a 14mer DNA duplex. The same approach should be equally applicable to numerous other problems, including the measurement of J(H-Cd) couplings in cadmium-ligated proteins, or (3)J(CH) couplings in other selectively enriched compounds.