DETERMINATION OF 3-BOND (1)H3'-P-31 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 pr otein-nucleic acid complexes. The method is based on measuring the cha nge 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-nucl eic acid complexes where the protein is C-13-labeled but the nucleic a cid 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. T he 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 pro teins, or (3)J(CH) couplings in other selectively enriched compounds.