Observation and measurement of internucleotide (2)J(NN) coupling constantsbetween N-15 nuclei with widely separated chemical shifts

Scalar coupling correlations between hydrogen bonded N-15 nuclei in non Wat son-Crick base pairs is a critical step in the structure determination of u nusual nucleic acids. For observing the (2)J(NN) coupling constant between far upfield N2,N6 (amino) nitrogens and far downfield (N1,N3,N7) nitrogens (separated by 150-160 ppm), the HNN-COSY experiment (Dingley and Grzesiek, 1998) is rather insensitive, due to technical difficulties associated with simultaneous excitation of both extremes of the N-15 spectrum. These nuclei may be correlated by treating them in a pseudo-heteronuclear manner, using N-15 selective pulses. The wide chemical shift separation allows accurate measurement of the (2)J(NN) coupling constant using spin-echo difference me thods. Pulse sequences for observation and measurement of (2)J(NN) coupling constants between amino and N7 nuclei are presented and demonstrated on an A-A mismatch segment of the uniformly (N-15,C-13) labelled DNA sample, d(G GAGGAT)(2).