Studies of physicochemical properties of N-H center dot center dot center dot N hydrogen bonds in DNA, using selective N-15-labeling and direct N-15 1D NMR

N-15-N-15 scalar coupling constants across base pair hydrogen bonds ((2h)J( NN)) were studied using residue- and atom-specifically N-15 labeled DNA oli gomers. The N3 atom selectively N-15 enriched 2'-deoxycytidine and thymidin e, and the uniformly N-15 enriched 2'-deoxyadenosine and 2'-deoxyguanosine, were chemically prepared and incorporated into two DNA oligomers, d(CGCGAA TTCGCG)(2) and d(CGCAAAAAGCG).d(CGCTTTTTGCG). This isotope labeling enabled us to determine the (2h)J(NN) value from the splitting of the N-15 1D spec trum. Additionally, it enabled the determination of (2h)J(NN) in D2O quite easily and highly quantitatively. The temperature and DNA sequence dependen ce were examined for these oligomers. The sequence dependence was not clear ; however, a significant decrease of (2h)J(NN) was observed by elevating th e temperature. This temperature dependence was not due to the hydrogen exch ange, since the addition of 20 mM NH3 did not change the (2h)J(NN) values. The (2h)J(NN) values in D2O were somewhat smaller than those in H2O. As com pared to our N-15 1D method, the quantitative HNN-COSY method gave systemat ically smaller (2h)J(NN) values in our system, due to the lower N-15 fracti on of our sample (79 and 88% for dA and the other nucleotides, respectively ) and the insufficient power of the N-15 RF pulse (B-1=6.6 kHz). These syst ematic differences were recovered by theoretical correction of the N-15 iso tope fraction contribution, by using the composite N-15 180 degrees pulse i n a quantitative HNN-COSY experiment.