Studies of physicochemical properties of N-H•••N hydrogen bonds in DNA, using selective 15N-labeling and direct 15N 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 oligomers. The N3 atom selectively N-15 enriched 2'-deoxycytidine and thymidine, and the uniformly N-15 enriched 2'-deoxyadenosine and 2'-deoxyguanosine, were chemically prepared and incorporated into two DNA oligomers, d(CGCGAATTCGCG)(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 spectrum. Additionally, it enabled the determination of (2h)J(NN) in D2O quite easily and highly quantitatively. The temperature and DNA sequence dependence were examined for these oligomers. The sequence dependence was not clear; however, a significant decrease of (2h)J(NN) was observed by elevating the temperature. This temperature dependence was not due to the hydrogen exchange, 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 compared to our N-15 1D method, the quantitative HNN-COSY method gave systematically smaller (2h)J(NN) values in our system, due to the lower N-15 fraction 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 systematic differences were recovered by theoretical correction of the N-15 isotope fraction contribution, by using the composite N-15 180 degrees pulse in a quantitative HNN-COSY experiment.