NMR Detection of NH···OC Hydrogen Bonds in 13C,15N-Labeled Nucleic Acids

We describe the first direct observation of N H···O C hydrogen bonding in nucleic acids via the four-bond 4h J N i N j coupling within an N1 i H1 i ···O6 j C6 j −N1 j segment of a G·G·G·G tetrad. The experiment, two-dimensional (2D) HN(N)-TOCSY, makes use of band-selective 15 N isotropic mixing to transfer magnetization exclusively via the 4h J NN couplings to yield correlations between N1H i (ω 2 ) and N1 j (ω 1 ) across the hydrogen-bonded segment. In a complementary experiment, 2D cross-polarization (CP)-H(N)CO−(NN)-TOCSY, employing band-selective heteronuclear 15 N− 13 C cross-polarization sequences on either side of the 15 N− 15 N TOCSY period, correlations are obtained between N1H i (ω 2 ) and C6 j (ω 1 ) nuclei across the hydrogen bond. The symmetric A 2 X 2 -type coupling topology of the four−spin nitrogen system in the G·G·G·G tetrad permits accurate measurement of these couplings by a new procedure that fits the experimental data with known analytical isotropic mixing transfer functions. The techniques are demonstrated on a G·G·G·G tetrad formed within a novel dimeric quadruplex fold of a uniformly 13 C/ 15 N-labeled d(G-G-G-T-T-C-A-G-G) DNA sequence. The value of the 4h J NN coupling constant for this system is estimated to be 0.136 ± 0.021 Hz.