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A 13C, 15N-labeled DNA hairpin molecule of the sequence d(GCGAAGC) = d(G1C2G3A4A5G6C7) is investigated by NMR spectroscopy to determine one-bond and two-bond NMR spin-spin coupling constants 1J(X, H) (X= C, N), 1J(C, X) (X= C, N), and 2J(X,H) (X=C,N). Measured J values for the Watson-Crick (WC) base-pairs G1C7, G6C2, the mismatched base-pair G3A5 and the unpaired base A4 were compared with calculated J values to verify sign and magnitude. For the J-calculations, coupled perturbed density functional theory in connection with the B3LYP hybrid functional and basis sets (9s5p1d/5s,1p)[6s,4p,1d/3s,1p] as well as (11s,7p,2d/6s,2p)[7s,6p,2d/4s,2p] was employed to determine diamagnetic spin-orbit, paramagnetic spin-orbit, Fermi contact, and spin-dipolar contributions to the total isotropic coupling constant J. Coupling constants 1J(C,H) and 2J(N,H) turn out to be very sensitive to the position of C and N in the pyrimidine or purine rings and, therefore, can be used for rapid structure determination. Coupling constant 1J(N1,C6) in G of GC clearly reflects the impact of H-bonding by an increase from -6.5 to -10.6 Hz. The direct investigation of H-bonding via the 2J(N,N') coupling constants reveals that these parameters depend on the distance R(N-N'), the bending angle N-H..N', and the degree of planarity at the H-donor group. Different types of H-bonding were identified. H-bonding is weaker in AG and therefore, leads to smaller changes in the J values of the bases A and G upon pairing than in the case of the WC base-pair GC.
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