Nuclear magnetic resonance (NMR) and differential scanning calorimetry (DSC) have been used to investigate the helix-to-coil transition of the duplex formed by the self-complementary dodecamer d(C1G2T3G4A5A6T6T5C4G3C2G1) (henceforth called 12-mer GT duplex) containing dG·dT interactions at position 3. These results are compared with the corresponding d(CGCGAATTCGCG) dodecamer duplex (henceforth called 12-mer GC duplex) containing standard Watson-Crick dG·dC base pairs at position 3 [Patel, D.J., Kozlowski, S.A., Marky, L.A., Broka, C., Rice, J.A., Itakura, K., & Breslauer, K.J. (1981) Biochemistry (first paper of four in this issue)]. The dG·dT interaction in the 12-mer GT duplex was monitored at the imino protons of both bases in the pair, the nonexchangeable protons of the thymidine residue, and the phosphate resonances at the modification site. We demonstrate dG·dT wobble base pair formation involving two imino proton-carbonyl group hydrogen bonds, based on a nuclear Overhauser effect between the adjacent interstrand guanosine and thymidine imino protons in the pair. Furthermore, the base pairing on either side of the dG·dT wobble base pair remains intact. The wobble base pair formation results in a small decrease in stacking of the thymidine residue with adjacent base pairs and a possible conformational change at two phosphodiester linkages. The nonexchangeable base proton markers at the dG·dT wobble pair at position 3 and the nonterminal Watson-Crick base pairs at positions 2, 4, 5, and 6 of the 12-mer GT duplex exhibit a common transition midpoint of −52 °C in 0.1 M phosphate solution. Differential scanning calorimetry also demonstrates that replacing two dG·dC base pairs with two dG·dT base pairs reduces the melting temperature of the dodecanucleotide duplex by −20 °C. Furthermore, we measure a calorimetric enthalpy change of 106 kcal (mol of double strand)−1 for the 12-mer GT duplex in 0.1 M NaCl. This enthalpy change is of similar magnitude to the corresponding value measured for the 12-mer GC duplex. These calorimetric results are consistent with the dG·dT wobble pair stacking with adjacent pairs in the 12-mer GT duplex to about the same extent as a regular dG·dC base pair. The model-dependent van't Hoff enthalpies are found to be much smaller than the calorimetrically measured values, thereby indicating that the transition from duplex to single strand involves intermediate states. The size of the cooperative melting unit remains unchanged at 9 ± 1 base pairs despite the wobble base pair induced destabilization of the 12-mer GT duplex relative to the 12-mer GC duplex.
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