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Duplexes are the most fundamental elements of nucleic acid folding. Although it has become increasingly clear that duplex formation can be associated with a significant change in heat capacity (deltaC(p)), this parameter is typically overlooked in thermodynamic studies of nucleic acid folding. Analogy to protein folding suggests that base stacking events coupled to duplex formation should give rise to a deltaC(p) due to the release of waters solvating aromatic surfaces of nucleotide bases. In previous work, we showed that the deltaC(p) observed by isothermal titration calorimetry (ITC) for RNA duplex formation depended on salt and sequence [Takach, J. C., Mikulecky, P. J., and Feig, A. L. (2004) J. Am. Chem. Soc. 126, 6530-6531]. In the present work, we apply calorimetric and spectroscopic techniques to a series of designed DNA duplexes to demonstrate that both the salt dependence and sequence dependence of deltaC(p)s observed by ITC reflect perturbations to the same fundamental phenomenon: stacking in the single-stranded state. By measuring the thermodynamics of single strand melting, one can accurately predict the deltaC(p)s observed for duplex formation by ITC at high and low ionic strength. We discuss our results in light of the larger issue of contributions to deltaC(p) from coupled equilibria and conclude that observed deltaC(p)s can be useful indicators of intermediate states in nucleic acid folding phenomena.

Heat Capacity Changes Associated with DNA Duplex Formation: Salt- and Sequence-Dependent Effects