Coupling of Sequential Transitions in a DNA Double Hairpin: Energetics, Ion Binding, and Hydration

In an effort to evaluate the relative contributions of sequence, ion binding, and hydration to the thermodynamic stability of nucleic acids, we have investigated the melting behavior of a double hairpin and that of its component single hairpins. Temperature-dependent UV absorption and differential scanning calorimetry techniques have been used to characterize the helix-coil transitions of three deoxyoligonucleotides: d(GTACT₅GTAC), d(GCGCT₅GCGC), and d(GCGCT₅GCGCGTACT₅GTAC). The first two oligomers melt with transition temperatures equal to 28 and 69 °C, respectively, in 10 mM dibasic sodium phosphate at pH 7.0. The T_m’s are independent of strand concentration, strongly indicating the presence of singlestranded hairpin structures at low temperatures. The third oligomer, with a sequence corresponding to the joined sequences of the first two oligomers, melts with two apparently independent monomolecular transitions with T_m’s of 41 and 69 °C. These transitions correspond to the melting of a double hairpin. In the salt range of 10-100 mM in NaCl, we obtain average enthalpies of 24 and 38 kcal/mol for the transitions in the single-hairpin molecules. Each transition in the double hairpin has an enthalpy of 32 kcal/mol. In addition, dt_m/d log [Na⁺] for the transitions are 4.1 and 4.7 °C for the single hairpins and 12.6 and 11.2 °C for each transition in the double hairpin. The differential ion binding parameter between the double hairpin and that of the sum of single hairpins is roughly equal to 1.1 mol of Na⁺ ions/mol of double hairpin and is consistent with an increase in the electrostatic behavior of the stem phosphates of this molecule. Thermodynamic profiles at 5 °C reveal that each thermodynamic parameter (ΔG°, ΔH°, and ΔS°) for the formation of the double hairpin is equal to the sum of the thermodynamic parameters of the two single hairpins. Combined measurements of ultrasound velocities and densities at 5 °C allows us to determine the apparent molar volume and the apparent molar adiabatic compressibility of each molecule. The values for the double hairpin correspond roughly to the sum of the individual contributions of the single-hairpin molecules and agree with the additivity of our CD spectra. A close inspection of the parameters for the transitions in the double hairpin relative to the corresponding transitions in the single hairpins indicates that the stabilization of the first transition is accompanied by a more favorable enthalpy together with an increase in the amount of bound sodium ions, while the second transition is accompanied by a more favorable entropy. The apparent molar volume and apparent molar adiabatic compressibility values for each single hairpin indicate that the hairpin with the GTAC/CATG stem is more hydrated than the hairpin with GCGC/CGCG stem.