Origins of the "nucleation" free energy in the hybridization thermodynamics of short duplex DNA.

Thermodynamic parameters deltaH(cal), deltaS(cal), and deltaG(cal) of the melting transitions for 19 short DNA/DNA duplexes ranging in length from 6 to 35 base pairs were systematically evaluated by differential scanning calorimetry melting experiments carried out at four salt concentrations from 85 mM to 1.0 M [Na+]. As expected, thermodynamic stabilities of the DNA duplexes increased with length and increasing [Na+]. From plots of deltaG25 versus duplex length, extrapolation to N = 0 provided estimates on values of deltaG(cal)25 (N = 0) as a function of [Na+], corresponding to the free-energy of the "hypothetical duplex" having zero base pairs, but occupying precisely the same molar volume as the fully base paired duplex. The values obtained for deltaG(cal)25 (N = 0) were 3.68, 5.59, 7.86, and 8.68 kcal/mol in 1.00, 0.60, 0.30, and 0.085 M Na+, respectively. These values are in reasonable agreement with published values of the nucleation or initiation free-energy, attributed to formation of the first base pair in a short duplex compared to formation of the remaining base pairs. A statistical thermodynamic formulation of the association of two strands accounting for displaced solvent was utilized to relate [Na+]-dependent deltaG(cal)25 (N = 0) values to configuration integrals for both single and duplex strands. Relative differences between two single strands in their standard states and the duplex (in its standard state), and solvent displaced during the annealing process was taken into account. This analysis provides a new vantage point to view what has historically been referred to as the helix initiation or nucleation parameter and provides an alternate interpretation and mechanism for the nucleation complex in duplex formation.

Influence of buffer species on the thermodynamics of short DNA duplex melting: sodium phosphate versus sodium cacodylate.

Thermodynamic parameters of the melting transitions of 53 short duplex DNAs were experimentally evaluated by differential scanning calorimetry melting curve analysis. Solvents for the DNA solutions contained approximately 1 M Na+ and either 10 mM cacodylate or phosphate buffer. Thermodynamic parameters obtained in the two solvent environments were compared and quantitatively assessed. Thermodynamic stabilities (deltaG(o) (25 degrees C)) of the duplexes studied ranged from quite stable perfect match duplexes (approximately -30 kcal/mol) to relatively unstable mismatch duplexes (approximately -9 kcal/mol) and ranged in length from 18 to 22 basepairs. A significant difference in stability (average free energy difference of approximately 3 kcal/mol) was found for all duplexes melted in phosphate (greater stability) versus cacodylate buffers. Measured effects of buffer species appear to be relatively unaffected by duplex length or sequence content. The popular sets of published nearest-neighbor (n-n) stability parameters for Watson-Crick (w/c) and single-base mismatches were evaluated from melting studies performed in cacodylate buffer (SantaLucia and Hicks, Annu. Rev. Biophys. Biomol. Struct. 2004, 33, 415). Thus, when using these parameters to make predictions of sequence dependent stability of DNA oligomers in buffers other than cacodylate (e.g., phosphate) one should be mindful that in addition to sodium ion concentration, the type of buffer species also provides a minor but significant contribution to duplex stability. Such considerations could potentially influence results of sequence dependent analysis using published n-n parameters and impact results of thermodynamic calculations. Such calculations and analyses are typically employed in the design and interpretation of DNA multiplex hybridization experiments.