THERMODYNAMICS OF DNA HAIRPINS - CONTRIBUTION OF LOOP SIZE TO HAIRPIN STABILITY AND ETHIDIUM BINDING

A combination of calorimetric and spectroscopic techniques was used to evaluate the thermodynamic behavior of a set of DNA hairpins with the sequence d(GCGCT(n)GCGC), where n = 3, 5 and 7, and the interaction of each hairpin with ethidium. All three hairpins melt in two-state monomolecular transitions, with t(m)'s ranging from 79.1-degrees-C (T3) to 57.5-degrees-C (T7), and transition enthalpies of approximately 38.5 kcal mol-1. Standard thermodynamic profiles at 20-degrees-C reveal that the lower stability of the T5 and T7 hairpins corresponds to a DELTAG-degrees term of + 0.5 kcal mol-1 per thymine residue, due to the entropic ordering of the thymine loops and uptake of counterions. Deconvolution of the ethidium-hairpin calorimetric titration curves indicate two sets of binding sites that correspond to one ligand in the stem with binding affinity, K(b), Of approximately 1.8 x 10(6) M-1, and two ligands in the loops with K(b) of approximately 4.3 x 10(4) M-1. However, the binding enthalpy, DELTAH(b), ranges from - 8.6 (T3) to - 11.6 kcal mol-1 (T7) for the stem site, and - 6.6 (T3) to - 12.7 kcal mol-1 (T7) for the loop site. Relative to the T3 hairpin, we obtained an overall thermodynamic contribution (per dT residue) of DELTADELTAH(b) = DELTA(TDELTAS(b)) = - 0.7(5) kcal mol-1 for the stem sites and DELTADELTAH(b) = DELTA(TDELTAS(b)) = - 1.5 kcal mol-1 for the loop sites. Therefore, the induced structural perturbations of ethidium binding results in a differential compensation of favorable stacking interactions with the unfavorable ordering of the ligands.