Thermodynamic investigation of the association of ethidium, propidium and bis-ethidium to DNA hairpins.

We have used a combination of calorimetric and spectroscopic techniques to investigate the association of the bis-intercalator ethidium homodimer (bis-ethidium) to short DNA hairpins with sequences: d(GCGCT5GCGC) and d(CGCGT5CGCG). The helix-coil transition of each hairpin, investigated by UV and calorimetric melting protocol, takes place in monomolecular two-state transitions with characteristic enthalpies of approximately 37 kcal mol-1 for disrupting the four dG-dC base pairs of the hairpin stems. Deconvolution of the bis-ethidium-hairpin calorimetric titration curves indicate that each hairpin contains two distinct binding sites for the ligand: a high affinity site in the stem (Kb approximately 10(7)) that accommodates one bis-ethidium molecule and a lower affinity site (Kb approximately 10(6)) located probably at the loop that accommodates two bis-ethidium molecules. The overall stoichiometries of three ligands per hairpin are in agreement with those obtained in continuous variation experiments using visible spectroscopy. The interaction of bis-ethidium for each type of sites results in enthalpy driven reactions, with average binding enthalpies, delta Hb, of -13.1 and -12.1 kcal mol-1 for the stem and loop sites, respectively. Comparison to the thermodynamic profiles of ethidium and propidium binding reveals that the bis-ethidium binding to the stem site of each hairpin has a more favorable free energy term of -1.4 kcal mol-1 and more favorable enthalpy of -4.2 kcal mol-1. These suggest that only one phenanthridine ring of bis-ethidium intercalates in the stem, while the second planar ring is exposed to solvent or weakly associated to the surface of DNA.