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.
Rentzeperis
D
D
Department of Chemistry, New York University, New York 10003, USA.
Medero
M
M
Marky
L A
LA
D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry*; D004996:Ethidium / Q000031:analogs & derivatives* / Q000737:chemistry*; D007364:Intercalating Agents / Q000737:chemistry*; D008024:Ligands; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011419:Propidium / Q000737:chemistry*; D013329:Structure-Activity Relationship; D013816:Thermodynamics
D007364:Intercalating Agents; D008024:Ligands; D009838:Oligodeoxyribonucleotides; D011419:Propidium; C018533:ethidium homodimer; D004247:DNA; D004996:Ethidium