Specific binding of hoechst 33258 to the d(CGCAAATTTGCG) 2 duplex: calorimetric and spectroscopic studies

Abstract

Fluorescence spectroscopy and high-sensitivity isothermal titration calorimetry (ITC) techniques have been used to examine the binding characteristics of Hoechst 33258 with the extended AT-tract DNA duplex d(CGCAAATTTGCG) 2 in aqueous solution. The method of continuous variation reveals a 1:1 binding stoichiometry. Fluorescence equilibrium studies carried out at three different, but fixed, ligand concentrations show that the binding isotherm shifts towards higher [DNA] as the concentration of ligand is increased. The data show tight binding with K b = 3.2(±0.6) × 10 8M(duplex) −1 at 25°C in solutions containing 200 mM Na +. Based on UV studies of duplex melting, which show that strand separation starts at ∼35°C and has a T m at 54°C in 300 mM NaCl, binding enthalpies were determined by ITC in the 10 to 30°C range. Binding is endothermic at all temperatures examined, with Δ H values ranging from +10.24(±0.18) to +4.2(±0.10) kcal mol(duplex) −1 at 9.4°C and 30.1°C, indicating that the interaction is entropically driven. The temperature dependence of Δ H shows a binding-induced change in heat capacity (Δ C p) of −330(±50) cal mol −1 K −1. This value is similar to that predicted from a consideration of the effects of hydrophobic and hydrophilic solvent-accessible surface burial on complexation. This result, almost entirely dictated by a removal from exposure of the non-polar reactant surfaces, represents the first demonstration of such behavior in a DNA-drug system. The salt dependence of the binding constant was examined using reverse-salt fluorescence titrations, with a value of 0.99 determined for the δln K/δln[Na +] parameter. These data provide a detailed thermodynamic profile for the interaction that enables a dissection of Δ G obs into the component free energy terms. Analysis of data obtained at 25°C reveals that Δ G obs is dominated by the free energy for hydrophobic transfer of ligand from solution to the DNA binding site. Molecular interactions, including H-bonding and van der Waals contacts, are found to play only a minor role in stabilizing the resulting complex, a somewhat surprising finding given the emphasis placed on such interactions from structural studies.