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

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) x 10(8) M(duplex)(-1) at 25 degrees C in solutions3.2(+/-0.6) x 10(8) M(duplex)(-1) at 25 degrees C in solutions containing 200 mM Na+. Based on UV studies of duplex melting, which show that strand separation starts at similar to 35 degrees C and has a T-m at 54 degrees C in 300 mM NaCl, binding enthalpies were determined by ITC in the 10 to 30 degrees C range. Binding is endothermic at all temperatures examined, with Delta H values ranging from +10.24(+/-0.18) to +4.2(+/-0.10) kcal mol(duplex)(-1) at 9.4 degrees C and 30.1 degrees C, indicating that the interaction is entropically driven. The temperature dependence of Delta H shows a binding-induced change in heat capacity (Delta 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 delta lnK/delta ln[Na+] parameter. These data provide a detailed thermodynamic profile for the interaction that enables a dissection of Delta G(obs) into the component free energy terms. Analysis of data obtained at 25 degrees C reveals that Delta 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. (C) 1997 Academic Press Limited.