SPECIFIC BINDING OF HOECHST-33258 TO THE D(CGCAAATTTGCG)(2) DUPLEX - CALORIMETRIC AND SPECTROSCOPIC STUDIES

Fluorescence spectroscopy and high-sensitivity isothermal titration ca lorimetry (ITC) techniques have been used to examine the binding chara cteristics of Hoechst 33258 with the extended AT-tract DNA duplex d(CG CAAATTTGCG)(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 concentr ation 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 m M Na+. Based on UV studies of duplex melting, which show that strand s eparation starts at similar to 35 degrees C and has a T-m at 54 degree s C in 300 mM NaCl, binding enthalpies were determined by ITC in the 1 0 to 30 degrees C range. Binding is endothermic at all temperatures ex amined, 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, indicati ng that the interaction is entropically driven. The temperature depend ence 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 pr edicted from a consideration of the effects of hydrophobic and hydroph ilic solvent-accessible surface burial on complexation. This result, a lmost entirely dictated by a removal from exposure of the non-polar re actant surfaces, represents the first demonstration of such behavior i n a DNA-drug system. The salt dependence of the binding constant was e xamined using reverse-salt fluorescence titrations, with a value of 0. 99 determined for the delta lnK/delta ln[Na+] parameter. These data pr ovide a detailed thermodynamic profile for the interaction that enable s a dissection of Delta G(obs) into the component free energy terms. A nalysis of data obtained at 25 degrees C reveals that Delta G(obs) is dominated by the free energy for hydrophobic transfer of ligand from s olution to the DNA binding site. Molecular interactions, including H-b onding and van der Waals contacts, are found to play only a minor role in stabilizing the resulting complex, a somewhat surprising finding g iven the emphasis placed on such interactions from structural studies. (C) 1997 Academic Press Limited.