Jurij Lah, Marjan Bežan and Gorazd Vesnaver
Faculty of Chemistry and Chemical Technology, University of Ljubljana, Aškerčeva 5, 1000 Ljubljana, Slovenia
 

Abstract

Thermodynamics of berenil binding to poly[d(AT)]×poly[d(AT)] and poly[d(A)]×poly[d(T)] duplexes in neutral buffer solutions was studied using a combination of calorimetric and spectroscopic techniques. By titration calorimetry we found that at 25 oC the enthalpies of berenil binding to poly[d(AT)]·poly[d(AT)] and poly[d(A)]·poly[d(T)] are –20.4 and 10.3 kJ/mol of bound drug, respectively. Our CD results show that berenil binding to both duplexes is at low berenil/DNA ratios slightly positively cooperative whereas at higher binding densities it is characterized by a constant affinity and a binding site size, n, of 4 base pairs/berenil molecule. Berenil binding to both polynucleotides was also followed by performing UV titrations of berenil solutions with DNA solutions at 25 oC and by measuring the UV melting curves at different berenil/DNA molar ratios. From Scatchard plots the best fit of experimental data with the predictions of the neighbor exclusion model was obtained with K = 1.3×107 and n = 3.5 for poly[d(AT)]×poly[d(AT)] and K = 1.5×107 and n = 3.8 for poly[d(A)]×poly[d(T)]. UV melting experiments showed for both polynucleotides that above their saturation with bound berenil they exhibit monophasic melting accompanied by a large increase in Tm, whereas at low drug/base pair ratios their melting is biphasic. Berenil binding constants determined at 25 oC using the Tm data and the neighbor exclusion model are K = 2.3×107 for poly[d(AT)]×poly[d(AT)] and K = 7.4×106 for poly[d(A)]×poly[d(T)]. From these K values the standard free energies of binding were determined and combined with the measured enthalpies of binding to obtain the corresponding entropies of binding. The resulting thermodynamic binding profiles show that berenil binding to poly[d(AT)]×poly[d(AT)] is governed by about equal enthalpic and entropic forces whereas its binding to poly[d(A)]×poly[d(T)] is overwhelmingly entropy driven.