Competition of Some Minor Groove Binders for a Single DNA Binding Site†
Jurij Lah*, Nejc Carl, Igor Drobnak, Boštjan Šumiga and Gorazd Vesnaver*
University of Ljubljana, Faculty of Chemistry and Chemical Technology, Askerceva 5, 1000 Ljubljana, Slovenia.
We employed circular dichroism (CD) and isothermal titration calorimetry (ITC) to characterize binding of netropsin (NET) and distamycin A (DST) to the hairpin (D) formed from the 5′-CGAATTGACGTCTCCGTCAATTCG-3′ oligonucleotide. From fitting the appropriate model to the CD titration curves describing NET and DST binding to the D and CD and ITC titration curves describing the displacement of NET bound to D by the added DST and vice versa we were able to determine the corresponding binding and displacement constants at 25 °C. The displacement constant (K12) determined for 2DST + NET-D ↔ DST2-D + NET process is in good agreement with the corresponding value calculated from the individual binding events (NET + D ↔NET-D, k1 = 1.6 106 M-1; DST + D ↔DST-D, k1 = 1.0 107 M-1 and DST + DST-D ↔DST2-D, k2 = 0.9 104 M-1). ITC results reveal that the free energy of displacement ∆G12° = -29 kJ mol-1 in combination with the enthalpy of displacement ∆H12° = -74 kJ mol-1 results in the corresponding entropy contribution T∆S12° = -45 kJ mol-1. Evidently, the 2DST + NET-D ↔DST2-D + NET displacement is a strongly enthalpy driven process. What we find important with the displacement studies is the observation that NET entirely displaces DST from one half of its 1:1 complexes with the hairpin in spite of the fact that the 1:1 binding constant of NET is about one order of magnitude lower than the corresponding 1:1 constant of DST. We propose that the driving force of this displacement is strong binding of DST molecules that are displaced as a result of the overall NET-DST-D equilibrium to the available DST-hairpin 1:1 complexes.
Key words: DNA, drug binding, netropsin, distamycin, isothermal titration calorimetry, thermodynamics, circular dichroism