Calorimetric and spectroscopic studies of Hoechst 33258: self-association and binding to non-cognate DNA

Sequence and structure-specific molecular recognition of DNA by small molecules is an important goal in biophysical chemistry and drug discovery. Many candidate ligands possess flat aromatic surfaces and other molecular features that allow them to self-associate. In addition, non-specific binding to the target is a complicating feature of these interactions. Therefore, multiple equilibria are present and need to be accounted for in data analysis in order to obtain meaningful thermodynamic parameters. In order to address these issues we have systematically examined the bis-benzimidazole dye Hoechst 33258 (H33258) in terms of self-aggregation and binding to DNA oligonucleotides lacking any cognate minor groove A.T sites. This model system has been interrogated using isothermal titration calorimetry (ITC), circular dichroism (CD), fluorescence spectroscopy and pulsed gradient spin echo NMR. Three distinct binding events and ligand self-aggregation have been identified and, where possible, quantified. H33258 self-aggregation involves a step-wise aggregation mechanism, driven by stacking interactions. The DNA binding process includes two specific binding modes and non-specific DNA-templated H33258 stacking. We have written novel ITC data-fitting software (IC-ITC; freely available to the biophysics community), which simultaneously fits ligand aggregation and ligand-DNA binding. Here, this numerical analysis, which uses simulated annealing of complex calorimetric data representing multiple coupled equilibria, is described.