Bi and tri-substituted phenyl rings containing bisbenzimidazoles bind differentially with DNA duplexes: a biophysical and molecular simulation study

Recently synthesis of programmable DNA ligands which can regulate transcription factors have increased the interest of researchers on the functional ability of DNA interacting compounds. A series of DNA interacting compounds are being designed which can differentiate between GC and AT rich DNA. In this study, we have studied the specificity of a few novel bisbenzimidazoles having different bi/tri-substituted phenyl rings, with DNA duplexes using spectroscopic methods. This study entails an integrative approach where we combine biophysical methods and molecular dynamics simulation studies to establish suitable scaffolds to target A/T DNA. We have designed a few analogues of Hoechst 33342 viz.; dimethoxy (DMA), trimethoxy (TMA), dichloro (DCA) and difluoro (DFA) functionalities and performed molecular docking of newly designed analogues with biologically relevant AT and GC rich DNA sequences. The docking studies, along with molecular dynamics (MD) simulations of d(ATATATATATATATAT)2, d(GA4T4C)2, d(GT4A4C)2 and GC rich sequence: d(GCGCGCGCGCGCGCGC)2 complexed with DMA, TMA and DFA, showed that these molecules have higher binding affinity towards AT rich DNA. None of these compounds exhibited an affinity to GC rich DNA rather we observed that these compounds destabilize GC rich DNA. The binding was characterized by strong stabilization of the polynucleotides against thermal strand separation in thermal melting experiments. New insights into the molecules binding to DNA have emerged from these studies. All the DNA binding ligands stabilized d(GA4T4C)2 and d(GT4A4C)2 more out of the five oligomers used for the study, suggesting that these ligands bind 'A4T4' and 'T4A4' strongly as compared to 'ATAT' base pairs.