This journal is c The Royal Society of Chemistry 2013 Mol. BioSyst., 2013, 9, 2541--2553 2541 Cite this: Mol. BioSyst., 2013, 9, 2541 Bi and tri-substituted phenyl rings containing bisbenzimidazoles bind differentially with DNA duplexes: a biophysical and molecular simulation study† Manish Singh,z a Souvik Sur,z b Gaurav Kumar Rastogi, b B. Jayaram c and Vibha Tandon* b 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(GA 4 T 4 C) 2 , d(GT 4 A 4 C) 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(GA 4 T 4 C) 2 and d(GT 4 A 4 C) 2 more out of the five oligomers used for the study, suggesting that these ligands bind ‘A 4 T 4 ’ and ‘T 4 A 4 ’strongly as compared to ‘ATAT’ base pairs. 1. Introduction The regular helical grooves of DNA offer no clues about how small molecules recognize specific sequences. When viewed at the individual base-pair level, however, it is apparent that the edges of the base pairs offer arrays of hydrogen-bond donors and acceptors that have details determined by the DNA sequence. From a theoretical point of view, the problems of developing a rigorous view of DNA recognition arise first from the challenge of modeling polyanionic DNA at an atomic level in an explicit aqueous solution including the mobile counter ions and second from the difficulties in computing binding free energies and the corresponding enthalpic and entropic compo- nents for large bimolecular systems. The convergence of which can require very long computation times. The binding modes of several biologically important bisbenzimidazole derivatives to DNA have been studied and summarized by several workers. 1–5 Rich, Dervan, Wemmer, Lown and other workers have described netropsin, distamycin A, and Hoechst-33258, etc. as DNA minor groove binding drugs. 6–10 Recently Wilson et al. have shown that polyamides binds with high sequence specificity 11 and clusters of water molecules around DNA play a key structural role in a Dr. B. R. Ambedkar Center for Biomedical Research, Delhi, India b Department of Chemistry, University of Delhi, Delhi-110007, India. E-mail: vtandon@acbr.du.ac.in c Supercomputing Facility for Bioinformatics, Indian Institute of Technology, New Delhi-110016, India † Electronic supplementary information (ESI) available: Fig. S1–S4, UV absorption spectra of bis-benzimidazole (DFA, DCA, DMA, TMA & Hoechst 33342) alone and in the presence of synthetic oligomers; Fig. S5–S8, CD scans of oligomers with increasing concentrations of ligands; Fig. S9–S12, fluorescence-detected binding of oligomers with ligands; Fig. S13, MD simulation protocols; Fig. S14–S17, ligand-docked structure of Hoechst-33342, DMA, TMA and DFA with (i) d(ATATATATATATATAT) 2 , (ii) d(GAAAATTTTC) 2 and (iii) d(GTTTTAAAAC) 2 ; Fig. S18, RMSD plots of free-DNA duplexes up to 25 ns simulation; Fig. S19 and S20, correlation diagrams of experimental and calculated data. See DOI: 10.1039/c3mb70169g ‡ First and second authors have equal contribution to work. Received 28th April 2013, Accepted 10th July 2013 DOI: 10.1039/c3mb70169g www.rsc.org/molecularbiosystems Molecular BioSystems PAPER Published on 10 July 2013. Downloaded by University of Delhi on 02/09/2013 11:02:57. View Article Online View Journal | View Issue