INTERACTION OF MINOR-GROOVE-BINDING DIAMIDINE LIGANDS WITH AN ASYMMETRIC DNA DUPLEX NMR AND MOLECULAR MODELING STUDIES

The aromatic diamidines berenil and propamidine bind reversibly to A+T -rich sites in the minor groove of B-form DNA duplexes. Based on exten sive solution and crystallographic information we have designed a non- self-complementary double-stranded DNA sequence, d(GCAATGAGCG) d(CGCTC ATTGC), that should contain a near-ideal binding site for berenil and a poorer site for the larger propamidine molecule, viz, d(AAT) d(ATT). H-1-NMR studies show that both ligands bind with 1:1 stoichiometry to the embedded 5'-AAT site, and induce numerous shifts of NMR resonance s of DNA protons located in the minor groove. In addition, interaction s with each strand can be distinguished by NOE spectroscopy due to the inherent asymmetry of the DNA. Detailed modelling based on experiment al data show that no significant distortion of the B-DNA duplex is ind uced by either ligand. Sufficient NOE data were obtained to determine the position of the bound ligands in each complex. These conclusions a re in agreement with predictions from molecular modelling calculations that provide a microscopic energy profile for interaction with the mi nor groove tract. Such calculations reveal an unexpected heterogeneous 5'-ATGA binding site that includes a spanned guanosine. This secondar y binding site accounts for the extensive chemical shift perturbation induced by these ligands. The structures of the free DNA and the rever sible complexes formed with each ligand molecule have been refined usi ng an NOE-restrained isothermal annealing procedure. These structures confirm that the introduced ligands effect minimal perturbation of the helix, with binding to the 5'-AAT base sequence largely determined by specific non-bonded interactions.