Nuclear Magnetic Resonance Comparison of the Binding Sites of Mithramycin and Chromomycin on the Self-complementary Oligonucleotide d(ACCCGGGT) 2: Evidence that the Saccharide Chains have a role in Sequence Specificity

Abstract

A comprehensive two-dimensional 1H nuclear magnetic resonance spectral analysis of the ternary 4:2:1 mithramycin-Mg 2+-d(A 1C 2C 3C 4G 5G 6G 7T 8) 2 complex and the ternary 2:1:1 chromomycin-Mg 2+-d(A 21C 2C 3C 4C 4G 5G 6G 7T 8) 2 complex is presented. The self-complementary oligonucleotide is found to bind two dimers of mithramycin in two identical off-center binding sites such that the twofold symmetry of the oligonucleotide is retained. In contrast, the same oligonucleotide binds only one dimer of chromomycin in a single but distinct of-center binding site. Two-dimensional nuclear Overhauser spectroscopy experiments show that the aglycone binding site of the drug dimer in each complex extends over almost four base-pairs and is similar in length to other complexes between chromomycin or mithramycin and oligonucleotides. The data demonstrate that the chromomycin dimer binding site is offset by one base-pair step from the dimer binding site in the mithramycin complex. This preferred binding site prevents two dimers of chromomycin binding to d(ACCCGGGT) 2 for steric reasons and lends further support to previous work that showed the 5′-CG base-pair site is less favored by these drugs compared to the 5′GC and 5′-GG, 5′-CC sites. Evidence is presented that suggests mithramycin may occupy either of two distinct binding sites on d(ACCCGGGT) 2 when the drug concentration is not saturating. The nuclear magnetic resonance data demonstrate that the saccharide chains of this family of drugs do have a role in determining the binding site on nucleotides and as a consequence the CDE trisaccharide chain may alter its conformation to fulfil this role. Titration of mithramycin up to a drug-duplex ratio of 7:1 reveals further association of mithramycin with the complex but no new drug-oligonucleotide nuclear Overhauser enhancement contacts.