This application focuses on the structure and dynamics at and adjacent to the binding site of antitumor drug-DNA oligomer complexes in solution. solution structures of the complexes will be defined by a combination of NMR and molecular dynamics calculations including iterative relaxation refinement while individual base pair opening parameters will be deduced from nucleic acid imino proton hydrogen exchange measurements as a function of added catalyst concentration. The first family of complexes include the intercalators nogalamycin and actinomycin, as well as covalent anthracycline intercalating agents. The emphasis here is to study truncated analogs lacking specific sugars attached to the intercalating aglycones and estimate the contribution of individual components to sequence specificity and complex stability. We shall define the nature of the conformational transitions in both the drug and the DNA oligomer that relieve potential steric clashes of the peptide side chains when two intercalators bind at adjacent sites on the duplex. Antitumor agents covalently bound to the DNA by a sugar linked tether offer the opportunity to precisely position the intercalating component within duplexes, triplexes and duplex-triplex junctions. The second family of complexes range from the bicyclic peptide bisintercalator UK- 63052 to the cyclic peptide bisintercalator quinaldopeptin. We shall define the hierarchy of sequence and position dependent bisintercalation sites on the DNA and identify the structural transitions on the cyclic peptides (cis-trans peptide bond isomerization) and the nucleic acid (Watson-Crick and Hoogsteen pairing alignments) which accompany complex formation. The nature of the pairing alignments and the base pair opening kinetics will be measured at A-T rich segments flanking the bisintercalation site and the modulation of this affect will be monitored as a function of distance from the binding site. the third family of complexes focus on minor groove binders which include covalent duocarmycin adducts, mitomycin cross-links and Mg(II)-coordinated mithramycin dimer complexes. We will generate covalent minor groove antitumor drug adducts in DNA triplexes to assess the potential groove cross-talk between the drug positioned in the minor groove and the third strand positioned in the major groove of the DNA triplex. A direct comparison will address structural features and base pair opening parameters for antitumor drugs that form intrastrand and interstrand cross-links. For sugar containing antibiotics we plan to elucidate additional features of saccharide-minor groove recognition and evaluate how differences in saccharide stereochemistry and substitutions modulate sequence specificity and stability. In the longer run, these studies will be extended to antitumor drug recognition by other higher order DNA structures.
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