This project will explore the molecular, thermodynamic, and biochemical properties associated with the interactions of the potent antitumor antibiotics m-AMSA and actinomycin D with nucleic acids. Specifically, these research efforts will be designed to explore the structural and functional properties of native and synthetic DNAs subsequent to drug binding utilizing both equilibrium and covalent modes of ligand interactions and correlate the relationships of drug structure with the biophysical parameters associated with complex formation. Experiments will be designed to extend the binding isotherms of m-AMSA to very low concentrations of bound drug, providing a more accurate description of the physical chemical nature of its chemotherapeutic effect. Thermodynamic, kinetic, and stoichiometric measurements will be obtained by spectroscopic, equilibrium dialysis, and phase partition techniques. The phase partition method will be particularly useful for obtaining accurate binding isotherms at very low concentrations of bound drug. The technique of photoaffinity labeling will be extended to the actinomycin D system. The capacity for covalent attachment will greatly aid in the study of target site specificities, cooperativity, and/or drug clustering phenomena. Through chemical modification of the parent actinomycin D with a photoreactive substituent (azido moiety), functional analogs of the parent molecule will be generated having equilibrium binding properties identical to those of the parent compound (in the absence of light) yet with the capacity for covalent attachment upon photolytic activation. Properties associated with the covalent nature of the actinomycin D - nucleic acid adduct will be examined in detail by a variety of biochemical and biophysical techniques. Restriction endonuclease activity will be used to monitor binding site specificities and the nucleic acid structural perturbations resulting from adduct formation. Similarly, the geometry and molecular orientation of the actinomycin D - DNA adduct will be determined at the oligonucleotide level by high field nuclear magnetic resonance spectroscopy. These studies on both m-AMSA and actinomycin D will increase the understanding of drug binding properties, the nature of the drug - DNA complex and/or adduct, and provide a powerful photoaffinity probe for further definition of the structural and functional properties of nucleic acids.
