The objectives of this research project are to study the interaction of nucleic acids with antitumor bifunctional alkylating agents, and to obtain a clearer understanding of the kinetics and mechanisms involved in the crosslinking of nucleic acids at the molecular level. To investigate the kinetics associated with interstrand DNA crosslink formation and the influence of nucleic acid structure on crosslinking processes, a comparative analysis of the dynamics of interstrand crosslinking by the nitrogen mustards and nitrosoureas to native DNAs and polynucleotides of defined sequence will be undertaken using well established spectrophotometric and bioanalytical procedures. Preliminary studies on the interstrand crosslinking of native DNAs by the nitrogen mustard prototype mechlorethamine, HN2, using a spectrofluorimetric crosslink assay method have provided direct measurements of the specific rate constants and activation energy associated with interstrand DNA crosslinking by HN2. Additional studies with synthetic deoxyribopolynucleotide copolymers of defined sequence using an alkaline gel electrophoresis assay in combination with the fluorimetric method have provided direct evidence for base-specific interactions at the HN2 crosslink site. This research proposal describes further work on studies of the interstrand DNA crosslinking processes associated with the nitrogen mustard and nitrosourea class of antitumor drugs. Further characterization of the kinetics and the influence of nucleic acid structure on crosslinking may lead to a better understanding of the mechanisms of action of a wide variety of antitumor DNA alkylating agents, and may ultimately provide a rationale for the interpretation of their diverse biochemical and pharmacological properties at a molecular level.