Direct Synthesis and Identification of Benzo[a]pyrene Diol Epoxide-Deoxyguanosine Binding Sites in Modified Oligodeoxynucleotides

Adducts derived from the reaction of the benzo[a]pyrene metabolite model compound (+)-anti-7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+)-BPDE] with the single-stranded oligodeoxynucleotide 5′-d(TATGCGTAT) were obtained according to direct synthesis techniques described earlier [Cosman, M., Ibanez, V., Geacintov, N. E., and Harvey, R. G. (1990) Carcinogenesis 11,1667-1672]. Four major adducts, involving trans and cis addition (trans/cis adduct ratio ≈ 4.5) of (+)-BPDE to the exocyclic amino groups of guanines G₄ and G₆ (the numbers denote the positions of the guanines counted from the 5′-side) were obtained. These adducts can be separated from one another by reverse-phase highperformance liquid chromatography methods. The site of BPDE binding on either G₄ or G₆ can be determined from the electrophoresis band patterns on 20% polyacrylamide gels of the BPDE-modified oligonucleotides subjected to the G+A and G Maxam-Gilbert strand cleavage reactions [Maxam, A. M., and Gilbert, W. (1980) Methods. Enzymol. 65, 499-560]. The electrophoresis gel band patterns are different for unmodified DNA and the two different BPDE-modified oligonucleotides because (1) the strand cleavage fragments bearing BPDE residues migrate slower than the corresponding fragments derived from the unmodified oligonucleotide and (2) strand cleavage tends to be inhibited on the 5′-sides of BPDE-modified guanines in the G+A, but not the G reaction.