Conformational studies of the (+)- trans, (−)- trans, (+)- cis, and (−)- cis adducts of anti-benzo[ a]pyrene diolepoxide to N 2-dG in duplex oligonucleotides using polyacrylamide gel electrophoresis and low-temperature fluorescence spectroscopy

Abstract

Using polyacrylamide gel electrophoresis (PAGE) and low-temperature, laser-induced fluorescence line narrowing (FLN) and non-line narrowing (NLN) spectroscopic methods, the conformational characteristics of stereochemically defined and site-specific adducts derived from the binding of 7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo[ a]pyrene ( anti-BPDE, a metabolite of the environmental carcinogen benzo[ a]pyrene), to DNA were studied. The focus of these studies was on the four stereochemically distinct anti-BPDE modified duplexes 5′-d(CCATCGCTACC) · (GGTAGCGATGG), where G denotes the lesion site derived from trans or cis addition of the exocyclic amino group of guanine to the C10 position of either (+) or (−)- anti-BPDE. PAGE experiments under non-denaturing conditions showed that the (+)- trans adduct causes a significantly greater retardation in the electrophoretic mobility than the other three adducts, probably the result of important adduct-induced distortions of the duplex structure. Low-temperature fluorescence studies in frozen aqueous buffer matrices showed that the (+)- trans adduct adopts primarily an external conformation with only minor interactions with the helix, but a smaller fraction (∼ 25%) appears to exists in a partially base-stacked conformation. The (−)- trans adduct exists almost exclusively (∼ 97%) in an external conformation. Both cis adducts were found to be intercalated; strong electron-phonon coupling observed in their FLN spectra provided additional evidence for significant π-π stacking interactions between the pyrenyl residues and the bases. FLN spectroscopy is shown to be suitable for distinguishing between trans and cis adducts, but lesions with either (+)- or (−)- trans, or (+)- or (−)- cis stereochemical characteristics showed very similar vibrational patterns. Addition of glycerol (50%, v/v) to the matrix caused a partial disruption of the chromophore-base stacking interactions for most adducts, but the (−)- cis isomer showed a strong blue-shift and unusual vibrational frequencies. Low-temperature fluorescence spectroscopy techniques are most suitable for distinguishing between different conformational benzo[ a]pyrene diol epoxide-DNA adduct types; because of the sensitivity of these methods, they may provide important information necessary for an understanding of the biological effects of the stereochemically distinct BPDE-guanine lesions.