Site-specifically modified oligonucleotides were obtained in milligram quantities by reacting racemic 3t,4r-dihydroxy-1,2t-epoxy-1,2,3,4-tetrahydrobenzo[c]phenanthrene (B[c]PhDE-2, or anti-B[c]PhDE) with the single deoxyadenosine (dA) residue in the oligodeoxynucleotide d(CTCTCACTTCC). Enzyme digestion of the covalently modified oligonucleotides with the exonuclease spleen phosphodiesterase yielded covalently linked B[c]PhDE-N6-deoxyadenosyl monophosphate (dAMP) adducts. Comparisons of the reverse phase HPLC retention times and CD spectra of these B[c]PhDE-3'-dAMP mononucleotide adducts, with those of standards derived from the reaction of the enantiomers (+)- and (—)-anti-B[c]PhDE with 3'-dAMP, show that two major oligonucleotide adducts (I and II) were obtained upon reacting racemic anti-B[c]PhDE with d(CTCTCACTTCC). In oligonucleotide adduct I, the lesion is a (+)-trans-anti-B[c]PhDE-N6-dA residue, and in oligonucleotide adduct II it is a (-)-trans-anti-B[c]PhDE-N6-dA residue. These assignments were further confirmed using a standard 32P postlabeling assay of B[c]PhDE-3'-dAMP mononucleotide adducts obtained from the digestion of oligonucleotides I and II by spleen phosphodiesterase. The melting points (Tm) of duplexes of modified oligonucleotides I and II and their natural complementary strands are not affected significantly by the presence of the covalently bound benzo[c]phenanthrenyl residues. Opposite stereoselective resistance to enzyme digestion by the exonucleases snake venom phosphodiesterase and spleen phosphodiesterase is exhibited by the stereoisomeric (+)-trans- and (-)-trans-anti-B[c]PhDE-modified oligonucleotide adducts I and II; these results are consistent with the intercalative insertion of the benzo[c]phenanthrenyl residues on the 5'-side of the modified dA residue in adduct I, and its insertion on the 3'-side of the dA residue in adduct II, as observed in the duplexes by high resolution NMR techniques [Cosman et al. (1993) Biochemistry 32, 12488–12497, and Cosman et al., Biochemistry, in press].
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