Spectroscopic studies of the trans adducts derived from (+)- and (-)-anti-benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide and the oligonucleotide 5'-d(CCTATAGATATCC).

Abstract

The oligonucleotide 5'-d(CCTATAGATATCC) has been reacted with the (+)- or (-)-enantiomers of trans-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+)- and (-)-anti-BPDE respectively]. Consistent with previous studies employing single-stranded oligonucleotides, adduct formation of both anti-BPDE enantiomers preferentially involved trans-addition of the C10 position of the diol-epoxide to the exocyclic nitrogen of deoxyguanosine [in the following abbreviated as (+)-BPDEt-N2-G and (-)-BPDEt-N2-G adducts respectively]. The unmodified or (+)-BPDEt-N2-G-modified oligonucleotide was allowed to form duplexes with the complementary sequence 5'-d(GGATATCTATAGG) or sequences in which C has been replaced with T, G or A and analysed with regard to thermal stability. The presence of a (+)-BPDEt-N2-G adduct in oligonucleotide duplexes substantially decreased the value of the melting point relative to the corresponding unmodified duplex. In mismatched complexes containing the (+)-BPDEt-N2-G adduct, a further decrease in thermal stability was observed. The presence of a (+)-BPDEt-N2-G adduct did not seem to change the extent of hyperchromicity (approximately 20%) upon melting. 5'-d(GGATATCTATAGG) or strands in which C was replaced with T, G or A were gradually added to (+)- or (-)-BPDEt-N2-G-modified oligonucleotides and the fluorescence emission intensity was determined. In all cases with (+)-BPDEt-N2-G, except when C was replaced with A in the complement, the fluorescence intensity steadily decreased and became constant at equal strand concentrations. When a strand containing A in place of C was gradually added to the (+)-BPDEt-N2-G oligonucleotide, a marked increase in the fluorescence intensity was observed (> 3-fold). In contrast, addition of strands containing A, T or G to the (-)-BPDEt-N2-G-modified oligonucleotide increased the fluorescence intensity from 1.5- to > 5-fold. Addition of the fully complementary sequence to the (-)-BPDEt-N2-G-containing oligonucleotide resulted in reduced fluorescence, however less pronounced than with the (+)-BPDEt-N2-G-modified analogue. Significant changes in spectral properties of the adducts were observed in the duplexes. The absorption and fluorescence excitation maxima of the single-stranded (+)-BPDEt-N2-G-modified oligonucleotide were at 353 nm. Insertion of C or A opposite the adduct caused a significant shift of these maxima to shorter wavelengths (347-348 nm). Addition of acrylamide, a fluorescence quencher, reduced the fluorescence intensity in all cases, but to variable extents.(ABSTRACT TRUNCATED AT 400 WORDS)