Multiple Fluorescence Lifetimes for Oligonucleotides Containing Single, Site-Specific Modifications at Guanine and Adenine Corresponding to Trans Addition of Exocyclic Amino Groups to (+)-(7R,8S,9S,10R)- and (-)-(7S,8R,9R,10S)-7,8-Dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene

Abstract

Fluorescence decay profiles of four oligonucleotide duplexes, [table: see text] ((+)- and (-)-trans-1) and [table: see text] ((+)- and (-)-trans-2), in which an exocyclic amino group of deoxyadenosine (A*) or deoxyguanosine (G*) has been alkylated by trans opening at C-10 of the epoxide group of either the (+)-(R,S,S,R)- or (-)-(S,R,R,S)-enantiomer of (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10- tetrahydrobenzo[a]pyrene (BPDE in which the benzylic 7-hydroxy group and the epoxide oxygen are trans), exhibit more than one fluorescence lifetime. Decay profiles of the oligomers, measured at 15 degrees C with excitation and emission wavelengths of 335 and 400 nm, respectively, have been analyzed using a triple-exponential decay law. Results for (+)- and (-)-trans-1 and -2 have been compared with results for the modified, single-stranded oligonucleotides ((+)- and (-)-trans-SS-1, and (+)- and (-)-trans-SS-2) and for the cis and trans opened products formed on alkylation at the 6-amino group of 2'-deoxyadenosine 5'-phosphate by (+)-(R,S,S,R)-BPDE ((+)-trans- and (+)-cis-A). The profiles of (+)-trans- and (+)-cis-A are well represented by single-exponential decay laws with lifetimes of 86 and 110 +/- 3 ns, respectively. For the single- and double-stranded oligomer adducts, which exhibit at least three fluorescence lifetimes, two of the lifetimes are short (0.5-14 +/- 1 ns) and one is long (35-59 +/- 3 ns). The fluorescence lifetimes and the amplitudes of the long-lived components in the decay profiles of the double-stranded oligomer adducts are generally smaller than those for the corresponding single-stranded adducts. The data provide evidence that the double-stranded oligomer adducts exist as multiple conformations. Previously reported NMR results suggest that the short lifetime fluorescence components are due to major adduct conformations in which the pyrenyl group is intercalated ((+)- and (-)-trans-1) or lies in the minor groove ((+)- and (-)-trans-2). The observation of long lifetime fluorescence species for the double-stranded oligomers is consistent with the presence of minor conformations (approximately 1-5%) in which the double-stranded oligomer either is locally denatured or is a mixture of locally denatured double-stranded conformations and equilibrium concentrations of single-stranded oligomers.

Synthesis and Characterization of Adducts Derived from the syn-Diastereomer of Benzo[a]pyrene 7,8-Dihydrodiol 9,10-Epoxide and the 5‘-d(CCTATAGATATCC) Oligonucleotide

“…The differences observed for some adducts between absorption and excitation maxima are outside the error limits and indicate a certain heterogeneity in the adduct distribution (cf. refs and ).…”

“…Previous studies have shown that anti-BPDE derived adducts present in single stranded DNA or oligonucleotides exhibit a maximal absorption at about 350-352 nm, a shift of 7-9 nm relative to the absorption at 343 nm of pyrenyl chromophores free in solution (22)(23)(24). The difference in absorption maxima has been attributed to base stacking interactions with the BPDE chromophore (39,43,44). In fully complementary duplexes, type II complexes (trans adducts of anti-BPDE) show absorption maxima at 346-348 nm, whereas type I complexes (cis adducts of anti-BPDE) typically exhibit maxima at 350-352 nm (21,29).…”

Molecular Modeling of Four Stereoisomers of the Major B[a]PDE Adduct (at N2-dG) in Five Cases Where the Structure Is Known from NMR Studies: Molecular Modeling Is Consistent with NMR Results

“…[+ta]-B[ a ]P-N 2 -dG also adopted the BPmi5 conformation in a 5‘-C G C-3‘ sequence context in fully duplex DNA (); however, when the base complementary to the adducted dG was removed, the conformation became base displaced (discussed in ref 35 ). Fluorescence studies also suggest that [+ta]-B[ a ]P-N 2 -dG can adopt multiple conformations in duplex DNA, although the conformations are not determinable by this technique ( − ). Furthermore, four stereoisomers of the B[ a ]P-N 2 -dG adduct were studied and each was in a different conformation as determined by NMR, and in several cases minor species were also observed ( 34 , 39−41 ; reviewed in 35 ; see below).…”

Probing murine methyltransfease Dnmt3a interactions with benzo[a]pyrene‐modified DNA by fluorescence methods

“…In the case of the (+)- or (-)- trans -B[ a ]PDE- N 2 -dG-containing duplexes, the low fluorescence intensity is attributed to an electron-transfer mechanism [39, 41, 42] and to the high accessibility of the B[ a ]P residue to the polar microenvironment (water molecules) [22, 36, 37]. The quenching of the fluorescence of intercalative B[ a ]P residues in A C G c+ C/T GM G, G C G c+ C/C GM G, G c- C G C/C GM G and A t+ C G C/T GM G duplexes is mostly due to the quenching effect of the neighbouring bases [35, 40, 44, 45]. Also, the fluorescence observed in the case of the A t+ C G C/T GM G sequence is quenched because the aromatic B[ a ]PDE residue is accessible to the polar microenvironment [22, 36, 37].…”