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.