Fluorescence Probing of Aminofluorene-Induced Conformational Heterogeneity in DNA Duplexes

Abstract

Fluorescence spectroscopy was used to study carcinogen-induced conformational heterogeneity in DNA duplexes. The fluorophore 2-aminopurine (AP) was incorporated adjacent (5′) to the lesion (G*) in eight different DNA duplexes [d(5′-CTTCTPG*NCCTC-3′):d(5′-GAGGNXTAGAAG-3′), G* = FAF adduct, P = AP, N = G, A, C, T, and X = C, A] modified by FAF [N-(2′-deoxyguanosin-8-yl)-7-fluoro-2-aminofluorene], a fluorine-tagged model DNA adduct derived from the potent carcinogen 2-aminofluorene. Steady-state measurements showed that fluorescence intensity and Stern-Volmer constants (K sv ) derived from acrylamide quenching experiments decreased for all carcinogen-modified duplexes relative to the controls, which suggests greater AP stacking in the duplex upon adduct formation. Conformation-specific stacking of AP with the neighboring adduct was evidenced by a sequence-dependent variation in fluorescence intensity, position of emission maximum, degree of emission quenching by acrylamide, and temperature-dependent spectral changes. The magnitude of stacking was in the order of FAF residue in base-displaced stacked (S) > minor groove wedged (W) > major groove B type (B). This work represents a novel utility of AP in probing adduct-induced conformational heterogeneities in DNA duplexes.

C8-Heteroaryl-2′-deoxyguanosine Adducts as Conformational Fluorescent Probes in the NarI Recognition Sequence

“…Quenching constants reflect the extent of adduct accessibility toward the quencher. 62 Examination of the K sv values determined for the quenching of modified duplexes by KI reveals that the values are much smaller than those determined for the corresponding adducts, reflecting a lower accessibility of the fluorophores to the quencher when located in the helical environment. The K sv values also show that when base-paired to C, the fluorophore is more accessible to the aqueous environment than when mismatched to G, as larger K sv values were derived for the modified oligonucleotides hybridized to NarI′(C) than when hybridized to NarI′(G).…”

“…Slopes of the plots derived from KI quenching experiments provide the Stern–Volmer quenching constant for different adducts, K sv , which are listed in Table . Quenching constants reflect the extent of adduct accessibility toward the quencher . Examination of the K sv values determined for the quenching of modified duplexes by KI reveals that the values are much smaller than those determined for the corresponding adducts, reflecting a lower accessibility of the fluorophores to the quencher when located in the helical environment.…”

“…59 Collisional quenching, using quenchers such as acrylamide and deoxyuridine (neutral), iodide ion (negatively charged), and cesium ion (positively charged) has been used to provide insight into the conformation of proteins, 59,60 benzopyrene diol epoxide (BPDE)−DNA adducts 61 and 2AP. 62 Collisional quenching experiments were first conducted with the free C 8 -heteroaryl−dG adducts, using KI as quencher. Plots of 6.…”

“…Collisional quenching has proved useful for study of aqueous accessibility of fluorescent groups, with changes in sensitivity, exposure and accessibility following ligand binding indicative of a change in conformation of the fluorophore . Collisional quenching, using quenchers such as acrylamide and deoxyuridine (neutral), iodide ion (negatively charged), and cesium ion (positively charged) has been used to provide insight into the conformation of proteins, , benzopyrene diol epoxide (BPDE)–DNA adducts and 2AP …”

α,β-Methylene-2′-deoxynucleoside 5′-Triphosphates as Noncleavable Substrates for DNA Polymerases: Isolation, Characterization, and Stability Studies of Novel 2′-Deoxycyclonucleosides, 3,5′-Cyclo-dG, and 2,5′-Cyclo-dT

“…As part of our ongoing research program on sequence effects on arylamine-induced conformational heterogeneity, we sought the isolation of ternary polymerase intermediates involving a lesion-containing template-primer and dNTP. Strategies to trap such catalytic complex entities, include utilizing a 3′-dideoxy-terminated primer (i .…”