Chemical mutagens with an aromatic ring system may be enzymatically transformed to afford aryl radical species that preferentially react at the C8-site of 2′-deoxyguanosine (dG). The resulting carbon-linked C8-aryl-dG adduct possesses altered biophysical and genetic coding properties compared to the precursor nucleoside. Described herein are structural and in vitro mutagenicity studies of a series of fluorescent C8-aryl-dG analogues that differ in aryl ring size and are representative of authentic DNA adducts. These structural mimics have been inserted into a hotspot sequence for frameshift mutations, namely, the reiterated G3-position of the NarI sequence within 12mer (NarI(12)) and 22mer (NarI(22)) oligonucleotides. In the NarI(12) duplexes, the C8-aryl-dG adducts display a preference for adopting an anti-conformation opposite C, despite the strong syn preference of the free nucleoside. Using the NarI(22) sequence as a template for DNA synthesis in vitro, mutagenicity of the C8-aryl-dG adducts was assayed with representative high-fidelity replicative versus lesion bypass Y-family DNA polymerases, namely, Escherichia coli pol I Klenow fragment exo− (Kf−) and Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4). Our experiments provide a basis for a model involving a two-base slippage and subsequent realignment process to relate the miscoding properties of C-linked C8-aryl-dG adducts with their chemical structures.

The optical, redox, and electronic properties of C(8)-heteroaryl-2'-deoxyguanosine (dG) adducts with C(8)-substituents consisting of furyl ((Fur)dG), pyrrolyl ((Pyr)dG), thienyl ((Th)dG), benzofuryl ((Bfur)dG), indolyl ((Ind)dG), and benzothienyl ((Bth)dG) are described. These adducts behave as fluorescent nucleobase probes with emission maxima from 379 to 419 nm and fluorescence quantum yields (Φ(fl)) in the 0.1-0.8 range in water at neutral pH. The probes exhibit quenched fluorescence with increased solvent viscosity and decreased solvent polarity. The (Fur)dG, (Bfur)dG, (Ind)dG, and (Bth)dG derivatives were incorporated into the G(3) position of the 12-mer oligonucleotide 5'-CTCG(1)G(2)CG(3)CCATC-3' that contains the recognition sequence of the NarI Type II restriction endonuclease. This sequence is widely used to study the biological activity (mutagenicity) of C(8)-arylamine-dG adducts with adduct conformation (anti vs syn) playing a critical role in the biological outcome. The modified NarI(X = (Fur)G, (Ind)G, (Bfur)G, or (Bth)G) oligonucleotides were hybridized to the complementary strand containing either C (NarI'(C)) or G (NarI'(G)) opposite the probe. The duplex structures were characterized by UV melting temperature analysis, fluorescence spectroscopy, collisional fluorescence quenching studies, and circular dichroism (CD). The emission of the probes showed sensitivity to the opposing base in the duplex, and suggested the utility of fluorescence spectroscopy to monitor probe conformation.

To study the structural and biological impact of 8-aryl-2'-deoxyguanosine adducts, an efficient protocol is required to incorporate them site-specifically into oligonucleotide substrates. Traditional phosphoramidite chemistry using 5'-O-DMT protection can be limiting because 8-aryl-dG adducts suffer from greater rates of acid-catalyzed depurination than dG and are sensitive to the acidic deblock conditions required to remove the DMT group. Herein we show that the 5'-O-2,7-dimethylpixyl (DMPx) protecting group can be used to limit acid exposure and improve DNA synthesis efficiency for DNA substrates containing 8-aryl-dG adducts. Our studies focus on 8-aryl-dG adducts with 8-substituents consisting of furyl ((Fur)dG), phenyl ((Ph)dG), 4-cyanophenyl ((CNPh)dG), and quinolyl ((Q)dG). These adducts differ in ring size and sensitivity to acid-promoted deglycosylation. A kinetic study for adduct hydrolysis in 0.1 M aqueous HCl determined that (Fur)dG was the most acid-sensitive (55.2-fold > dG), while (Q)dG was the most resistant (5.6-fold > dG). The most acid-sensitive (Fur)dG was chosen for optimization of solid-phase DNA synthesis. Our studies show that the 5'-O-DMPx group can provide a 4-fold increase in yield compared to 5'-O-DMT for incorporation of (Fur)dG into DNA substrates critical for determining adduct impact on DNA synthesis and repair.