Enhancing Bulge Stabilization through Linear Extension of C8-Aryl-Guanine Adducts to Promote Polymerase Blockage or Strand Realignment to Produce a C:C Mismatch.

Aryl radicals can react at the C8-site of 2'-deoxyguanosine (dG) to produce DNA adducts with a C8-C linkage (denoted C-linked). Such adducts are structurally distinct from those possessing a flexible amine (N-linked) or ether (O-linked) linkage, which separates the C8-aryl moiety from the guanine nucleobase. In the current study, two model C-linked C8-dG adducts, namely, C8-benzo[b]thienyl-dG ([BTh]G) and C8-(pyren-1-yl)-dG ([Py]G), were incorporated into the NarI (12mer, NarI(12) and 22mer, NarI(22)) hotspot sequence for frameshift mutations in bacteria. For the first time, C-linked C8-dG adducts are shown to stabilize the -2 deletion duplex within the NarI sequence. Primer-elongation assays employing Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) demonstrates the influence of C8-aryl ring size and shape in promoting Dpo4 blockage or strand realignment to produce a C:C mismatch downstream of the adduct site. Molecular dynamics simulations of the -2 deletion duplex suggest that both anti and syn adduct structures are energetically accessible. These findings provide a rationale for describing the biochemical outcome induced by C-linked C8-dG adducts when processed by Dpo4.

Chlorine functionalization of a model phenolic C8-guanine adduct increases conformational rigidity and blocks extension by a Y-family DNA polymerase.

Certain phenoxyl radicals can attach covalently to the C8-site of 2'-deoxyguanosine (dG) to afford oxygen-linked C8-dG adducts. Such O-linked adducts can be chemically synthesized through a nucleophilic displacement reaction between a phenolate and a suitably protected 8-Br-dG derivative. This permits the generation of model O-linked C8-dG adducts on scales suitable for insertion into oligonucleotide substrates using solid-phase DNA synthesis. Variation of the C8-aryl moiety provides an opportunity to derive structure-activity relationships on adduct conformation in duplex DNA and replication bypass by DNA polymerases. In the current study, the influence of chlorine C8-dG functionalization on in vitro DNA replication by Klenow fragment exo(-) (Kf(-)) and the Y-family polymerase (Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4)) has been determined. Model O-linked C8-dG adducts derived from the pentachlorophenoxyl radical ([PCP]G) and 2,4,6-trichlorophenoxyl radical ([TCP]G) were inserted into the reiterated G3-position of the NarI sequence (12-mer, NarI(12); and 22-mer, NarI(22)), which is a known hotspot for frameshift mutations mediated by N-linked polycyclic C8-dG adducts in bacterial mutagenesis. Within the NarI(12) duplex, the unsubstituted C8-phenoxy-dG ([PhO]G) adduct adopts a minimally perturbed B-form helix. Chlorination of [PhO]G to afford [PCP]G does not significantly change the adduct conformation within the NarI(12) duplex, as predicted by molecular dynamics simulations. However, when using NarI(22) for DNA synthesis in vitro, the chlorinated [PCP]G and [TCP]G lesions significantly block DNA replication by Kf(-) and Dpo4, whereas [PhO]G is readily bypassed. These findings highlight the impact that chlorine substituents impart to bulky C8-dG lesions.

Positional impact of fluorescently modified G-tetrads within polymorphic human telomeric G-quadruplex structures.

Emissive C8-aryl-2'-deoxyguanosines placed within G-tetrads of G-quadruplex structures are useful probes for distinguishing G-quadruplexes from duplex structures using fluorescence spectroscopy. Here, we report the positional impact of C8-furyl-dG ((Fur)dG) on G-quadruplex folding in the human telomere 22-mer oligonucleotide (HTelo22, (d[AG3(T2AG3)3])). The (Fur)dG probe was inserted into four different positions within the three unique G-tetrads of HTelo22, and G-quadruplex folding was monitored by UV-vis thermal denaturation, circular dichroism, and fluorescence spectroscopy. Our studies demonstrate the impacts of C8-aryl-dG adduct formation on G-quadruplex polymorphism in K(+) solution and in the presence of the additives and cosolutes, CH3CN, polyethylene glycol (PEG-600), and N-methyl mesoporphyrin IX (NMM). Our experiments predict that C8-aryl-dG derivatives can serve as useful tools for various in vitro studies aimed at understanding both the implications of DNA adduct formation within G-quadruplex structures and the unique implications imposed by various folding topologies on biological function/recognition.

Structural and biochemical impact of C8-aryl-guanine adducts within the NarI recognition DNA sequence: influence of aryl ring size on targeted and semi-targeted mutagenicity.

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.

Harnessing G-tetrad scaffolds within G-quadruplex forming aptamers for fluorescence detection strategies.

G-tetrads are essential structural components required for the formation of G-quadruplexes. Replacement of G nucleobases within G-tetrads with fluorescent 8-aryl-dG residues provides diagnostic handles that are universally applicable to antiparallel G-quadruplex aptamers, as they can stabilize quadruplex folding and maintain aptamer function.

Utility of 5'-O-2,7-dimethylpixyl for solid-phase synthesis of oligonucleotides containing acid-sensitive 8-aryl-guanine adducts.

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.

Structural influence of C8-phenoxy-guanine in the NarI recognition DNA sequence.

Phenoxyl radicals can covalently attach to the C8 site of 2'-deoxyguanosine (dG) to generate oxygen-linked biaryl ether C8-dG adducts. To assess the structural impact of an O-linked C8-dG adduct in duplex DNA, C8-phenoxy-G ((PhO)G) and C8-4-fluorophenoxy-G ((4FPhO)G) were incorporated into the G(3) position of the 12-mer NarI recognition sequence (5'-CTCGGCXCCATC, where X = G, (PhO)G, or (4FPhO)G) using solid-phase DNA synthesis with O-linked C8-dG phosphoramidites. The modified strands were hybridized to six different complementary strands that include regular base pairing to C [NarI'(C)], mismatches with G, A, T [NarI'(N)], and an abasic site [NarI'(THF)], and a 10-mer sequence to model a -2 deletion duplex [NarI'(-2)]. All duplex structures were characterized using UV-vis thermal melting temperature analysis, and in each instance, the O-linked C8-phenoxy-G adducts were found to destabilize the duplex relative to the unmodified controls. The most stable duplex structures match the O-linked C8-dG adduct against C and a G mismatch, which are comparable in terms of stability. These duplexes were further characterized using circular dichroism, dynamic (19)F nuclear magnetic resonance experiments, and molecular dynamics simulations. On the basis of these findings, (PhO)dG adopts the B conformation opposite C, with the phenoxy moiety residing in the solvent-exposed major groove. However, opposite the G mismatch, (PhO)dG adopts a "W-type" wedge conformation with the phenoxy group residing in the minor groove. These studies predict that the O-linked C8-dG lesion (PhO)G will have a weak mutagenic effect, as determined for the corresponding single-ringed nitrogen-linked C8-dG adduct derived from aniline.

C8-heteroaryl-2'-deoxyguanosine adducts as conformational fluorescent probes in the NarI recognition sequence.

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.