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

Abstract

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.

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

“…Structural parameters for the free nucleoside adducts were determined using DFT calculations at the B3LYP/6–31G(d) level. Fully optimized anti and syn minima and transition states on the potential energy surface for the PhG and QG nucleoside adducts are displayed in Supplementary Figures S1 and S2, whereas the corresponding data for FurG ( 49 ) and CNPhG ( 50 ) is published elsewhere. C8-Aryl ring size governs the degree of syn preference ( E anti - E syn = 19.3 for FurG versus 29.5 kJ mol −1 for QG) and the degree of twist ( θ ) between the nucleobase and the C8-aryl substituent ( θ = 343.8º for FurG versus 305.6º for QG, Table 2 ).…”

“…C8-aryl = furyl (FurG), phenyl (PhG), 4-cyanophenyl (CNPhG) and quinolyl (QG), Figure 1b ) chosen for this study differ in their ring size (5-membered ring in FurG versus 6-membered ring in PhG and CNPhG versus fused ring in QG), type of substituent (PhG versus CNPhG) and ring type (carbocyclic rings in PhG and CNPhG versus heterocyclic rings in FurG and QG). This series of adducts was designed on the basis of their structural similarities to natural carcinogen DNA-adducts, but having interesting fluorescence properties that we could exploit to define their conformations in DNA ( 49 – 51 ) and establish the relationship between adduct structure and mutagenicity in vitro . Among these adducts, PhG is a known adduct derived from the carcinogen phenylhydrazine ( vide supra ).…”

Structural and energetic characterization of the major DNA adduct formed from the food mutagen ochratoxin A in the NarI hotspot sequence: influence of adduct ionization on the conformational preferences and implications for the NER propensity

“…To explore the preferred conformations of OTB-dG adducted DNA, the present work undertakes an extensive molecular modeling study, including molecular dynamics (MD) simulations and post-processing free energy calculations, to determine the conformational preferences of OTB-dG adducted DNA in three protonation states at the G 1 , G 2 or G 3 sites in the Nar I sequence (5′–CTC G 1 G 2 CG 3 CC ATC–3′) opposite complementary cytosine. The center of this sequence (underlined) is the recognition site of the Nar I Type II restriction endonuclease, which is the most vulnerable hotspot for deletion mutations induced by carcinogenic N-linked C8-dG adducts ( 45 ), and the site-specific incorporation of a variety of C8-linked adducts has been studied in this sequence ( 41 , 46 – 50 ). Since OTA exposure has been linked to an increase in the frequency of deletion mutations at carcinogenic target sites ( 20 , 26 ), we chose the Nar I sequence to study the conformational preferences of the OTB-dG lesion.…”

Positional Impact of Fluorescently Modified G-Tetrads within Polymorphic Human Telomeric G-Quadruplex Structures

“…For the free Fur dG nucleoside, the syn-conformation is more stable than the anti-structure by ~ 19 kJ mol −1 , based on DFT calculations. 21 Consequently, Fur dG stabilized an antiparallel GQ formed by the 15-mer thrombin-binding aptamer (TBA) when substituted for a syn-G (increase in T m by ~ 9 °C compared to native TBA in K + buffer), but destabilized the GQ when substituted for an anti-G (decrease in T m by ~ −11 °C). 19 The positions chosen for modification within HTelo22 included G3, G4, G8 and G10.…”