Harnessing a 4-Formyl-Aniline Handle to Tune the Stability of a DNA Aptamer-Protein Complex via Fluorescent Surrogates.

Interactions between DNA aptamers and protein targets hold promise for the development of pharmaceuticals and diagnostics. As such, the utilization of fluorescent nucleobase surrogates in studying aptamer-protein interactions is a powerful tool due to their ability to provide site-specific information through turn-on fluorescence. Unfortunately, previously described turn-on probes serving as nucleobase replacements have only been strongly disruptive to the affinity of aptamer-protein interactions. Herein, we present a modified TBA15 aptamer for thrombin containing a fluorescent surrogate that provides site-specific turn-on emission with low nanomolar affinity. The modification, referred to as AnBtz, was substituted at position T3 and provided strong turn-on emission (Irel ≈ 4) and brightness (ε·Φ > 20 000 cm-1 M-1) with an apparent dissociation constant (Kd) of 15 nM to afford a limit of detection (LOD) of 10 nM for thrombin in 20% human serum. The probe was selected through a modular "on-strand" synthesis process that utilized a 4-formyl-aniline (4FA) handle. Using this platform, we were able to enhance the affinity of the final aptamer conjugate by ∼30-fold in comparison with the initial conjugate design. Molecular dynamics simulations provide insight into the structural basis for this phenomenon and highlight the importance of targeting hydrophobic protein binding sites with fluorescent nucleobase surrogates to create new contacts with protein targets.

Isomer-Specific Solvatochromic and Molecular Rotor Properties of ESIPT-Active Push-Pull Fluorescent Chalcone Dyes.

Aromatic chromophores possessing intramolecular hydrogen-bonds that can undergo excited-state intramolecular proton transfer (ESIPT) are critical tools for chemosensing/biosensing applications because they create large Stokes-shifted fluorescence with no overlap with the absorption spectrum to limit back-ground interferences. Classic ESIPT-active fluorophores, such as the 2-(2'-hydroxyphenyl) benzazole (HBX) series (X = NH, O, S), favor a ground-state (GS) enol (E) form that undergoes ESIPT to afford an excited-state (ES) keto (K) tautomer that generates red-shifted fluorescence. Herein, we have attached the HBX moiety to 6-methoxy-indanone (6MI) to create isomeric (ortho and para) ESIPT-active chalcone dyes and have characterized their photophysical properties in polar protic solvents (MeOH and glycerol (Gly)/MeOH mixtures) and a nonpolar aprotic (1,4-dioxane) solvent for comparison. The chalcones favor a GS E structure, which undergoes ESIPT in MeOH, Gly/MeOH mixtures, and dioxane to exclusively afford K emission with large Stokes shifts. The o-isomers possess expanded π-conjugation compared to their p-isomer counterparts, which diminishes their tendency to generate twisted intramolecular charge transfer (TICT) states. Consequently, the o-isomers have greater quantum yields and lack molecular rotor (MR) character with little K emission response to increased solvent viscosity. However, they possess strong positive solvatochromism, displaying significant blue wavelength shifts coupled with turn-on K emission in moving from polar protic MeOH to nonpolar dioxane. In contrast, the p-isomers display MR character with turn-on K emission in 75:25 Gly/MeOH compared to their emission in MeOH (up to 14-fold) due to a strong tendency for TICT. Mechanistic insight into the observed isomer-specific photophysical properties of the ESIPT-active chalcones was obtained through density functional theory (DFT) calculations. Implications for DNA biosensing applications are discussed.

Unraveling the Chemosensing Mechanism by the 7-(Diethylamino)coumarin-hemicyanine Hybrid: A Ratiometric Fluorescent Probe for Hydrogen Peroxide.

The hemicyanine hybrid containing the 7-(diethylamino)coumarin (ACou) donor attached to the cationic indolenium (Ind) acceptor through a vinyl linkage (ACou-Ind) represents a classic ratiometric fluorescent probe for detecting nucleophilic analytes, such as cyanide and reactive sulfur species (RSS), through addition reactions that disrupt dye conjugation to turn off red internal charge transfer (ICT) fluorescence and turn on blue coumarin emission. The chemosensing mechanism for RSS detection by ACou-Ind suggested in the literature has now been revised. Our studies demonstrate that thiolates react with ACou-Ind through conjugate addition to afford C4-SR adducts that lack coumarin fluorescence due to photoinduced electron transfer quenching by the electron-rich enamine intermediate. Thus, ACou-Ind serves as a turn-off probe through loss of red ICT fluorescence upon RSS addition. The literature also suggests that blue coumarin emission of thiolate adducts is enhanced in the presence of reactive oxygen species (ROS) due to ROS-mediated cellular changes. Our studies predict that such a scenario is unlikely and that thiolate adducts undergo oxidative deconjugation in the presence of H2O2, the pervasive ROS. Under basic conditions, H2O2 also reacts directly with ACou-Ind to generate intense coumarin fluorescence through an epoxidation process. The relevance of our chemosensing mechanism for ACou-Ind was assessed within live zebrafish, and implications for the utility of ACou-Ind for unraveling the interplay between RSS and ROS are discussed.

On-Strand Knoevenagel Insertion of a Hemicyanine Molecular Rotor Loop Residue for Turn-On Fluorescence Detection of Pb-Induced G-Quadruplex Rigidity.

We demonstrate the ability to distinguish Pb2+ from K+ within the central cavity of the antiparallel G-quadruplex (GQ) DNA produced by the thrombin binding aptamer (TBA) using an internal molecular rotor fluorescent probe. An indole-aldehyde containing an acyclic N-glycol group was first employed in the on-strand Knoevenagel condensation with five different heterocyclic quaternary cationic acceptors to assess the molecular rotor character of the resulting cyanine-styryl dyes within duplex DNA. An indole-pyridinium (4PI) nucleobase surrogate displayed the greatest turn-on emission response to duplex formation and was thus inserted into the loop residues of TBA to monitor GQ-folding in the presence of Pb2+ versus K+. TBA-4PI exhibits turn-on emission upon Pb2+-binding with a brightness (ε·Φfl) of 9000 cm-1 M-1 compared to K+-binding (ε·Φfl ∼ 2000 cm-1 M-1) due to Pb2+-induced GQ rigidity with 4PI-G-tetrad stacking interactions. The Pb2+-bound TBA-4PI GQ also provides energy-transfer (ET) fluorescence with a diagnostic excitation at 310 nm for distinguishing Pb2+ from K+ within the antiparallel GQ. The TBA-4PI GQ affords the desired turn-on fluorescence response for detecting Pb2+ ions with an apparent dissociation constant (Kd) of 63 nM and a limit of detection (LOD) of 19 nM in an aqueous buffer. It can also distinguish Pb2+ (230 nM) from K+ (1.5 mM, 6500-fold excess) in an antiparallel GQ recognition motif without topology twitching.

Screening Internal Donor-Acceptor Biaryl Nucleobase Surrogates for Turn-On Fluorescence Affords an Aniline-Carboxythiophene Probe for Protein Detection by G-Quadruplex DNA.

Donor-acceptor biaryls serve as microenvironment fluorescent sensors with highly quenched intramolecular charge transfer (ICT) emission in polar protic solvents that turns on in aprotic media. In DNA, canonical donor-acceptor fluorescent base analogs can be prepared through on-strand Suzuki-Miyaura cross-coupling reactions involving 8-bromo-2'-deoxyguanosine (8-Br-dG) with an acceptor aryboronic acid. Herein, we demonstrate that replacement of 8-Br-dG with N-methyl-4-bromoaniline (4-Br-An) containing an acyclic N-glycol group can be employed in the on-strand Suzuki-Miyaura reaction to afford new donor-acceptor biaryl nucleobase surrogates with a 40-fold increase in emission intensity for fluorescent readout within single-strand oligonucleotides. Screening the best acceptor for turn-on fluorescence upon duplex formation afforded the carboxythiophene derivative [COOTh]An with a 7.4-fold emission intensity increase upon formation of a single-bulged duplex (-1) with the surrogate occupying a pyrimidine-flanked bulge. Insertion of the [COOTh]An surrogate into the lateral TT loops produced by the antiparallel G-quadruplex (GQ) of the thrombin binding aptamer (TBA) afforded a 4.1-fold increase in probe fluorescence that was accompanied by a 20 nm wavelength shift to the blue upon thrombin binding. The modified TBA afforded a limit of detection of 129 nM for thrombin and displayed virtually no emission response to off-target proteins. The fluorescence response of [COOTh]An to thrombin binding highlights the utility of the thienyl-aniline moiety for monitoring DNA-protein interactions.

Temperature Sensing of Thiolate Addition by Phenolate Merocyanine Dyes: Importance of the Quinone Methide Resonance Structure.

Merocyanine (MC) dyes containing an aromatic donor vinyl linked to a cationic acceptor serve as chemosensors for analyte detection. Their electrophilicity permits anion detection through addition reactions that disrupt dye conjugation. Herein, we demonstrate the temperature influence on thiolate addition to MCs containing the N-methylbenzothiazolium (Btz) acceptor. The zwitterionic phenolate dye (PhOBtz) displays impressive temperature sensitivity to thiolate addition, with the brightly colored phenolate favored upon heating and the colorless thiolate adduct favored upon cooling. In contrast, MC dyes containing neutral donors (PhOMeBtz and PhNMe2Btz) display only moderate temperature sensitivity to thiolate capture and release. Extraction of thermodynamic parameters demonstrates a strong enthalpic driving force for thiolate addition to PhOBtz that is absent for PhOMeBtz and PhNMe2Btz. Variable temperature 1H NMR studies demonstrate that PhOBtz adopts the para-quinone methide (p-QM) resonance structure. Thus, thiolate addition to PhOBtz resembles 1,6-conjugate addition to p-QMs which is accompanied by a large increase in the π-stabilization energy upon adduct formation. Manipulation of PhOBtz electrophilicity by attaching chlorine substituents to the phenolate caused the thiolate adducts to dissipate over time for p-QM regeneration. Our work provides new design ideas for the utility of phenolate MC dyes, given that they are carriers of the p-QM electrophile.

Lighting Up the Thrombin-Binding Aptamer G-Quadruplex with an Internal Cyanine-Indole-Quinolinium Nucleobase Surrogate. Direct Fluorescent Intensity Readout for Thrombin Binding without Topology Switching.

Fluorescent nucleobases represent an important class of molecular reporters of nucleic acid interactions. In this work, the advantages of utilizing a noncanonical fluorescent nucleobase surrogate for monitoring thrombin binding by the 15-mer thrombin binding aptamer (TBA) is presented. TBA folds into an antiparallel G-quadruplex (GQ) with loop thymidine (T) residues interacting directly with the protein in the thrombin-TBA complex. In the free GQ, T3 is solvent-exposed and does not form canonical base-pairs within the antiparallel GQ motif. Upon thrombin binding, T3 interacts directly with a hydrophobic protein binding pocket. Replacing T3 with a cyanine-indole-quinolinium (4QI) hemicyanine dye tethered to an acyclic 1,2-propanediol linker is shown to have minimal impact on GQ stability and structure with the internal 4QI displaying a 40-fold increase in emission intensity at 586 nm (excitation 508 nm) compared to the free dye in solution. Molecular dynamics (MD) simulations demonstrate that the 4QI label π-stacks with T4 and T13 within the antiparallel GQ fold, which is supported by strong energy transfer (ET) fluorescence from the GQ (donor) to the 4QI label (acceptor). Thrombin binding to 4QI-TBA diminishes π-stacking interactions between 4QI and the GQ structure to cause a turn-off emission intensity response with an apparent dissociation constant (Kd) of 650 nM and a limit of detection (LoD) of 150 nM. These features highlight the utility of internal noncanonical fluorescent surrogates for monitoring protein binding by GQ-folding aptamers in the absence of DNA topology switching.

Structure of an Unusual Tetracyclic Deoxyguanosine Adduct: Implications for Frameshift Mutagenicity of ortho-Cyano Nitroanilines.

Nitroaromatic compounds represent a major class of industrial chemicals that are also found in nature. Polycyclic derivatives are regarded as potent mutagens and carcinogens following bioactivation to produce nitrenium electrophiles that covalently modify DNA to afford N-linked C8-2'-deoxyguanosine (C8-dG) lesions that can induce frameshift mutations, especially in CpG repeat sequences. In contrast, their monocyclic counterparts typically exhibit weak mutagenicity or a lack thereof, despite also undergoing bioactivation to afford N-linked C8-dG adducts. Recently, it has been reported that cyano substitution can greatly increase the mutagenicity of nitroaniline derivatives that are components of azo dyes. The basis of this "cyano effect" may be rooted in the formation of a novel polycyclic adduct arising from initial formation of the N-linked C8-dG adduct followed by a cyclization process involving N7 of dG and the ortho-CN group of the attached C8-aryl moiety to generate a quinazolinimine ring as part of a fused tetracyclic C8,N7-dG adduct structure. The present work structurally characterizes this novel cyclic adduct using a combination of optical spectroscopies, NMR analysis, density functional theory (DFT) calculations, and molecular dynamics (MD) simulations. Our data indicate that this highly fluorescent cyclic adduct adopts the promutagenic syn conformation and can stabilize the slipped mutagenic intermediate (SMI) within the CpG repeat of the NarI sequence, which is a hotspot for frameshift mutagenesis mediated by polycyclic N-linked C8-dG adducts. In contrast, the open para-CN (4-aminobenzontrile-derived) N-linked C8-dG adduct is less likely to disrupt the canonical B-form. Together, our results provide a rationale for the potent mutagenicity of cyano-substituted nitroaniline derivatives recently reported in frameshift-sensitive tester strains.

Ratiometric fluorescent sensing of the parallel G-quadruplex produced by PS2.M: implications for K+ detection.

Fluorescent ligands that selectively bind to a specific G-quadruplex (GQ) topology (antiparallel, hybrid or parallel) are highly sought after for aptasensor development and nanodevice construction. The coumarin-benzothiazole hybrid (BnBtC) is an internal charge transfer (ICT) ratiometric fluorescent probe, which displays two well-resolved emission bands at ∼450 nm for the coumarin component and ∼650 nm for the ICT band. The red ICT emission of BnBtC displays turn-on responses to protic solvent polarity and upon binding GQ structures, especially those produced by the hemin binding aptamer (PS2.M). In the present work, BnBtC was found to exhibit enhanced ICT emission upon binding the parallel GQ topology of PS2.M that is selectively produced in the presence of K+. This ability to discriminate K+ from other cationic metal ions through a turn-on ratiometric fluorescent response demonstrates the potential utility of the BnBtC probe for biosensor applications.

Ligand-Induced G-Quadruplex Polymorphism: A DNA Nanodevice for Label-Free Aptasensor Platforms.

G-Quadruplexes (GQs) serve as popular recognition elements for DNA aptasensors and are incorporated into a DNA nanodevice capable of controlled conformational changes to activate a sensing mechanism. Herein we highlight the utility of a GQ-GQ nanodevice fueled by GQ-specific ligands as a label-free aptasensor detection strategy. The concept was first illustrated utilizing the prototypical polymorphic human telomeric repeat sequence (H-Telo22, d[AG3(T2AG3)3]) that can undergo ligand-induced topology changes between antiparallel, parallel, or hybrid GQ structures. The H-Telo22-ligand interactions served as a model of the GQ-GQ nanodevice. The utility of the device in a real aptasensor platform was then highlighted utilizing the ochratoxin A (OTA) binding aptamer (OTABA) that folds into an antiparallel GQ in the absence and presence of target OTA. Three cationic fluorogenic ligands served as GQ-specific light-up probes and as potential fuel for the GQ-GQ nanodevice by producing an inactive GQ topology (parallel or hybrid) of OTABA. Our findings demonstrate efficient OTA-mediated dye displacement with excellent emission sensitivity for OTA detection when the fluorogenic dyes induce a topology change in OTABA (parallel or hybrid). However, when the fluorogenic dye fails to induce a conformational change in the antiparallel fold of OTABA, subsequent additions of OTA to the aptamer-dye complex results in poor dye displacement with weak emission response for OTA detection. These results are the first to exemplify a ligand-induced GQ-GQ nanodevice as an aptasensor mechanism and demonstrate diagnostic applications for topology-specific GQ binders.

Adduct Fluorescence as a Tool to Decipher Sequence Impact on Frameshift Mutations Mediated by a C-Linked C8-Biphenyl-Guanine Lesion.

Aromatic chemicals can undergo metabolic activation to afford electrophilic species that react at the C8-site of 2'-deoxyguanosine (dG) to generate bulky C8-dG adducts as a basis of initiating carcinogenesis. These DNA lesions have served as models to understand the mechanism of frameshift mutagenesis, especially within CG-dinucleotide repeat sequences, such as NarI (5'-GGCXCC-3', where X = C8-dG adduct), however there is still limited capacity to predict the likelihood of mutation arising within particular contexts, and hence chemistry-based strategies are needed for probing relationships between nucleic acid sequence and structure with replication errors. In the NarI sequence, certain C8-dG adducts may trigger in the course of DNA synthesis the formation of a slipped mutagenic intermediate (SMI) that contains a two nucleotide (XC) bulge in the template strand that can form upstream of the polymerase active site. This distortion facilitates polymerization but affords a GC dinucleotide deletion product (-2 frameshift mutation). In the current study, incorporating the fluorescent C-linked 4-fluorobiphenyl-dG (FBP-dG) adduct into two 22-mer templates containing CG-dinucleotide repeats ( NarI: 3'-CXCGGC-5' and CG3: 3'-CXCGCG-5', X = FBP-dG) and performing primer extension reactions using DNA polymerase I, Klenow fragment exo- (Kf-) revealed a dramatic sequence-based difference in polymerase bypass efficiency. Primer extension past FBP-dG within the NarI sequence was strongly blocked, whereas Kf- extended the primer past FBP-dG within a CG3 template to afford a full-length product and the GC dinucleotide deletion. To model the nucleotide insertion steps in the fully paired (FP) versus the slipped mutagenic (SM) translesion pathways, adducted template:primer duplexes were constructed and characterized by UV thermal denaturation and fluorescence spectroscopy. The emission intensity of the FBP-dG lesion exhibits sensitivity to SMI formation (turn-on) versus a FP duplex (turn-off), permitting insight into adduct base-pairing within the template:primer duplexes. This fluorescence sensitivity provides a rationale for sequence impact on -2 frameshift mutations mediated by the C-linked FBP-dG lesion.

Acceptor Influence on Thiolate Sensing by Hemicyanine Dyes.

Promoting selective interactions between a nucleophile and electrophilic dye in complex environments is a central goal in nucleophilic chemosensor development. Commonly employed dyes are hemicyanines containing either the N-methylbenzothiazolium (Btz) or the N-methyl-3,3-dimethylindolium (Ind) acceptors. The dyes are related to α,ß-unsaturated carbonyls and contain two sites of reactivity (C2 vs C4) with the C2-site directly attached to the quaternary nitrogen possessing greater electrophilicity. We demonstrate the regioselectivity between reactions of sodium thiomethoxide (NaSMe) with two electrophilic hemicyanine dyes bearing Btz (1) or Ind (2) in dipolar aprotic solvent-water mixtures. Adduct complexation was followed by NMR spectroscopy, and structures were optimized in the gas phase to estimate relative adduct stability. The key results include finding a preference for thiolate attachment at the C4-site to generate an enamine adduct with no evidence for attachment at the more electrophilic C2-position. Equilibration between NaSMe and water also affords NaOH that displays a thermodynamic preference for C2-attachment. Dye 1 containing the Btz moiety exhibits greater selectivity for the thiolate addition, with dye 2 being more reactive toward adventitious water to generate OH-adducts. Our data affords diagnostic 1H/13C NMR adduct signals, regioselectivity for various dye/nucleophile combinations, and suggests use of the Btz acceptor for direct thiolate detection.

Molecular Dynamics Simulations of Mismatched DNA Duplexes Associated with the Major C8-Linked 2'-Deoxyguanosine Adduct of the Food Mutagen Ochratoxin A: Influence of Opposing Base, Adduct Ionization State, and Sequence on the Structure of Damaged DNA.

Exposure to ochratoxin A (OTA) is associated with chronic renal diseases and carcinogenesis. The deleterious effects of OTA have been linked to its covalent binding at the C8 position of guanine (G) to form a DNA adduct (OT-G), which causes various mutations. To contribute toward understanding the complex mutagenic profile of OTA, the present work uses a robust computational approach to characterize postreplication DNA structures containing OT-G mismatched with canonical nucleobases. Our MD simulations provide insight into the effects of the opposing base, adduct ionization state, and flanking base on duplex structural features for the competing (major groove (B-type), wedge (W), and stacked (S)) conformers. For the B-type duplexes, our data suggest that significantly more stable lesion-site hydrogen bonding may lead to preferential insertion of an opposing cytosine (C) if the OT moiety is directed toward the major groove at the replication fork. Although the W conformation is consistently predicted to be less stable than the B conformer, a G mismatch is likely the most stable and least distorted replication outcome when the bulky moiety is directed into the DNA minor groove. These findings directly correlate with the limited contribution of substitution mutations to the overall mutagenic profile of OTA and suggest that the dominant mutations are G → C transversions. In contrast, stable S conformers that are known precursors to small (one- or two-base) deletion mutations are found when the lesion is opposite cytosine, adenine, or thymine, which directly correlates with the large number of deletion mutations previously reported for animals exposed to OTA. Nevertheless, the predicted sequence and ionization-dependent distortion of the S conformer points toward the dependence of the repair propensity on the cellular environment, which rationalizes the reported tissue specific OTA-induced toxicity.

Conformational Preference and Fluorescence Response of a C-Linked C8-Biphenyl-Guanine Lesion in the NarI Mutational Hotspot: Evidence for Enhanced Syn Adduct Formation.

Aromatic chemical carcinogens can undergo enzymatic transformations to produce a range of electrophilic species that attach covalently to the C8-site of 2'-deoxyguanosine (dG) to afford C8-dG adducts. The most studied C8-dG adducts are formed from arylamines and contain a N-linkage separating the dG from the C8-aryl moiety. Other carcinogenic species result in direct aryl ring attachment to the dG moiety, resulting in C-linked adducts. The resulting C-linked adducts have reduced conformational flexibility compared to the corresponding N-linked C8-dG adducts, which can alter their orientation in the DNA duplex. Described herein are structural studies of a fluorescent C-linked 4-fluorobiphenyl-dG (FBP-dG) that has been incorporated into the reiterated G3-postion of the 12-mer NarI sequence and those containing other 5'-flanking nucleobases. FBP-dG displays a strong preference for adopting a syn conformation in the fully paired NarI duplex to produce an intercalated structure that exhibits stacking interactions between the C-linked biphenyl and the flanking bases. FBP-dG is also shown to significantly stabilize the slippage mutagenic intermediate (SMI) duplex containing the lesion and 5'-flanking base within a 2-base bulge. FBP-dG exhibits fluorescence sensitivity to SMI duplex formation that can readily distinguish it from the fully paired duplex. Molecular dynamics simulations and optical spectroscopy for the NarI oligonucleotides containing the C-linked FBP-dG predict increased rigidity of the biphenyl in the syn conformation. The greater propensity to generate the promutagenic syn conformation for the C-linked FBP-dG adduct compared to the N-linked 4-aminobiphenyl-dG adduct (ABP-dG) suggests greater mutagenicity for the C-linked analogue. These results highlight the effect of the adduct linkage type on the conformational properties of adducted DNA. The turn-on emission response of FBP-dG in the SMI duplex may be a powerful tool for monitoring SMI formation in the NarI sequence upon synthesis with DNA polymerases.

Manipulation of a DNA aptamer-protein binding site through arylation of internal guanine residues.

Chemically modified aptamers have the opportunity to increase aptamer target binding affinity and provide structure-activity relationships to enhance our understanding of molecular target recognition by the aptamer fold. In the current study, 8-aryl-2'-deoxyguanosine nucleobases have been inserted into the G-tetrad and central TGT loop of the thrombin binding aptamer (TBA) to determine their impact on antiparallel G-quadruplex (GQ) folding and thrombin binding affinity. The aryl groups attached to the dG nucleobase vary greatly in aryl ring size and impact on GQ stability (∼20 °C change in GQ thermal melting (Tm) values) and thrombin binding affinity (17-fold variation in dissociation constant (Kd)). At G8 of the central TGT loop that is distal from the aptamer recognition site, the probes producing the most stable GQ structure exhibited the strongest thrombin binding affinity. However, within the G-tetrad, changes to the electron density of the dG component within the modified nucleobase can diminish thrombin binding affinity. Detailed molecular dynamics (MD) simulations on the modified TBA (mTBA) and mTBA-protein complexes demonstrate how the internal 8-aryl-dG modification can manipulate the interactions between the DNA nucleobases and the amino acid residues of thrombin. These results highlight the potential of internal fluorescent nuclobase analogs (FBAs) to broaden design options for aptasensor development.

Understanding the Mutagenicity of O-Linked and C-Linked Guanine DNA Adducts: A Combined Experimental and Computational Approach.

The formation of DNA adducts by the attack of intermediates derived from toxic substances at C8 of 2'-deoxyguanosine (dG) is a common damaging event. Although the majority of studies on C8-dG adducts have focused on lesions containing a C8-N-C tether between the bulky moiety and the nucleobase, the formation of O-linked lesions with a similar tether topology and C-linked adducts involving direct C8-C connectivity have also been uncovered. Several studies have been done to try to better understand the structural impact and mutagenicity of O-linked and C-linked aryl C8-dG adducts, including lesions arising from unsubstituted and chloro-substituted phenols and the food mutagen ochratoxin A (OTA). Information about the structural preferences of the adducts in duplexes containing the NarI sequence has been gained from optical spectroscopy (UV-vis, CD, and fluorescence), 19F NMR spectroscopy, and computational chemistry (density functional theory calculations at the nucleobase, nucleoside, and nucleotide levels and molecular dynamics simulations of adducted duplexes). The replication of select adducts has also been investigated using primer-elongation assays, and model high-fidelity and Y-family polymerases. Although the (unsubstituted) O-linked phenoxy-dG adduct preferentially induces a single duplex conformation and is replicated as per natural dG, chloro substitution blocks DNA replication. In contrast, the unsubstituted C-linked phenyl-dG adduct induces mismatches, while the C-linked ortho- and para-phenoxy-dG lesions lead to conformational heterogeneity of adducted DNA indicative of strong mutagenic potential. Finally, the C-linked OTA-derived lesion exhibits the greatest conformational flexibility in duplexes, which provides structural explanations for observed outcomes in OTA-exposed cells. Overall, the variation in the conformational preferences of DNA containing O-linked and C-linked aryl-dG adducts highlights the fact that the type of C8 linkage, the presence and location of functional groups in the bulky moiety, the adduct ionization state, and the sequence context can have profound effects on the conformational outcomes of adducted DNA, which directly dictate the activity of the original toxin.

Molecular Modeling of the Major DNA Adduct Formed from Food Mutagen Ochratoxin A in NarI Two-Base Deletion Duplexes: Impact of Sequence Context and Adduct Ionization on Conformational Preference and Mutagenicity.

Exposure to ochratoxin A (OTA), a possible human carcinogen, leads to many different DNA mutations. As a first step toward understanding the structural basis of OTA-induced mutagenicity, the present work uses a robust computational approach and a slipped mutagenic intermediate model previously studied for C8-dG aromatic amine adducts to analyze the conformational features of postreplication two-base deletion DNA duplexes containing OT-dG, the major OTA lesion at the C8 position of guanine. Specifically, a total of 960 ns of molecular dynamics simulations (excluding trial simulations) were carried out on four OT-dG ionization states in three sequence contexts within oligomers containing the NarI recognition sequence, a known hotspot for deletion mutations induced by related adducts formed from known carcinogens. Our results indicate that the structural properties and relative stability of the competing "major groove" and "stacked" conformations of OTA adducted two-base deletion duplexes depend on both the OTA ionization state and the sequence context, mainly due to conformation-dependent deviations in discrete local (hydrogen-bonding and stacking) interactions at the lesion site, as well as DNA bending. When the structural characteristics of the OT-dG adducted two-base deletion duplexes are compared to those associated with previously studied C8-dG adducts, a greater understanding of the effects of the nucleobase-carcinogen linkage, and size of the carcinogenic moiety on the conformational preferences of damaged DNA is obtained. Most importantly, our work predicts key structural features for OT-dG-adducted deletion DNA duplexes, which in turn allow us to develop hypotheses regarding OT-dG replication outcomes. Thus, our computational results are valuable for the design and interpretation of future biochemical studies on the potentially carcinogenic OT-dG lesion.

Optimization of fluorescent 8-heteroaryl-guanine probes for monitoring protein-mediated duplex → G-quadruplex exchange.

In this study, we describe the thermal and optical properties of the thrombin binding aptamer (TBA) that has been modified at syn-G-tetrad positions with fluorescent 8-heteroaryl-2'-deoxyguanosine derivatives consisting of pyrrolyl ((Pyr)dG), furyl ((Fur)dG), thienyl ((Th)dG), benzofuryl ((Bfur)dG), indolyl ((Ind)dG) and benzothienyl ((Bth)dG). Insertion of the modified base into the syn-G5 position of TBA decreases duplex stability, but enhances stability of the antiparallel G-quadruplex (GQ) structure produced by TBA in the presence of K(+) ion and its molecular target, thrombin. The resulting modified TBA (mTBA) oligonucleotides have been employed in duplex → GQ exchange to monitor thrombin binding affinity and rates of GQ formation driven by thrombin binding. Our studies demonstrate that 8-heteroaryl-dG bases can be inserted into syn-G-tetrad positions of TBA without perturbing thrombin binding affinity and that the 8-thienyl-dG ((Th)dG) analog is particularly useful as an emissive probe for monitoring duplex → GQ exchange due to its heightened emissive sensitivity to change in DNA topology compared to the other 8-heteroaryl-dG analogs. The positional impact of a single (Th)dG probe versus multiple (Th)dG incorporation at syn-G sites of TBA highlight an advantage for di-substituted mTBA oligonucleotides for increased emission intensity and rates of duplex → GQ exchange that can be vital for diagnostics through aptamer detection strategies.

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.

The nephrotoxic food mutagen ochratoxin A (OTA) produces DNA adducts in rat kidneys, the major lesion being the C8-linked-2'-deoxyguanosine adduct (OTB-dG). Although research on other adducts stresses the importance of understanding the structure of the associated adducted DNA, site-specific incorporation of OTB-dG into DNA has yet to be attempted. The present work uses a robust computational approach to determine the conformational preferences of OTB-dG in three ionization states at three guanine positions in the NarI recognition sequence opposite cytosine. Representative adducted DNA helices were derived from over 2160 ns of simulation and ranked via free energies. For the first time, a close energetic separation between three distinct conformations is highlighted, which indicates OTA-adducted DNA likely adopts a mixture of conformations regardless of the sequence context. Nevertheless, the preferred conformation depends on the flanking bases and ionization state due to deviations in discrete local interactions at the lesion site. The structural characteristics of the lesion thus discerned have profound implications regarding its repair propensity and mutagenic outcomes, and support recent experiments suggesting the induction of double-strand breaks and deletion mutations upon OTA exposure. This combined structural and energetic characterization of the OTB-dG lesion in DNA will encourage future biochemical experiments on this potentially genotoxic lesion.

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.