Oxidation Of Adenine Research Articles

Electrochemical study of the interaction between dsDNA and copper(I) using carbon paste and hanging mercury drop electrode

The interaction of copper(I) with double-stranded (ds) calf thymus DNA was studied in solution and at the electrode surface by means of transfer voltammetry using a carbon paste electrode (CPE) as working electrode in 0.2 M acetate buffer solution (pH 5.0). As a result of the interaction of Cu(I) between the base pairs of the dsDNA, the characteristic peaks of dsDNA, due to the oxidation of guanine and adenine, increased and after a certain concentration of Cu(I) a new peak at +1.37 V appeared, probably due to the formation of a purine–Cu(I) complex (dsDNA–Cu(I) complex). Accordingly, the interaction of copper(I) with calf thymus dsDNA was studied in solution as well as at the electrode surface using hanging mercury drop electrode (HMDE) by means of alternating current voltammetry (AC voltammetry) in 0.3 M NaCl and 50 mM sodium phosphate buffer (pH 8.5) as supporting electrolyte. Its interaction with DNA is shown to be time dependent. Significant changes in the characteristic peaks of dsDNA were observed after addition of higher concentration of Cu(I) to a solution containing dsDNA, as a result of the interaction between Cu(I) and dsDNA. All the experimental results indicate that Cu(I) can bind to DNA by electrostatic binding and form an association complex.

Synthesis and Properties of DNA Oligomers Containing 2‘-Deoxynucleoside N-Oxide Derivatives

Cytosine and adenine N-oxide derivatives have long been known as products resulting from the oxidative damage of DNA by peroxides such as hydrogen peroxide. Although the synthesis and properties of 2'-deoxynucleoside N-oxide derivatives have been well described, little has been reported about the chemical and biochemical behavior of initially formed DNA oligomers containing these N-oxide bases. In this study, we established a convenient method for the solid-phase synthesis of oligodeoxynucleotides incorporating 2'-deoxycytidine N-oxide (dC O) or 2'-deoxyadenosine N-oxide (dA O) by using the postsynthetic oxidation of N-protected DNA oligomers except for the target dC or dA site with m-CPBA in MeOH in a highly selective manner. In this strategy, the benzoyl, phthaloyl, and (4-isopropylphenoxy)acetyl groups proved to serve as base protecting groups to avoid oxidation of adenine, cytosine, and guanine, respectively, at the unmodified sites.

Electrochemical studies on the oxidation of guanine and adenine at cyclodextrin modified electrodes

An electrochemical sensor for guanine and adenine using cyclodextrin-modified poly(N-acetylaniline) (PNAANI) on a carbon paste electrode has been developed. The oxidation mechanism of guanine and adenine on the surface of the electrode was investigated by cyclic voltammetry. It was found that the electrode processes are irreversible, pH dependent, and involve several reaction products. The electron transfer process occurs in consecutive steps with the formation of a strongly adsorbed intermediate on the electrode surface. Also, a new method for estimating the apparent formation constants of guanine and adenine with the immobilized cyclodextrins, through the change of surface coverage of studied analytes has been reported. Both guanine and adenine showed linear concentrations in the range of 0.1-10 microM by using differential pulse voltammetry, with an experimental limit of detection down to 0.05 microM. Linear concentration ranges of 2-150 microM for guanine and 6-104 microM for adenine have been found when cyclic voltammetry was used for determination of both analytes.

Pinecone shape hydroxypropyl-β-cyclodextrin on a film of multi-walled carbon nanotubes coated with gold particles for the simultaneous determination of tyrosine, guanine, adenine and thymine

A conductive composite film containing functionalized multi-walled carbon nanotubes ( fMWCNTs), gold nanoparticles (Au) with hydroxypropyl-β-cyclodextrin (HPβCD) as catalysts have been synthesized on glassy carbon, gold and on indium tin oxide electrodes by potentiostatic methods. The presence of fMWCNTs and HPβCD in the composite film enhances the active surface coverage concentration of Au by 397.0%. The presence of nanoparticles of gold catalyst in the film enhances the functional properties and produces an overall increase in the sensitivity of the modified electrodes. These modified electrodes exhibit promising electrocatalytic activity towards the oxidation of tyrosine (TYR), guanine (GU), adenine (AD) and thymine (THY) present in pH 7.4 aqueous solutions. Well separated voltammetric peaks are obtained between TYR and GU (80 mV), GU and AD (290 mV), AD and THY (185 mV) present in the analyte mixture. The sensitivity values of the composite films from cyclic voltammetry (CV) and semi derivative differential pulse voltammetry (DPV) show that, the composite film modified electrodes are efficient and they could be applied in biosensor devices. However, a detailed comparison between the sensitivities obtained using CV and semi derivative DPV shows that, the sensitivity obtained in semi derivative DPV technique is higher than CV. Electrochemical quartz crystal microbalance, scanning electrochemical microscope and scanning electron microscope techniques have been used for the electrochemical characterizations and surface morphology studies.

Oxidative DNA Damage Defense Systems in Avoidance of Stationary-Phase Mutagenesis in Pseudomonas putida

Oxidative damage of DNA is a source of mutation in living cells. Although all organisms have evolved mechanisms of defense against oxidative damage, little is known about these mechanisms in nonenteric bacteria, including pseudomonads. Here we have studied the involvement of oxidized guanine (GO) repair enzymes and DNA-protecting enzyme Dps in the avoidance of mutations in starving Pseudomonas putida. Additionally, we examined possible connections between the oxidative damage of DNA and involvement of the error-prone DNA polymerase (Pol)V homologue RulAB in stationary-phase mutagenesis in P. putida. Our results demonstrated that the GO repair enzymes MutY, MutM, and MutT are involved in the prevention of base substitution mutations in carbon-starved P. putida. Interestingly, the antimutator effect of MutT was dependent on the growth phase of bacteria. Although the lack of MutT caused a strong mutator phenotype under carbon starvation conditions for bacteria, only a twofold increased effect on the frequency of mutations was observed for growing bacteria. This indicates that MutT has a backup system which efficiently complements the absence of this enzyme in actively growing cells. The knockout of MutM affected only the spectrum of mutations but did not change mutation frequency. Dps is known to protect DNA from oxidative damage. We found that dps-defective P. putida cells were more sensitive to sudden exposure to hydrogen peroxide than wild-type cells. At the same time, the absence of Dps did not affect the accumulation of mutations in populations of starved bacteria. Thus, it is possible that the protective role of Dps becomes essential for genome integrity only when bacteria are exposed to exogenous agents that lead to oxidative DNA damage but not under physiological conditions. Introduction of the Y family DNA polymerase PolV homologue rulAB into P. putida increased the proportion of A-to-C and A-to-G base substitutions among mutations, which occurred under starvation conditions. Since PolV is known to perform translesion synthesis past damaged bases in DNA (e.g., some oxidized forms of adenine), our results may imply that adenine oxidation products are also an important source of mutation in starving bacteria.

Hole Transfer in DNA and Photosensitized DNA Damage: Importance of Adenine Oxidation

Photosensitized DNA damage reactions were investigated for two well-known DNA-damaging photosensitizers (Sens), naphthalimide (NI) and napthaldiimide (NDI), which have similar photophysical properties but differ in their redox properties. NI and NDI derivatives (NIN, NDIN), which have cationic side chains and electrostatically binding to DNA due to favorable electrostatic interactions between the negatively charged phosphate groups of DNA and cationic groups, and NIP and NDIP, which possess phosphate groups and do not bind to DNA, were synthesized. NIN and NDIN can oxidize A and G via their singlet excited state, and NDIP oxidizes A and G via its triplet excited state, whereas NIP oxidizes only G. A combination of laser flash photolysis kinetic studies and quantitative HPLC analyses of photosensitized DNA damage was performed for several DNA sequences in the presence of Sens. NIN, NDIN, and NDIP, which oxidizes A, caused significant DNA damage upon photoirradiation, and DNA damage yield increased with the length of the consecutive A stretch. In contrast, NIP, which oxidizes only G, caused only moderate damage to DNA and showed no preference for the consecutive A sequences. These results clearly demonstrate the importance of A-oxidation, especially in consecutive A sequences, which triggers the rapid hole transfer between A's.

Electrochemical behaviors of native and thermally denatured fish DNA in the presence of cytosine derivatives and porphyrin by cyclic voltammetry using boron-doped diamond electrode

The electrochemical behaviors of native and thermally denatured fish DNA was investigated using boron-doped diamond (BDD) film electrode by cyclic voltammetry. The BDD electrode afforded us to measure weak current less than μA for the DNA solution in 100 μl. The mixture of acetic acid and sodium acetate solution (0.2 M) was used as a supporting electrolyte. Two oxidation peaks were observed at about + 1.1 V and + 1.3 V at pH 4.6 for thermally denatured fish DNA. This is due to the oxidation of guanine and adenine in the denatured fish DNA, respectively. In contrast, the native fish DNA showed ill-defined peaks at + 1.1 V. Furthermore, the electrochemical behaviors of thermally denatured fish DNA were studied in the presence of cytosine, cytidine, cytidine-5-monophosphate, tetrakis(1-methypyridinium-4-yl)porphyrin (H 2(TMPyP) 4+) and Ru II(TMPyP) 4+. The oxidation peak intensity at + 1.1 V gradually decreased with the increase of the concentrations of the above compounds. Based on the above studies, electrochemical behaviors of the thermally denatured fish DNA at BDD electrode is discussed.

Electrochemical behaviour of 2,8-dihydroxyadenine at a glassy carbon electrode

The electrochemical behaviour of 2,8-dihydroxyadenine (2,8-DHA)– the main adenine oxidation product– has been investigated over a wide pH range at a glassy carbon electrode (GCE) using cyclic, differential pulse and square wave voltammetry. The oxidation of 2,8-DHA is a quasi-reversible process, pH dependent and occurs with the formation of a main oxidation product, P 2,8-DHA, that strongly adsorbs on the electrode surface. The reduction of 2,8-DHA also occurs and is a reversible process in the absence of molecular oxygen. In electrolytes with pH between 4 and 9 two consecutive reversible charge transfer reactions were identified. However, it was observed that O 2 interfered with the reductive electron transfer process of 2,8-DHA and that, in the presence of oxygen, the reduction of 2,8-DHA occurs at less negative potentials than in the absence of oxygen.

Graphite Composite Electrode in Voltammetry

Graphite composite electrode (CCE30), composed from 30% of graphite powder and 70% of epoxy resin, was tested. Determination of aminocompounds, oxidation of adenine and guanine, and determination of manganese by differential pulse voltammetry (DPV) and direct current voltammetry (DCV) was tested in positive potential area.

Rigid carbon composites: a new transducing material for label-free electrochemical genosensing

A rigid carbon–polymer composite material as a transducer for the electrochemical determination of label-free DNA based on differential pulse voltammetry (DPV) is reported. Graphite–epoxy composites (GEC) have an uneven surface allowing DNA, oligonucleotides and free DNA bases to be adsorbed using a simple and fast wet-adsorption procedure. In contrast with other transducers commonly used for electrochemical genosensing, the oxidation potentials are much lower when GEC is used. Free guanine base is oxidized at +0.35 V while adenine oxidation occurs at +0.63 V (vs Ag|AgCl). Cytosine and inosine free bases show no peaks within the experimental potential range. The oxidation of DNA guanine moieties occurs at a potential of +0.55 V while DNA adenine bases are oxidized at +0.85 V. A novel label-free hybridization genosensor using GEC as an electrochemical transducer for the specific detection of a sequence related with Salmonella spp. is also reported. This approach relies on the wet adsorption of the 23-mer inosine-substituted probe. The extent of hybridization onto the GEC surface between the probe and the target has been determined by using the oxidation signal of guanine coming from the target in connection with DPV. DNA hybridization has been determined in a target concentration of 10 μg/ml in 15 min of hybridization time. The hybridization event has also been detected in co-existing salmon testes DNA (stDNA) as interference. The features of this device are discussed and compared with state-of-the-art of label free DNA detection methods.

Oxidation of adenine and adenosine derivatives by dimethyldioxirane (DMDO) using halogenated metalloporphyrins as catalysts

In an effort to evaluate the reactivity of nucleobases with halogenated metalloporphyrins, adenine and adenosine derivatives were oxidized by dimethyldioxirane (DMDO) as oxygen atom donor using Mn[(Cl16)TDMPP]Cl and Mn[(Cl8)TDCPP]Cl porphyrins as catalysts. The role of hydrogen bonding interactions in the selectivity of the reaction was investigated through the oxidation of adenine and adenosine derivatives bearing hydrogen bond donors on the sugar moiety or on the N-9 side chain. This procedure is a useful synthetic tool for the selective C-8 versus N-1 oxidation of purine derivatives.

First principles calculations of the pKa values and tautomers of isoguanine and xanthine.

The accurate replication of DNA requires the formation of complementary hydrogen bonds between a template base and the base moiety of an incoming deoxynucleotide-5'-triphosphate. Recent structural studies suggest that some DNA polymerases contribute additional constraints by interrogating the minor groove face of the incoming and template bases. Therefore, the hydrogen bond-donating or -accepting properties of the base pairing as well as minor groove faces of the bases could be important determinants of correct base selection. In this paper, we investigate two purines that could arise by endogenous damage of the normal DNA bases: isoguanine (which can be generated by the oxidation of adenine) and xanthine (which can be generated by the deamination of guanine). In both cases, the potential exists for the placement of a proton in the N3 position, converting the N3 position from a hydrogen bond acceptor to a donor. In this paper, we use first principles quantum mechanical methods (density functional theory using the B3LYP functional and the 6-31G++Gbasis set) to predict the ionization and tautomeric equilibria of both isoguanine and xanthine in the gas phase and aqueous solution. For isoguanine, we find that the N1H and N3H neutral tautomeric forms are about equally populated in aqueous solution, while the enol tauotomers are predominant in the gas phase. In contrast, we find that xanthine displays essentially no tautomeric shifts in aqueous solution but is nearly equally populated by both an anionic and a neutral form at physiological pH. To obtain these results, we carried out an extensive examination of the tautomeric and ionic configurations for both xanthine and isoguanine in solution and in the gas phase. The potential hydrogen-bonding characteristics of these damaged purines may be used to test predictions of the important components of base selection by different DNA polymerases during DNA replication.

UV laser photolysis of DNA: effect of duplex stability on charge-transfer efficiency.

The distribution of the final base damage was determined within isolated DNA exposed to pulses of 266 nm laser light. Studied lesions included oxidation products arising from biphotonic ionization of DNA bases and pyrimidine dimeric photoproducts arising from monophotonic processes. The distribution of the latter class of damage was found to be correlated with the stability of the DNA duplex. The quantum yield for formation of 8-oxo-7,8-dihydroguanine was much higher than that of other oxidized nucleosides arising from the degradation of thymine and adenine. This observation, together with the shape of the intensity dependence curves, provided evidence for the occurrence of charge-transfer processes within DNA. In addition, increase in the ionic strength of the irradiated DNA and stabilization of the DNA duplex were found to induce a drastic decrease in the yield of thymine and adenine oxidation products. Concurrently, an increase in the yield of 8-oxo-7,8-dihydroguanine was observed. This was rationalized in terms of an increase in the overall charge-transfer efficiency. Therefore, it may be concluded that stabilization of the double-helix favors charge-transfer process toward guanine bases.

A novel colorimetric assay and activity staining for cytokinin oxidase based on the copper amine oxidase property of the enzyme.

A rapid and sensitive colorimetric assay for cytokinin oxidase (CKO) was developed based on the copper amine oxidase property of the enzyme from cucumber cotyledons. The assay involved oxidation of isopentenyl adenine by CKO in the presence of phenazine methosulphate as an electron acceptor, resulting in reduction of nitroblue tetrazolium to formazon. The formation of formazon was linear with time for at least 20 min and could be followed spectrophotometrically at A490. CKO could also be specifically stained on polyacrylamide gels using the same principle.

Simultaneous determination of guanine and adenine contents in DNA, RNA and synthetic oligonucleotides using a chemically modified electrode

Simultaneous determination of guanine and adenine contents in DNA, RNA and synthetic oligonucleotides on a Nafion–ruthenium oxide pyrochlore modified electrode is described. This chemically modified electrode shows very pronounced electrocatalytic effects towards the oxidation of guanine and adenine. The experimental parameters for individual determinations of guanine and adenine on the modified electrode were first optimised. Excellent detection limits (S/N = 3) of 0.86 ng ml–1 (5.7 nM) and 2.7 ng ml–1 (20 nM) for guanine and adenine, respectively, were obtained. Simultaneous determination of guanine and adenine in a mixture was also optimised. The denatured calf thymus DNA and yeast RNA showed oxidation peaks corresponding to guanine and adenine with a linear calibration line up to 5 µg ml–1 in pH 4.0 phosphate buffer. The detection limit estimated for these nucleic acids were 11.5 ng ml–1 and 37.7 ng ml–1 for guanine and adenine, respectively. The new method provides an alternative to estimate the guanine and adenine contents more selectively and sensitively than currently applied techniques.

Determination of oxidative DNA base damage by gas chromatography-mass spectrometry. Effect of derivatization conditions on artifactual formation of certain base oxidation products

GC-MS is a widely used tool to measure oxidative DNA damage because of its ability to identify a wide range of base modification products. However, it has been suggested that the derivatization procedures required to form volatile products prior to GC-MS analysis can sometimes produce artifactual formation of certain base oxidation products, although these studies did not replicate previously-used reaction conditions, e.g. they failed to remove air from the derivatization vials. A systematic examination of this problem revealed that levels of 8-hydroxyguanine, 8-hydroxyadenine,5-hydroxycytosine and 5-(hydroxymethyluracil) in commercial calf thymus DNA determined by GC-MS are elevated by increasing the temperature at which derivatization is performed in our laboratory. In particular, 8-hydroxyguanine levels after silylation at 140°C were raised 8-fold compared to derivatization at 23°C. Experiments on the derivatization of each undamaged base revealed that the artifactual oxidation of guanine, adenine, cytosine and thymine respectively was responsible. Formation of the above products was potentiated by not purging with nitrogen prior to derivatization. Increasing the temperature to 140°C or allowing air to be present during derivatization did not significantly increase levels of the other oxidized bases measured.This work suggests that artifactual oxidation during derivatization is restricted to certain products (8-hydroxyguanine, 8-hydroxyadenine, 5-hydroxycytosine and 5-[hydroxymethyluracil]) and can be decreased by reducing the temperature of the derivatization reaction to 23°C and excluding as much air possible. Despite some recent reports, we were easily able to detect formamidopyrimidines in acid-hydrolyzed DNA. Artifacts of derivatization are less marked than has been claimed in some papers and may vary between laboratories, depending on the experimental procedures used, in particular the efficiency of exclusion of O2 during the derivatization process.

ChemInform Abstract: N‐1 Oxidation of Adenine Substituted at N‐9 by an Olefinic Chain.

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N-1 Oxidation of Adenine Substituted at N-9 by an Olefinic Chain

N-1 Oxidation of Adenine Substituted at N-9 by an Olefinic Chain

Oxidation chemistry of adenine and hydroxyadenines at pyrolytic graphite electrodes

The electrochemical oxidation of adenine and hydroxyadenines has been studied in aqueous solutions in the pH range 3.0–11.2 using a pyrolytic graphite electrode. The initial course of the electrode reaction has been deduced to involve a 2e, 2H+ reaction to give 2- and not 8-hydroxyadenine, further oxidation of which gives 2,8-dihydroxyadenine and then diimine species which undergo a series of chemical reactions to give different products. The major products of oxidation at pH 3.0 {urea, alloxan [2,4,5,6(1H,3H)-pyrimidinetetrone] and parabanic acid (imidazolidinetrione)} and at pH 7.0 [allantoin (5-ureidohydantoin)] have been isolated using HPLC and column chromatography and their structures elucidated by spectroscopic techniques. The probable EC mechanisms for their formation have also been suggested.

Gas chromatographic-mass spectrometric determination of isotopic enrichment of 6- 15NH 2 in adenine nucleotides

A gas chromatographic-mass spectrometric method for the determination of isotopic abundance in [6- 15NH 2]adenine nucleotides is described. The method involves formation of the di-t-butyldimethylsilyl (TBDMS) derivative of adenine following isolation of the nucleotide fraction with solid-phase ion-exchange chromatography and subsequent acid hydrolysis of nucleotides to free base. Mass spectra for both adenine-diTBDMS and [6- 15NH 2]adenine-diTBDMS were obtained to identify those ions containing the 6-NH 2 moiety. The base peak ( m z 306) was formed by loss of C 4H 9 (57) and constitutes approximately one-third of the total ion current. Using selected ion monitoring of the m z 306/ m/z 307 ratio, levels of isotopic abundance of 1.0–50.0 mol% excess could be measured reproducibly with the injection of 10–20 pmol of the adenine-diTBDMS derivative obtained from isolated rat hepatocytes. Confirmation that measured isotopic abundance was referable to labeling of the 6- 15NH 2 group was obtained by oxidation of adenine to hypoxanthine and determination of enrichment in the hypoxanthine-diTBDMS derivative. The method was used to study the formation of [6- 15NH 2]adenine nucleotides during the incubation of isolated rat hepatocytes with [ 15N]alanine. A level of approximately 6.0 mol% excess was observed at 60 min incubation.