Redox Couple of DNA on Multiwalled Carbon Nanotube Modified Electrode

It has been envisioned that carbon nanotubes could promote electron‐transfer reactions when used as electrode materials in electrochemical cells. In the present study, calf thymus DNA was electrochemically oxidized at an electrode modified with multiwalled carbon nanotubes. The potentials for DNA oxidation at pH 7.0 were found to be 0.71 and 0.81 V versus SCE, corresponding to the oxidation of guanine and adenine residues, respectively. An initial oxidation of adenine was observed in the first scan, which was followed by a quasi‐reversible redox process of the oxidation product in the subsequent scans.     

Electrochemical oxidation of underivatized-nucleic acids at highly boron-doped diamond electrodes

Boron-doped diamond (BDD) electrodes have been examined for the electrochemical oxidation of underivatized-nucleic acids in terms of single stranded and double stranded DNA. Cyclic voltammetry and square wave voltammetry have been used to study the oxidation reactions and to detect DNA without derivatization or hydrolysis steps. At the diamond electrode, at least two well-defined voltammetric peaks were observed for both single stranded and double stranded DNA. Diamond electrode is the first material to show a well-defined voltammetric peaks for adenine group oxidation directly in the helix structure of nucleic acid due to its wide potential window. For single stranded DNA, a third peak, related to the pyrimidine group oxidation was also observed. As-deposited diamond film with predominantly hydrogen-terminated surface exhibited superior performance over oxygen-terminated diamond in terms of sensitivity. However, by optimizing the ionic strength, sensitivity of O-terminated films could be improved. Linear calibration results have shown linearity of current with concentration in the range 0.1-8 microg mL(-1) for both guanine and adenine residues at as-deposited BDD. Detection limits (S/N = 3) of 3.7 and 10 ng mL(-1) for adenine and guanine residue in single stranded DNA, respectively, and 5.2 and 10 ng mL(-1) for adenine and guanine residue in double stranded DNA, respectively, were observed. This work shows the promising use of diamond as an electrochemical detector for direct detection of nucleic acids. The results also show the possibility of using the oxidation peak current of adenine group that is more sensitive for the direct detection of nucleicacids.     

Exocyclic carbons adjacent to the N6 of adenine are targets for oxidation by the Escherichia coli adaptive response protein AlkB

The DNA and RNA repair protein AlkB removes alkyl groups from nucleic acids by a unique iron- and α-ketoglutarate-dependent oxidation strategy. When alkylated adenines are used as AlkB targets, earlier work suggests that the initial target of oxidation can be the alkyl carbon adjacent to N1. Such may be the case with ethano-adenine (EA), a DNA adduct formed by an important anticancer drug, BCNU, whereby an initial oxidation would occur at the carbon adjacent to N1. In a previous study, several intermediates were observed suggesting a pathway involving adduct restructuring to a form that would not hinder replication, which would match biological data showing that AlkB almost completely reverses EA toxicity in vivo. The present study uses more sensitive spectroscopic methodology to reveal the complete conversion of EA to adenine; the nature of observed additional putative intermediates indicates that AlkB conducts a second oxidation event in order to release the two-carbon unit completely. The second oxidation event occurs at the exocyclic carbon adjacent to the N(6) atom of adenine. The observation of oxidation of a carbon at N(6) in EA prompted us to evaluate N(6)-methyladenine (m6A), an important epigenetic signal for DNA replication and many other cellular processes, as an AlkB substrate in DNA. Here we show that m6A is indeed a substrate for AlkB and that it is converted to adenine via its 6-hydroxymethyl derivative. The observation that AlkB can demethylate m6A in vitro suggests a role for AlkB in regulation of important cellular functions in vivo.     

DNA‐Directed Attachment of Carbon Nanotubes for Enhanced Label‐Free Electrochemical Detection of DNA Hybridization

Multi‐walled carbon nanotubes (MWNTs) were used as nanowires, which combined DNA molecules to a carbon paste electrode (CPE). The attachment of MWNT on the electrode surface was controlled by a hybridization assay between adenine and thymine containing oligonucleotides. The appearance of guanine oxidation signal after hybridization with target DNA greatly simplified the specific sequence DNA detection mechanism. Combination of sidewall‐ and end‐functionalization of MWNT provided a significant enhancement in the voltammetric signal of guanine oxidation in comparison with the signals obtained from only end‐oxidized MWNT modified CPE and a bare CPE. A control experiment involving adenine containing polynucleotide (poly(A)) instead of adenine probe modified MWNT was performed. The effect of target and noncomplementary DNA concentration on the guanine signal was also monitored. Discrimination against single‐base mismatch and noncomplementary DNA was achieved by surfactant containing washing solution. The promising conductivity of carbon nanotubes, and the creation of a larger surface area for DNA immobilization by sidewall‐ and end‐oxidation of MWNT provided a detection limit down to 10 pg/mL, which is compatible with the demand of the genetic tests.     

Direct electrochemistry of DNA,guanine and adenine at a nanostructured film-modified electrode

A nanostructured film electrode, a multi-wall carbon nanotubes (MWNT)-modified glassy carbon electrode (GCE), is described for the simultaneous determination of guanine and adenine. The properties of the MWNT-modified GCE were investigated by scanning electron microscopy (SEM) and cyclic voltammetry. The oxidation peak currents of guanine and adenine increased significantly at the MWNT-modified GCE in contrast to those at the bare GCE. The experimental parameters were optimized and a direct electrochemical method for the simultaneous determination of guanine and adenine was proposed. Using the MWNT-modified GCE, a sensitive and direct electrochemical technique for the measurement of native DNA was also developed, and the value of (G+C)/(A+T) of HCl-digested DNA was detected.     

Simultaneous determination of guanine and adenine in DNA using an electrochemically pretreated glassy carbon electrode

A simple and reliable method based on adsorptive stripping at an electrochemically pretreated glassy carbon electrode (GCE) was proposed for simultaneous or individual determination of guanine and adenine in DNA. The detection sensitivity of guanine and adenine was improved greatly by activating the GCE electrochemically. After accumulation on pretreated GCE at open circuit for 5 min or at the potential of +0.3 V for 120 s, guanine and adenine produced well-defined oxidation peaks at about +0.8 and +1.1 V, respectively in pH 5 phosphate buffer. The detection limit for individual measurement of guanine and adenine was 4.5 ng ml⁻¹ (3×10⁻⁸ mol l⁻¹) and 4 ng ml⁻¹ (3×10⁻⁸ mol l⁻¹), respectively. Acid-denatured DNA showed two oxidation peaks corresponding to guanine and adenine residues in the same buffer. The proposed method can be used to estimate the guanine and adenine contents in DNA with good selectivity in a linear range of 0.25-5 μg ml⁻¹.     

Electrochemical behavior of natural and biosynthetic polynucleotides at the pyrolytic graphite electrode A new probe for studies of polynucleotide structure and reactions

The electrochemical oxidation of natural and biosynthetic polynucleotides at a pyrolytic graphite electrode (PGE) has been studied under differential pulse voltammetric conditions. Denatured DNA, ribosomal and transfer RNA give two voltammetric peaks. The first (more negative peak, peak G) corresponds to electrochemical oxidation of guanine residues where-as the second, more positive peak (peak A) corresponds to electrochemical oxidation of adenine residues. Native DNA gives rise only to a small peak A, peak G being totally absent. Denatured DNA and its voltammetric oxidation product are both strongly adsorbed at the PGE. Differential pulse voltammetric oxidation of natural and biosynthetic polynucleotides may provide a valuable technique for probing A-T and G-C regions during structural and conformational changes of these molecules and for following their interactions with other solution species.     

基于BCNTs/GC电极的鸟嘌呤与腺嘌呤电化学行为及其同时检测

利用掺硼碳纳米管（BCNTs）/GC电极研究了鸟嘌呤（G）和腺嘌呤（A）的电化学氧化行为. 与GC和CNTs/GC电极相比,BCNTs/GC电极具有更强的电催化活性,且响应电流明显增加. 两混合样品在BCNTs/GC电极上的氧化峰间隔较大,可实现对A和G的同时检测.     

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.     

In situ evaluation of heavy metal-DNA interactions using an electrochemical DNA biosensor

Heavy metal ions, lead, cadmium and nickel, are well known carcinogens with natural different origins and their direct mode of action is still not fully understood. A dsDNA-electrochemical biosensor, employing differential pulse voltammetry, was used for the in situ evaluation of Pb2+, Cd2+ and Ni2+ interaction with dsDNA. The results confirm that Pb2+, Cd2+ and Ni2+ bind to dsDNA, and that this interaction leads to different modifications in the dsDNA structure. These modifications were electrochemically recognized as changes in the oxidation peaks of guanosine and adenosine bases. Using homopolynucleotides of guanine and adenine it has been proved that the interaction between Pb2+ and DNA causes oxidative damage and preferentially takes place at adenine-containing segments, with the formation of 2,8-dihydroxyadenine, the oxidation product of adenine residues and a biomarker of DNA oxidative damage. The Pb2+ bound to dsDNA can still undergo oxidation. The interaction of Cd2+ and Ni2+ causes conformational changes, destabilizing the double helix, which can enable the action of other oxidative agents on DNA.     

Novel electrochemical sensor based on functionalized graphene for simultaneous determination of adenine and guanine in DNA

A nano-material carboxylic acid functionalized graphene (graphene-COOH) was prepared and used to construct a novel biosensor for the simultaneous detection of adenine and guanine. The direct electrooxidation behaviors of adenine and guanine on the graphene-COOH modified glassy carbon electrode (graphene-COOH/GCE) were carefully investigated by cyclic voltammetry and differential pulse voltammetry. The results indicated that both adenine and guanine showed the increase of the oxidation peak currents with the negative shift of the oxidation peak potentials in contrast to that on the bare glassy carbon electrode. The electrochemical parameters of adenine and guanine on the graphene-COOH/GCE were calculated and a simple and reliable electroanalytical method was developed for the detection of adenine and guanine, respectively. The modified electrode exhibited good behaviors in the simultaneous detection of adenine and guanine with the peak separation as 0.334V. The detection limit for individual determination of guanine and adenine was 5.0×10(-8)M and 2.5×10(-8)M (S/N=3), respectively. Furthermore, the measurements of thermally denatured single-stranded DNA were carried out and the value of (G+C)/(A+T) of single-stranded DNA was calculated as 0.80. The biosensor exhibited some advantages, such as simplicity, rapidity, high sensitivity, good reproducibility and long-term stability.     

An Electrochemical Sensor Based on Gold Nanoparticles Incorporated in Mesoporous MFI Zeolite for Determination of Purine Bases in DNA

An electrochemical sensor was developed based on gold nanoparticles incorporated in mesoporous MFI zeolite for the determination of purine bases. Au nanoparticles (AuNPs) were incorporated into the mesoporous MFI zeolite (AuNPs/m‐MFI) by post‐grafting reaction. The composite materials were characterized by transmission electron microscopy (TEM), X‐ray photoelectron spectroscopy (XPS) and electrochemical methods. Au nanoparticles with a size of 5‐20 nm are uniformly dispersed in the pores of mesoporous MFI zeolite. And the morphology of MFI zeolite can be perfectly kept after pore expansion and Au nanoparticles incorporation. The electrocatalytic oxidation of purine bases (guanine and adenine in DNA) is investigated by cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The surface‐confined Au nanoparticles provide the good catalytic activity for oxidation of purine bases. The simultaneous detection of guanine and adenine can be achieved at AuNPs/m‐MFI composites modified glassy carbon electrode (GCE). The electrochemical sensor based on AuNPs/m‐MFI exhibits wide linear range of 0.5–500 μM and 0.8–500 μM with detection limit of 0.25 and 0.29 μM for guanine and adenine, respectively. Moreover, the electrochemical sensor is applied to evaluation of guanine and adenine in herring sperm DNA samples with satisfactory results.     

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 muA for the DNA solution in 100 microl. 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.     

Oxidation of DNA Bases Influenced by the Presence of Other Bases

Electrochemical detection of DNA is a highly important topic. Here we show that the electrochemical responses of one DNA base (guanine, adenine, cytosine or thymine), in terms of oxidation potential, current intensity, peak width and resolution can be highly influenced by the presence of other DNA bases at electrochemically reduced graphene oxide (ER‐GO) as well as standard glassy carbon electrode. We have observed that the effects were more significant for adenine base on ER‐GO and cytosine base on glassy carbon (GC) electrode. Differences in responses were generally low in a mixture of four different DNA bases but interestingly, deviations become significantly larger when only one or two other bases were present. Our findings are of paramount importance for future developments in DNA detection and analysis since individual DNA bases are not present in isolation in nature or in typical biosensing systems.     

Adsorption and oxidation of purine bases and their derivatives on electrodes modified with carbon nanotubes

The electrochemical behavior of nitrogen bases and their derivatives is studied on electrodes modified with carbon nanotubes. On these electrodes, the strong adsorption of purines and their oxidation is observed at potentials in the vicinity of +0.8 V for guanine and +1.0 V (vs. Ag/AgCl) for guanine nucleosides/nucleotides and adenine. At more positive potentials, the high background current prevents the detection of adenine nucleotides and pyrimidines. The peculiarities of oxidation of the most easily detectable DNA components, namely, guanine and deoxyguanosinemonophosphate, on modified electrodes are elucidated and the corresponding reaction scheme is proposed. The results can be used in the development of biosensors based on electroactive properties of nucleic acids and their components.     

Voltammetric sensing of guanine and adenine using a glassy carbon electrode modified with a tetraoxocalix[2]arene[2]triazine Langmuir-Blodgett film

We have prepared a new voltammetric sensor for guanine and adenine. It is based on a glassy carbon electrode modified with a Langmuir-Blodgett film made from tetraoxocalix[2]arene[2]triazine. The direct electro-oxidation of adenine and guanine was investigated and the results indicat that in contrast to a bare glassy carbon electrode both guanine and adenine cause an increase in the oxidation peak currents along with a negative shift of the oxidation potentials. The electrode enables the simultaneous determination of guanine and adenine using square wave voltammetry. Analysis of acid denatured calf thymus DNA was carried out and the value of (G + C)/(A + T) was correctly found to be 0.75.

Electrochemical oxidation of guanosine and adenosine: Two convergent pathways

We herein explore a novel route for oxidative stress in DNA by using electrochemistry as mimicking tool. Essentially, the electrochemical oxidation of guanine and adenine nucleosides and oligo-homo-nucleotides at pyrolytic graphite electrodes in neutral and alkaline aqueous solutions was studied. Under these experimental conditions all these compounds give rise to an adsorbed product not previously described, which was electrochemically and kinetically characterized. The virtually identical kinetic and electrochemical features exhibited by the oxidized compounds generated from all precursors strongly suggest a common species arising from both adenine and guanine derivatives. Supported by DFT calculations, we propose two convergent pathways for the electrochemical oxidation of adenosine and guanosine. Those calculations indicate that the common oxidized base product forms stable H-bond base pairs with both thymine and cytosine.     

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.     

In situ infrared spectroelectrochemical studies on adsorption and oxidation of nucleic acids at glassy carbon electrode

The adsorption and oxidation of yeast RNA and herring sperm DNA (HS DNA) at glass carbon (GC) electrode are studied by differential pulse voltammetry (DPV) and in situ FTIR spectroelectrochemistry. Two oxidation peaks of yeast RNA are obtained by DPV, whose peak potentials shift negatively with increasing pH. The peak currents decrease gradually in successive scans and no corresponding reduction peaks occur, thus indicating that the oxidation process of yeast RNA is completely irreversible. The IR bands in the 1200-1800 cm(-1) range, attributed to the stretching and ring vibrations of nucleic acid bases, show the main spectral changes when the potential is shifted positively, which gives evidence that the oxidation process takes place in the base residues. The oxidation process of HS DNA is similar to that of yeast RNA. The results both from DPV and in situ FTIR spectroelectrochemistry confirm that the guanine and adenine residues can be oxidized at the electrode surface, which is consistent with the oxidation mechanism of nucleic acids proposed previously.     

Studies on Model Interaction of Keggin-type Polyoxometalates with Nucleic Acid

Keggin anions behave differently from each other when they react with nucleic acids. The molybdenum series exhibits oxidative cleavage activity towards AMP and DNA. The mechanism of AMP damage and DNA cleavage caused by the molybdenum series is mainly oxidation and the oxidation sites are on the ribose parts other than on the adenine parts, while the hydrolysis probably makes significant contributions to the cleavage of DNA and to the damage of AMP caused by the tungsten series.