Analysis of DNA adducts of acetaldehyde by liquid chromatography-mass spectrometry

A highly sensitive method using liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) was developed for the analysis of DNA adducts of acetaldehyde (AA). AA, which is the primary oxidative metabolite of ethanol, is considered to possess carcinogenic activity. AA reacts with the exocyclic amino group of guanine in DNA to form N2-ethylguanine (Et-Gua) and 1,N2-propanoguanine (Pr-Gua) adducts. With the present method, such adducts were detected as the base forms from DNA chains using depurination in the pretreatment process. In our measurement with LC-ESI-MS, the limits of detection (LODs) of the Et-Gua and Pr-Gua adducts of the base forms were 3.0 x 10(-10) and 1.0 x 10(-9) M, respectively, and the LODs are about two orders of magnitude lower than those of the nucleoside forms. Calf thymus DNA samples treated with AA and NaBH3CN were analyzed by this method. Et-Gua was clearly detected and, in the absence of NaBH3CN, Pr-Gua was detected predominantly. Furthermore, the method was also applied to study whether or not these two adducts are formed in DNA of cultured HL-60 cells during exposure to AA for 24 h. Pr-Gua was clearly detected and traces of Et-Gua were also detected in the DNA of the cells. Although the sensitivity of this method is lower by at least oneorder of magnitude than the 32P-postlabeling assay, currently the most sensitive method, our method does not involve complex enzymatic reactions for the postlabeling and the use of troublesome radioactive materials. Furthermore, it enables structural identification of guanine adducts. The present method would be a useful tool for studies of Et-Gua and Pr-Gua adducts in connection with carcinogenesis.     

Thermochemical properties of some vinyl chloride-induced DNA lesions: detailed view from NBO & AIM analysis

Etheno-damaged DNA adducts such as 3,N ⁴-ethenocytosine, N ²,3-ethenoguanine, and 1,N ²-ethenoguanine are associated with carcinogenesis and cell death. These inevitable damages are counteracted by glycosylase enzymes, which cleave damaged nucleobases from DNA. Escherichia coli alkyl purine DNA glycosylase is the enzyme responsible for excising damaged etheno adducts from DNA in humans. In an effort to understand the intrinsic properties of these molecules, we examined gas-phase acidity values and proton affinities (PA) of multiple sites of these molecules as well as equilibrium tautomerization and base pairing properties by quantum mechanical calculations. We also used calculations to compare the acidic and basic properties of these etheno adduct with those of the normal bases—cytosine and guanine nucleobases. We hypothesize that alkyl DNA glycosylase may cleave certain damaged nucleobases as anions and that the active site may take advantage of a nonpolar environment to favor deprotonated cytosine or guanine as a leaving group versus damaged nucleobases.     

A quantum chemical study of reactions of DNA bases with sulphur mustard: a chemical warfare agent

Reactions of the sulphonium ion of sulphur mustard (SM⁺¹) at the N7, N3 and O6 sites of guanine, N7, N3 and N1 sites of adenine, O2 and N3 sites of cytosine and O2 and O4 sites of thymine were studied theoretically in gas phase and aqueous media employing density functional theory (DFT) and second order Møller–Plesset perturbation (MP2) theory. The B3LYP, B3PW91 and B1B95 functionals of DFT and the 6-31+G* and AUG-cc-pVDZ basis sets were used in the calculations. Basis set superposition error was treated using the counterpoise method by single point energy calculations at the B3LYP/6-31+G* level in gas phase. The present study explains the mechanism of alkylation of the DNA bases and shows that SM⁺¹ would form stable adducts at the endocyclic nitrogen sites of the DNA bases, and at the O6 site of guanine and the O2 site of cytosine. Formation of adducts at the N7 site of guanine and N3 site of adenine are found to be most favored and next most favored respectively, which agrees with experimental observations.     

Synthesis of fluorescently labeled alkylated DNA adduct standards and separation by capillary electrophoresis

DNA adducts are regarded as individual internal dosimeters for the exposure to chemical carcinogens. To date, the most sensitive method for DNA adduct analysis is the radioactive 32P-postlabeling method, which allows the detection of one adduct in 10(10) unmodified nucleotides in microg amounts of DNA. However, this technique suffers from disadvantages such as working with radioactive phosphorus and time-consuming chromatographic separation procedures. In addition, the simultaneous detection of adducts from different classes of carcinogens in a DNA sample is difficult. In order to overcome these drawbacks, we are developing a new detection method, comprising fluorescence labeling of DNA adducts, capillary electrophoretic (CE) separation, and on-line detection by monitoring laser-induced fluorescence (LIF). So far, we have evaluated the separation power and the detection limit of CE with fluorescently labeled standard compounds such as unmodified nucleotides or alkylated thymidines. For this purpose, we developed a universal method for labeling 5'-OH-mononucleosid-3'-dicyanoethyl-phosphates with fluorescent dyes based on the phosphoramidite technology for DNA synthesis. The separation of N3-methylated, N3-, O2- and O4-butylated thymidines from the unmodified nucleotide within a few minutes recommends CE-LIF as a powerful method for DNA adduct analysis.     

Acidity and proton affinity of hypoxanthine in the gas phase versus in solution: intrinsic reactivity and biological implications

Hypoxanthine is a mutagenic purine base that most commonly arises from the oxidative deamination of adenine. Damaged bases such as hypoxanthine are associated with carcinogenesis and cell death. This inevitable damage is counteracted by glycosylase enzymes, which cleave damaged bases from DNA. Alkyladenine DNA glycosylase (AAG) is the enzyme responsible for excising hypoxanthine from DNA in humans. In an effort to understand the intrinsic properties of hypoxanthine, we examined the gas-phase acidity and proton affinity using quantum mechanical calculations and gas-phase mass spectrometric experimental methods. In this work, we establish that the most acidic site of hypoxanthine has a gas-phase acidity of 332 +/- 2 kcal mol-1, which is more acidic than hydrochloric acid. We also bracket a less acidic site of hypoxanthine at 368 +/- 3 kcal mol-1. We measure the proton affinity of the most basic site of hypoxanthine to be 222 +/- 3 kcal mol-1. DFT calculations of these values are consistent with the experimental data. We also use calculations to compare the acidic and basic properties of hypoxanthine with those of the normal bases adenine and guanine. We find that the N9-H of hypoxanthine is more acidic than that of adenine and guanine, pointing to a way that AAG could discriminate damaged bases from normal bases. We hypothesize that AAG may cleave certain damaged nucleobases as anions and that the active site may take advantage of a nonpolar environment to favor deprotonated hypoxanthine as a leaving group versus deprotonated adenine or guanine. We also show that an alternate mechanism involving preprotonation of hypoxanthine is energetically less attractive, because the proton affinity of hypoxanthine is less than that of adenine and guanine. Last, we compare the acidity in the gas phase versus that in solution and find that a nonpolar environment enhances the differences in acidity among hypoxanthine, adenine, and guanine.     

Simultaneous determination of purine bases, ribonucleosides and ribonucleotides by capillary electrophoresis-electrochemistry with a copper electrode

Simultaneous determination of purine bases, ribonucleosides and ribonucleotides was achieved by coupling capillary electrophoresis (CE) with wall-jet amperometric detection. A 200 μm diameter copper disk electrode was applied at working potential, +0.65 V vs. saturated calomel electrode. The current response of high sensitivity and stability was obtained in strong basic solutions which were suitable for satisfactory CE separations. The calibration curve was linear over 2–3 orders of magnitude and the limits of detection for adenine, guanine, xanthine, uric acid, adenosine, guanosine, adenosine-5′-monophosphate and guanosine-5′-monophosphate were below 9 fmol (S/N=3). The use of this method for the separation and detection of compounds present in human plasma samples was reported.     

Differentiation of isomeric C8-substituted alkylaniline adducts of guanine by electrospray ionization and tandem quadrupole ion trap mass spectrometry

Product ion spectra from thirteen C8-substituted alkylaniline adducts of guanine and deoxyguanosine were generated using electrospray ionization and quadrupole ion trap mass spectrometry and studied to investigate the possibility of differentiating isomeric adduct structures based upon the relative abundances of fragment ions derived from the alkylaniline-modified guanine bases (BH₂⁺ ions). The structural discrimination of the BH₂⁺ ions formed by attachment of isomeric alkylanilines to the C8 position of guanine is a challenging problem because the ions tend to yield product ion spectra that are qualitatively identical upon collisional activation. In this study, a statistical method, referred to as a similarity index, was used to compare the product ion spectra of isomeric BH₂⁺ ions and differentiate their structures. All the adducts investigated could be distinguished from SIs calculated using 5–6 product ions. These results suggest that a searchable database of product ion spectra may be created and used to characterize DNA adducts from aromatic amines whenever they are detected at levels amenable to mass spectral analysis.     

DEB-FAPy-dG Adducts of 1,3-Butadiene: Synthesis,Structural Characterization,and Formation in 1,2,3,4-Diepoxybutane Treated DNA**

Metabolic activation of the human carcinogen 1,3-butadiene (BD) by cytochrome 450 monooxygenases gives rise to a genotoxic diepoxide, 1,2,3,4-diepoxybutane (DEB). This reactive electrophile alkylates guanine bases in DNA to produce N7-(2-hydroxy-3,4-epoxy-1-yl)-dG (N7-DE-dG) adducts. Because of the positive charge at the N7 position of the purine heterocycle, N7-DEB-dG adducts are inherently unstable and can undergo spontaneous depurination or base-catalyzed imidazole ring opening to give N⁶-[2-deoxy-D-erythro-pentofuranosyl]-2,6-diamino-3,4-dihydro-4-oxo-5-N-1-(oxiran-2-yl)propan-1-ol-formamidopyrimidine (DEB-FAPy-dG) adducts. Here we report the first synthesis and structural characterization of DEB-FAPy-dG adducts. Authentic standards of DEB-FAPy-dG and its ¹⁵N₃-labeled analogue were used for the development of a quantitative nanoLC-ESI⁺-HRMS/MS method, allowing for adduct detection in DEB-treated calf thymus DNA. DEB-FAPy-dG formation in DNA was dependent on DEB concentration and pH, with higher numbers observed under alkaline conditions.     

Handling and detection of 25 amol of near-infrared dye deoxynucleotide conjugates by capillary electrophoresis with laser-induced fluorescence detection

Near-infrared dyes are attractive as labeling reagents to enhance sensitivity in trace analysis largely because background fluorescence is low in this spectral region. Here we demonstrate, towards a goal of detecting DNA adducts in small biological samples, that some near-infrared (IR) dye-labeled deoxynucleotides can be separated and detected with high sensitivity by capillary electrophoresis (CE)-laser-induced fluorescence detection (LIF) in a realistic way (handling detection limit of 25 amol) for near-IR dye-labeled deoxynucleotides. This detection limit is achieved by polarity-switching injection of 2.0 microl from a volume of 5.0 microl, in which the compounds are 5 x 10(-12) mol/l in 50% aqueous methanol. Although the adenine and cytosine-containing conjugates co-migrated, the other three (guanine, N2-ethylguanine and thymine) were resolved.     

Carbon fiber nanoelectrodes applied to microchip electrophoresis amperometric detection of neurotransmitter dopamine in rat pheochromocytoma (PC12) cells

Microelectrodes have been adopted in electrochemical detection for CE or microchip CE in recent years. In this paper, the use of nanoelectrodes (with tip diameter of 100-300 nm) as the electrochemical detector in microchip CE is firstly reported. The experimental results indicated that both the sensitivity and resolution of microchip CE with the carbon fiber nanoelectrode (CFNE) amperometric detection have been improved markedly comparing with the traditional microelectrodes. The detection limit of dopamine (S/N = 3) is 5.9x10(-8) M, which is one or two orders of magnitude lower than that reported so far, and the resolution of dopamine (DA) and isoprenaline (IP) has also improved from 0.6 (using 7 mum carbon fiber microelectrodes, CFME) to 1.0. We assembled a novel and easily operated microchip CE system with end-column amperometric detection, which allows the convenient and fast replacement of the passivated electrodes. Under the optimized condition, the RSDs of peak height and migration time are 1.47 and 0.31%, respectively (n = 40), indicating that the system displays excellent reproducibility. The nanoelectrode-based microchip CE system has been successfully applied to the determination of DA in cultured rat pheochromocytoma (PC12) cells, and the average content of DA in an individual PC12 cell is 0.54 +/- 0.07 fmol, which is in good agreement with that reported in the literature.     

Nanopore detection of 8-oxo-7,8-dihydro-2'-deoxyguanosine in immobilized single-stranded DNA via adduct formation to the DNA damage site

The ability to detect DNA damage within the context of the surrounding sequence is an important goal in medical diagnosis and therapies, but there are no satisfactory methods available to detect a damaged base while providing sequence information. One of the most common base lesions is 8-oxo-7,8-dihydroguanine, which occurs during oxidation of guanine. In the work presented here, we demonstrate the detection of a single oxidative damage site using ion channel nanopore methods employing α-hemolysin. Hydantoin lesions produced from further oxidation of 8-oxo-7,8-dihydroguanine, as well as spirocyclic adducts produced from covalently attaching a primary amine to the spiroiminodihydantoin lesion, were detected by tethering the damaged DNA to streptavidin via a biotin linkage and capturing the DNA inside an α-hemolysin ion channel. Spirocyclic adducts, in both homo- and heteropolymer background single-stranded DNA sequences, produced current blockage levels differing by almost 10% from those of native base current blockage levels. These preliminary studies show the applicability of ion channel recordings not only for DNA sequencing, which has recently received much attention, but also for detecting DNA damage, which will be an important component to any sequencing efforts.     

Inter- and intrastrand DNA crosslinks by 2-fluoro-substituted pyrrolobenzodiazepine dimers: stability, stereochemistry and drug orientation

A 2-fluoro-substituted pyrrolo[2,1-c][1,4]benzodiazepine (PBD) dimer with a 1,4-di-n-propyl piperazine linker was studied with respect to its binding and crosslinking capability towards double-helical DNA targets. Duplex thermal stabilizations upon drug binding as measured by UV melting experiments suggest that two guanine bases separated by four AT base pairs constitute the favorable binding site for the PBD dimer. Large stabilizations were observed for the self-complementary duplex d(AACAATTGTT)(2) as well as for the non-self-complementary duplex d(AAGAATTGTT)·d(AACAATTCTT) with both guanines located on the same strand. Formation of interstrand and intrastrand crosslinks by the covalent binding of both PBD moieties of the dimer to the exocyclic 2-amino group of the two guanine bases within the duplex minor groove was confirmed by NMR structural studies. In both the symmetric and non-symmetric DNA-PBD adducts the newly created stereogenic center at C11 of the tricyclic PBD subunits favors an S configuration. Different orientations of the PBD aromatic A-ring with respect to the covalently modified guanine as observed in the non-symmetric complex are shown to result in characteristic changes of PBD H11 and H11a proton chemical shifts. Based on a compilation of available NMR data on various PBD complexes, these differences may be used as valuable probes for the identification of PBD orientational preferences in DNA-PBD adducts.     

Head-to-head cross-linked adduct between the antitumor unit bis(mu-N,N'-di-p-tolylformamidinato)dirhodium(II,II) and the DNA fragment d(GpG)

Reactions of the compound cis-[Rh2(DTolF)2(CH3CN)6](BF4)2, a formamidinate derivative of the class of antitumor compounds [Rh2(O2CR)4] (R=Me, Et, Pr), with 9-ethylguanine (9-EtGuaH) or the dinucleotide d(GpG) proceed by substitution of the acetonitrile groups, with the guanine bases spanning the Rh--Rh bond, in a bridging fashion, through sites N7/O6. In the case of 9-EtGuaH, both head-to-head (HH) and head-to-tail (HT) isomers are formed, whereas with the tethered bases in d(GpG), only one right-handed conformer HH1R [Rh2(DTolF)2{d(GpG)}] is present in solution. For both cis-[Rh2(DTolF)2(9-EtGuaH)2](BF4)2 and [Rh2(DTolF)2{d(GpG)}], the absence of N7 protonation at low pH and the substantial decrease of the pKa values for N1-H deprotonation, support N7/O6 binding of the bases to the dirhodium core. The N7/O6 binding of the bases is further corroborated by the downfield shift by Deltadelta approximately 4.0 ppm of the 13C NMR resonances for the C6 nuclei as compared to the corresponding resonances of the free ligands. The HH arrangement of the guanine bases in [Rh2(DTolF)2{d(GpG)}] is indicated by the intense H8/H8 ROE cross-peaks in the 2D ROESY NMR spectrum. Complete characterization of the [Rh2(DTolF)2{d(GpG)}] conformer by 2D NMR spectroscopy supports anti-orientation and N (C3'-endo) conformation for both deoxyribose residues. The N-pucker for the 5'-G base is universal in such cross-links, but it is very unusual for platinum and unprecedented for dirhodium HH cross-linked adducts to have both deoxyribose residues in the N-type conformation. The bulk, the nonlabile character, and the electron-donating ability of the formamidinate bridging groups spanning the dirhodium core affect the nature of the preferred dirhodium DNA adducts. Molecular modeling studies performed on [Rh2(DTolF)2{d(GpG)}] corroborate the structural features obtained by NMR spectroscopy.     

Head-to-head right-handed cross-links of the antitumor-active bis(mu-N,N'-di-p-tolylformamidinato)dirhodium(II,II) unit with the dinucleotides d(GpA) and d(ApG)

Reactions of cis-[Rh(2)(DTolF)(2)(NCCH(3))(6)](BF(4))(2) with the dinucleotides d(GpA) and d(ApG) proceed to form [Rh(2)(DTolF)(2){d(GpA)}] and [Rh(2)(DTolF)(2){d(ApG)}], respectively, with bridging purine bases spanning the Rh-Rh unit in the equatorial positions. Both dirhodium adducts exhibit head-to-head (HH) arrangement of the bases, as indicated by the presence of H8/H8 NOE cross-peaks in the 2D ROESY NMR spectra. The guanine bases bind to the dirhodium core at positions N7 and O6, a conclusion that is supported by the absence of N7 protonation at low pH values and the notable increase in the acidity of the guanine N1H sites (pK(a) approximately 7.4 in 4:1 CD(3)CN/D(2)O), inferred from the pH-dependence titrations of the guanine H8 proton resonances. In both dirhodium adducts, the adenine bases coordinate to the metal atoms through N6 and N7, which induces stabilization of the rare imino tautomer of the bases with a concomitant substantial decrease in the basicity of the N1H adenine sites (pK(a) approximately 7.0-7.1 in 4:1 CD(3)CN/D(2)O), as compared to the imino form of free adenosine. The presence of the adenine bases in the rare imino form is further corroborated by the observation of DQF-COSY H2/N1H and ROE N1H/N6H cross-peaks in the 2D NMR spectra of [Rh(2)(DTolF)(2){d(GpA)}] and [Rh(2)(DTolF)(2){d(ApG)}] in CD(3)CN at -38 degrees C. The 2D NMR spectroscopic data and the molecular modeling results suggest the presence of right-handed variants, HH1R, in solution for both adducts (HH1R refers to the relative base canting and the direction of propagation of the phosphodiester backbone with respect to the 5' base). Complete characterization of [Rh(2)(DTolF)(2){d(GpA)}] and [Rh(2)(DTolF)(2){d(ApG)}] by 2D NMR spectroscopy and molecular modeling supports anti-orientation of the sugar residues for both adducts about the glycosyl bonds as well as N- and S-type conformations for the 5'- and 3'-deoxyribose residues, respectively.     

Active-site clashes prevent the human 3-methyladenine DNA glycosylase from improperly removing bases

The human 3-methyladenine DNA glycosylase (AAG, MPG) removes a diverse array of damaged purines via a nucleotide-flipping mechanism. In the crystal structure of AAG bound to DNA containing 1,N(6) ethenoadenine, an asparagine (N169) occupies the active-site floor, in close proximity to the C-2 position of the flipped-out 1,N(6) ethenoadenine. We engineered site-specific AAG mutants to determine whether N169 prevents normal bases from mistakenly entering the active site. Substituting alanine or serine resulted in mutants that excised substrates at a faster rate than wild-type. Furthermore, these mutants acquired the ability to excise normal guanine within mispairs but not opposite cytosine. The results suggest that AAG can recognize helical deformations, such as mispairs. However, the active site then prevents the mistaken excision of bases, which prevents AAG from acquiring a mutator activity.     

Simultaneous determination of guanine,adenine, thymine and cytosine with a simple electrochemical method

A simple method for simultaneous detection of guanine, adenine, thymine and cytosine was set up by using a bare glassy carbon electrode in acetate buffer solution of pH 4.5. The peak current responses of these four DNA bases in this supporting electrolyte were significantly increased comparing with those in phosphate buffer solution and Tris-HCl, moreover, the peak current values were linearly dependent on the concentration of four DNA bases, respectively. Individual and simultaneous determinations of four bases were performed by controlling certain experimental conditions, and broad linear ranges and low detection limits (S/N = 3) were obtained. The assay processes do not need any separation or pretreatment steps. In addition, this method showed good selectivity, reproducibility, and stability and can be used for determination of the four bases content in real DNA sample.     

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.     

Integrated microfluidic device for the separation and electrochemical detection of catechol estrogen-derived DNA adducts

Catechol estrogen-derived DNA adducts are formed as a result of the reaction of catechol estrogen metabolites (e.g., catechol estrogen quinones) with DNA to form depurinating adducts. Developing a new methodology for the detection of various DNA adducts is essential for medical diagnostics, and to this end, we demonstrate the applicability of on-chip capillary electrophoresis with an integrated electrochemical system for the separation and amperometric detection of various catechol estrogen-derived DNA adducts. A hybrid PDMS/glass microchip with in-channel amperometric detection interfaced with in situ palladium decoupler is utilized and presented. The influence of buffer additives along with the effect of the separation voltage on the resolving power of the microchip is discussed. Calibration plots were constructed in the range 0.4–10 μM with r ² ≥ 0.999, and detection limits in the attomole range are reported. These results suggest that on-chip analysis is applicable for analyzing various DNA adducts as potential biomarkers for future medical diagnostics.     

Efficient syntheses of C(8)-aryl adducts of adenine and guanine formed by reaction of radical cation metabolites of carcinogenic polycyclic aromatic hydrocarbons with DNA

The synthesis of the C(8)-aryl adducts of adenine and guanine formed by reaction of the radical cation metabolites of carcinogenic polycyclic aromatic hydrocarbons (PAHs), such as benzo[a]pyrene (BP) and dibenzo[def,p]chrysene (DBC), with DNA is reported. The synthetic approach involves in the key step direct reaction of a PAH aldehyde with a di- or triamine precursor of a purine. The method is operationally simple, affords good yields of adducts, and is broad in its scope. The C(8)-aryl adducts of adenine and guanine derived from BP (6-BP-8-Ade and 6-BP-8-Gua) and DBC (10-DBC-8-Ade and 10-DBC-8-Gua) were synthesized in good yields by this method. Analogous C(8)-aryl adenine and guanine derivatives of other PAHs (anthracene, benz[a]anthracene, and chrysene) were also readily prepared via this approach. This method of synthesis is superior to the only method that is currently available. It entails direct reaction of short-lived PAH radical cations (generated electrochemically or chemically) with 2'-deoxyribonucleosides or the corresponding purine bases. It provides the adducts in low yields accompanied by complex mixtures of secondary products. An alternative synthesis that involves Pd-catalyzed Suzuki-Miyaura coupling of arylboronic acids with 8-bromopurine nucleosides was also investigated. Although the C(8)-purine adducts of PAHs, such as naphthalene, phenanthrene, pyrene, and chrysene, could be prepared by this method, analogous adducts of carcinogenic PAHs and other structurally related PAHs, e.g., anthracene, benz[a]anthracene, benzo[a]pyrene, and dibenzo[def,p]chrysene, could not be obtained. This difference was shown to be a consequence of the facility of competing hydrolytic deboronation of the corresponding arylboronic acids.     

Selective voltammetric and flow-injection determination of guanine and adenine on a glassy carbon electrode modified by a ruthenium hexachloroplatinate film

An inorganic film of ruthenium hexachloroplatinate (RuPtCl₆), deposited on the surface of a glassy carbon electrode, exhibits electrocatalytic activity in the oxidation of purine bases, such as adenine and guanine. Appropriate working conditions are found for fabricating a polymer film on the surface of glassy carbon and for recording the maximum electrocatalytic effect for the modified electrode. A method is developed for the selective voltammetric determination of guanine and adenine in their simultaneous presence on an electrode modified by a RuPtCl₆ film. A procedure is proposed for the amperometric detection of purine bases with this modified electrode under the conditions of flow-injection analysis. The dependence of the analytical signal on the concentration of guanine and adenine is linear up to 5 × 10^?6 and 5 × 10^?7 M in the stationary mode and to 5 × 10^?7 and 5 × 10^?8 M under flow conditions, respectively. The proposed method was tested in the analysis of calf thymus DNA for the concentrations of guanine and adenine.