Electroanalysis in Pharmacogenomic Studies: Mechanisms of Drug Interaction with DNA.

The review discusses electrochemical methods for analysis of drug interactions with DNA. The electroanalysis method is based on the registration of interaction-induced changes in the electrochemical oxidation potential of heterocyclic nitrogenous bases in the DNA molecule and in the maximum oxidation current amplitude. The mechanisms of DNA-drug interactions can be identified based on the shift in the electrooxidation potential of heterocyclic nitrogenous bases toward more negative (cathodic) or positive (anodic) values. Drug intercalation into DNA shifts the electrochemical oxidation potential to positive values, indicating thermodynamically unfavorable process that hinders oxidation of nitrogenous bases in DNA. The potential shift toward the negative values indicates electrostatic interactions, e.g., drug binding in the DNA minor groove, since this process does not interfere with the electrochemical oxidation of bases. The concentration-dependent decrease in the intensity of electrochemical oxidation of DNA bases allows to quantify the type of interaction and calculate the binding constants.

Pharmacogenomic Studies of Antiviral Drug Favipiravir.

In this work, we conducted a study of the interaction between DNA and favipiravir (FAV). This chemotherapeutic compound is an antiviral drug for the treatment of COVID-19 and other infections caused by RNA viruses. This paper examines the electroanalytical characteristics of FAV. The determined concentrations correspond to therapeutically significant ones in the range of 50-500 µM (R2 = 0.943). We have shown that FAV can be electro-oxidized around the potential of +0.96 V ÷ +0.98 V (vs. Ag/AgCl). A mechanism for electrochemical oxidation of FAV was proposed. The effect of the drug on DNA was recorded as changes in the intensity of electrochemical oxidation of heterocyclic nucleobases (guanine, adenine and thymine) using screen-printed graphite electrodes modified with single-walled carbon nanotubes and titanium oxide nanoparticles. In this work, the binding constants (Kb) of FAV/dsDNA complexes for guanine, adenine and thymine were calculated. The values of the DNA-mediated electrochemical decline coefficient were calculated as the ratio of the intensity of signals for the electrochemical oxidation of guanine, adenine and thymine in the presence of FAV to the intensity of signals for the electro-oxidation of these bases without drug (S, %). Based on the analysis of electrochemical parameters, values of binding constants and spectral data, intercalation was proposed as the principal mechanism of the antiviral drug FAV interaction with DNA. The interaction with calf thymus DNA also confirmed the intercalation mechanism. However, an additional mode of interaction, such as a damage effect together with electrostatic interactions, was revealed in a prolonged exposure of DNA to FAV.

The multifaceted role of proteases and modern analytical methods for investigation of their catalytic activity.

We discuss the diverse functions of proteases in the context of their biotechnological and medical significance, as well as analytical approaches used to determine the functional activity of these enzymes. An insight into modern approaches to studying the kinetics and specificity of proteases, based on spectral (absorption, fluorescence), mass spectrometric, immunological, calorimetric, and electrochemical methods of analysis is given. We also examine in detail electrochemical systems for determining the activity and specificity of proteases. Particular attention is given to exploring innovative electrochemical systems based on the detection of the electrochemical oxidation signal of amino acid residues, thereby eliminating the need for extra redox labels in the process of peptide synthesis. In the review, we highlight the main prospects for the further development of electrochemical systems for the study of biotechnologically and medically significant proteases, which will enable the miniaturization of the analytical process for determining the catalytic activity of these enzymes.

Electrochemical approach for the analysis of DNA degradation in native DNA and apoptotic cells.

The aim of this work was to develop an electrochemical approach for the analysis of DNA degradation and fragmentation in apoptotic cells. DNA damage is considered one of the major causes of human diseases. We analyzed the cleavage processes of the circular plasmid pTagGFP2-N and calf thymus DNA, which were exposed to restriction endonucleases (the restriction endonucleases BstMC I and AluB I and the nonspecific endonuclease I). Genomic DNA from the leukemia K562 cell line was used as a marker of the early and late (mature) stages of apoptosis. Registration of direct electrochemical oxidation of nucleobases of DNA molecules subjected to restriction endonuclease or apoptosis processes was proposed for the detection of these biochemical events. Label-free differential pulse voltammetry (DPV) has been used to measure endonuclease activities and DNA damage using carbon nanotube-modified electrodes. The present DPV technique provides a promising platform for high-throughput screening of DNA hydrolases and for registering the efficiency of apoptotic processes. DPV comparative analysis of the circular plasmid pTagGFP2-N in its native supercoiled state and plasmids restricted to 4 and 23 parts revealed significant differences in their electrochemical behavior. Electrochemical analysis was fully confirmed by means of traditional methods of DNA analysis and registration of apoptotic process, such as gel electrophoresis and flow cytometry.

Bielectrode Strategy for Determination of CYP2E1 Catalytic Activity: Electrodes with Bactosomes and Voltammetric Determination of 6-Hydroxychlorzoxazone.

We describe a bielectrode system for evaluation of the electrocatalytic activity of cytochrome P450 2E1 (CYP2E1) towards chlorzoxazone. One electrode of the system was employed to immobilize Bactosomes with human CYP2E1, cytochrome P450 reductase (CPR), and cytochrome b5 (cyt b5). The second electrode was used to quantify CYP2E1-produced 6-hydroxychlorzoxazone by its direct electrochemical oxidation, registered using square-wave voltammetry. Using this system, we determined the steady-state kinetic parameters of chlorzoxazone hydroxylation by CYP2E1 of Bactosomes immobilized on the electrode: the maximal reaction rate (Vmax) was 1.64 ± 0.08 min-1, and the Michaelis constant (KM) was 78 ± 9 µM. We studied the electrochemical characteristics of immobilized Bactosomes and have revealed that electron transfer from the electrode occurs both to the flavin prosthetic groups of CPR and the heme iron ions of CYP2E1 and cyt b5. Additionally, it has been demonstrated that CPR has the capacity to activate CYP2E1 electrocatalytic activity towards chlorzoxazone, likely through intermolecular electron transfer from the electrochemically reduced form of CPR to the CYP2E1 heme iron ion.

Catalytic and Electrocatalytic Mechanisms of Cytochromes P450 in the Development of Biosensors and Bioreactors.

Cytochromes P450 are a unique family of enzymes found in all Kingdoms of living organisms (animals, bacteria, plants, fungi, and archaea), whose main function is biotransformation of exogenous and endogenous compounds. The review discusses approaches to enhancing the efficiency of electrocatalysis by cytochromes P450 for their use in biotechnology and design of biosensors and describes main methods in the development of reconstituted and electrochemical catalytic systems based on the biochemical mechanism of cytochromes P450, as well as and modern trends for their practical application.

Effect of an NGR Peptide on the Efficacy of the Doxorubicin Phospholipid Delivery System.

This study is a continuation of an investigation into the effect of a targeted component, a peptide with an NGR, on the properties of the previously developed doxorubicin phospholipid delivery system. The NGR peptide has an affinity for aminopeptidase N (known as the CD13 marker on the membrane surface of tumor cells) and has been extensively used to target drug delivery systems. This article presents the results of a study investigating the physical properties of the phospholipid composition with and without the peptide chain: particle size, zeta potential, stability in fluids, and dependence of doxorubicin release from nanoparticles at different pH levels (5.0, 6.5, 7.4). The cytotoxic effect of the compositions has also been shown to depend on the dose of the drug used for incubation, the presence of the targeted component in the composition, and the time of incubation time of the substances. There was a significant difference in the cytotoxic effect on HT-1080 (CD13-positive) and MCF-7 (CD13-negative) cells. Cell death pathway analysis has shown that death occurred mainly by apoptosis. We also present data on the effect of doxorubicin embedded in phospholipid nanoparticles with the targeted peptide on DNA assessed by differential pulse voltammetry, the mechanism of action being electrostatic interactions. The interactions of native dsDNA with doxorubicin encapsulated in phospholipid nanoparticles with the targeted peptide were studied electrochemically by differential pulse voltammetry. Here, we have highlighted that the targeted peptide in the doxorubicin composition moved specific interaction of the drug with dsDNA from intercalative mode to electrostatic interactions.

Interaction of Doxorubicin Embedded into Phospholipid Nanoparticles and Targeted Peptide-Modified Phospholipid Nanoparticles with DNA.

The interactions of dsDNA with new targeted drug delivery derivatives of doxorubicin (DOX), such as DOX embedded into phospholipid nanoparticles (NPhs) and DOX with the NGR targeted peptide-modified NPhs were studied electrochemically by differential pulse voltammetry technique. Screen-printed electrodes (SPEs), modified with stable fine dispersions of carbon nanotubes (CNTs), were used for quantitative electrochemical investigations of direct electrochemical oxidation of guanine, adenine, and thymine heterocyclic bases of dsDNA, and their changes in the presence of DOX nanoderivatives. Analysing the shifts of peak potentials of nucleobases in the presence of drug, we have shown that the doxorubicin with NGR targeted peptide changed the mode of interaction in DNA-drug complexes from intercalative to electrostatic. Binding constants (Kb) of DNA-drug complexes were calculated in accordance with adenine, guanine, and thymine oxidation signals. Based on our experiments, we have proven that the surface modification of a drug delivery system with NGR targeted peptide dramatically changed the mechanism of interaction of drug with genetic material. DNA-mediated drug toxicity was calculated based on the concentration-dependent "response" of heterocyclic nucleobases on drug influence. DOX, DOX-loaded phospholipid nanoparticles (NPhs), and DOX with NGR addressed peptide-modified NPhs were moderately toxic in the concentration range of 0.5-290 µM.

Improving the Efficiency of Electrocatalysis of Cytochrome P450 3A4 by Modifying the Electrode with Membrane Protein Streptolysin O for Studying the Metabolic Transformations of Drugs.

In the present work, screen-printed electrodes (SPE) modified with a synthetic surfactant, didodecyldimethylammonium bromide (DDAB) and streptolysin O (SLO) were prepared for cytochrome P450 3A4 (CYP3A4) immobilization, direct non-catalytic and catalytic electrochemistry. The immobilized CYP3A4 demonstrated a pair of redox peaks with a formal potential of -0.325 ± 0.024 V (vs. the Ag/AgCl reference electrode). The electron transfer process showed a surface-controlled mechanism ("protein film voltammetry") with an electron transfer rate constant (ks) of 0.203 ± 0.038 s-1. Electrochemical CYP3A4-mediated reaction of N-demethylation of erythromycin was explored with the following parameters: an applied potential of -0.5 V and a duration time of 20 min. The system with DDAB/SLO as the electrode modifier showed conversion of erythromycin with an efficiency higher than the electrode modified with DDAB only. Confining CYP3A4 inside the protein frame of SLO accelerated the enzymatic reaction. The increases in product formation in the reaction of the electrochemical N-demethylation of erythromycin for SPE/DDAB/CYP3A4 and SPE/DDAB/SLO/CYP3A4 were equal to 100 ± 22% and 297 ± 7%, respectively. As revealed by AFM images, the SPE/DDAB/SLO possessed a more developed surface with protein cavities in comparison with SPE/DDAB for the effective immobilization of the CYP3A4 enzyme.

Electrochemical biosensor for trypsin activity assay based on cleavage of immobilized tyrosine-containing peptide.

In this work, we proposed a biosensor for trypsin proteolytic activity assay using immobilization of model peptides on screen-printed electrodes (SPE) modified with gold nanoparticles (AuNPs) prepared by electrosynthetic method. Sensing of proteolytic activity was based on electrochemical oxidation of tyrosine residues of peptides. We designed peptides containing N-terminal cysteine residue for immobilization on an SPE, modified with gold nanoparticles, trypsin-specific cleavage site and tyrosine residue as a redox label. The peptides were immobilized on SPE by formation of chemical bonds between mercapto groups of the N-terminal cysteine residues and AuNPs. After the incubation with trypsin, time-dependent cleavage of the immobilized peptides was observed by decline in tyrosine electrochemical oxidation signal. The kinetic parameters of trypsin, such as the catalytic constant (kcat), the Michaelis constant (KM) and the catalytic efficiency (kcat/KM), toward the CGGGRYR peptide were determined as 0.33 ± 0.01 min-1, 198 ± 24 nM and 0.0016 min-1 nM-1, respectively. Using the developed biosensor, we demonstrated the possibility of analysis of trypsin specificity toward the peptides with amino acid residues disrupting proteolysis. Further, we designed the peptides with proline or glutamic acid residues after the cleavage site (CGGRPYR and CGGREYR), and trypsin had reduced activity toward both of them according to the existing knowledge of the enzyme specificity. The developed biosensor system allows one to perform a comparative analysis of the protease steady-state kinetic parameters and specificity toward model peptides with different amino acid sequences.

Analysis of Single Biomacromolecules and Viruses: Is It a Myth or Reality?

The beginning of the twenty-first century witnessed novel breakthrough research directions in the life sciences, such as genomics, transcriptomics, translatomics, proteomics, metabolomics, and bioinformatics. A newly developed single-molecule approach addresses the physical and chemical properties and the functional activity of single (individual) biomacromolecules and viral particles. Within the alternative approach, the combination of "single-molecule approaches" is opposed to "omics approaches". This new approach is fundamentally unique in terms of its research object (a single biomacromolecule). Most studies are currently performed using postgenomic technologies that allow the properties of several hundreds of millions or even billions of biomacromolecules to be analyzed. This paper discusses the relevance and theoretical, methodological, and practical issues related to the development potential of a single-molecule approach using methods based on molecular detectors.

Approaches for increasing the electrocatalitic efficiency of cytochrome P450 3A4.

The electrochemically driven cytochrome P450 reactions have great promise as drug sensing device, new drug searching tool and bioreactor with broad synthetic application. In the present work, we proposed approaches for the increasing the efficiency of cytochrome P450 3A4 electrocatalysis, based on fine regulation and reproduction of nature hemeprotein catalytic cycle and electron transfer pathways on electrode. To analyze the comparative electrochemical and electrocatalytic activity, cytochrome P450 3A4 was immobilized on electrodes modified with a membrane-like synthetic surfactant, didodecyldimethylammonium bromide (DDAB). We used riboflavin, FMN and FAD as low molecular models of NADPH-dependent cytochrome P450 reductase for the improving and enhancement properties of catalytically responsible cytochrome P450 3A4-electrode. The efficiencies of electrocatalysis of erythromycin N-demethylation as well-known cytochrome P450 3A4 substrate in the case of riboflavin, FAD and FMN as electron transfer mediators were 135 ± 6, 171 ± 15 and 203 ± 10 %, respectively (in comparison with 100 ± 18 % erythromycin N-demethylation in the case of cytochrome P450 3A4-electrode as catalyst). Molecular modeling of cytochrome P450 3A4 complexes with riboflavin, FMN and FAD confirms possibility of binding isoalloxazine ring of riboflavin to the protein on the proximal side of hemeprotein, which is the place for binding of redox partners of the cytochrome P450.

Biotransformation of phenytoin in the electrochemically-driven CYP2C19 system.

The possibility of the detection of atypical kinetic profiles of drug biotransformation using electrochemical systems based on immobilized cytochromes P450 with phenytoin hydroxylation by cytochrome P450 2C19 (CYP2C19) as an example was evaluated for the first time. For this purpose, we developed an electrochemical system, where one of the electrodes was modified by didodecyldimethylammonium bromide (DDAB) and was used as an electron donor for reduction of heme iron ion of the immobilized CYP2C19 and initiation of the catalytic reaction, while the second electrode was not modified and served for an electrochemical quantitation of 4-hydroxyphenytoin, which is a metabolite of antiepileptic drug phenytoin, by its oxidation peak. It was revealed that the dependence of the rate of 4-hydroxyphenytoin formation on phenytoin concentration is described by the equation for two enzymes or two binding sites indicating the existing of high- and low-affinity forms of the enzyme. The atypical kinetics and the kinetic parameters of CYP2C19-mediated phenytoin hydroxylation in the electrochemical system correlate to the same characteristics obtained by other authors in an alternative enzymatic system. Our results demonstrate the possibility of electrochemical systems based on cytochromes P450 to be applied for the detection of atypical kinetic profiles of drug metabolism.

Predicting drug-drug interactions by electrochemically driven cytochrome P450 3A4 reactions.

OBJECTIVES: Human cytochrome P450 3A4 is the most abundant hepatic and intestinal Phase I enzyme that metabolizes approximately 60% marketed drugs. Simultaneous administration of several drugs may result in appearance of drug-drug interaction. Due to the great interest in the combination therapy, the exploration of the role of drug as "perpetrator" or "victim" is important task in pharmacology. In this work the model systems based on electrochemically driven cytochrome P450 3A4 for the analysis of drug combinations was used. We have shown that the analysis of electrochemical parameters of cytochrome P450 3A4 and especially, potential of the start of catalysis, Eonset, possess predictive properties in the determination of the leading ("perpetrator") properties of drug. Based on these experimental data, we concluded, that the more positive potential of the start of catalysis, Eonset, the more pronounced the role of drug as leading medication. METHODS: Electrochemically driven cytochrome P450 3A4 was used as probe and measuring tool for the estimation of the role of interacting drugs. RESULTS: It is shown that the electrochemical non-invasive model systems for monitoring the catalytic activity of cytochrome P450 3A4 can be used as prognostic devise in assessment of drug/drug interacting medications. CONCLUSIONS: Cytochrome P450 3A4 activity was studied in electrochemically driven system. Method was implemented to monitor drug/drug interactions. Based on the obtained experimental data, we can conclude that electrochemical parameter such as potential of onset of catalysis, Eonset, has predictive efficiency in assessment of drug/drug interacting medications in the case of the co-administration.

Electrochemical characterization of mutant forms of rubredoxin B from Mycobacterium tuberculosis.

Electron transfer in metalloproteins is a driving force for many biological processes and widely distributed in nature. Rubredoxin B (RubB) from Mycobacterium tuberculosis is a first example among [1Fe-0S] proteins that support catalytic activity of terminal sterol-monooxygenases enabling its application in metabolic engineering. To explore the tolerance of RubB to the specific amino acid changes we evaluated the effect of surface mutations on its electrochemical properties. Based on the RubB fold we also designed the mutant with a putative additional site for protein-protein interactions to further evaluate electron transfer and electrochemical properties. The investigation of redox properties of mutant variants of RubB was done using screen-printed graphite electrodes (SPEs) modified with stable dispersion of multi-walled carbon nanotubes (MWCNTs). The redox potentials (midpoint potentials, E0Ꞌ) of mutants did not significantly differ from the wild type protein and vary in the range of -264 to -231 mV vs. Ag/AgCl electrode. However, all mutations affect electron transfer rate between the protein and electrode. Notably, the modulation of the protein-protein interactions was observed for the insertion mutant suggesting the possibility of tailoring of rubredoxin for the selected redox-partner. Overall, RubB is tolerant to the significant modifications in its structure enabling rational engineering of novel redox proteins.

Electroanalysis of Biomolecules: Rational Selection of Sensor Construction.

Methods of electrochemical analysis of biological objects based on the reaction of electro-oxidation/electro-reduction of molecules are presented. Polymer nanocomposite materials that modify electrodes to increase sensitivity of electrochemical events on the surface of electrodes are described. Examples of applications electrochemical biosensors constructed with nanocomposite material for detection of biological molecules are presented, advantages and drawbacks of different applications are discussed.

A new twist of rubredoxin function in M. tuberculosis.

Electron transfer mediated by metalloproteins drives many biological processes. Rubredoxins are a ubiquitous [1Fe-0S] class of electron carriers that play an important role in bacterial adaptation to changing environmental conditions. In Mycobacterium tuberculosis, oxidative and acidic stresses as well as iron starvation induce rubredoxins expression. However, their functions during M. tuberculosis infection are unknown. In the present work, we show that rubredoxin B (RubB) is able to efficiently shuttle electrons from cognate reductases, FprA and FdR to support catalytic activity of cytochrome P450s, CYP124, CYP125, and CYP142, which are important for bacterial viability and pathogenicity. We solved the crystal structure of RubB and characterized the interaction between RubB and CYPs using site-directed mutagenesis. Mutations that not only neutralize single charge but also change the specific residues on the surface of RubB did not dramatically decrease activity of studied CYPs. Together with isothermal calorimetry (ITC) experiments, the obtained results suggest that interactions are transient and not highly specific. The redox potential of RubB is -264 mV vs. Ag/AgCl and the measured extinction coefficients are 9931 M-1cm-1 and 8371 M-1cm-1 at 380 nm and 490 nm, respectively. Characteristic parameters of RubB along with the discovered function might be useful for biotechnological applications. Our findings suggest that a switch from ferredoxins to rubredoxins might be crucial for M. tuberculosis to support CYPs activity during the infection.

Electrochemical studies of the interaction of rifampicin and nanosome/rifampicin with dsDNA.

The interactions of dsDNA with rifampicin (RF) or with rifampicin after encapsulation in phospholipid micelles (nanosome/rifampicin) (NRF) were studied electrochemically. Screen-printed electrodes (SPEs) modified by stable dispersions of multi-wolled carbon nanotubes (MWCNTs) in aqueous solution of poly(1,2-butadiene)-block-poly(2-(dimethylamino)ethyl methacrylate) (PB290-b-PDMAEMA240) diblock copolymer were used for quantitative electrochemical investigation of direct electrochemical oxidation of guanine at E = 0.591 V (vs. Ag/AgCl) and adenine at E = 0.874 V (vs. Ag/AgCl) of dsDNA and its change in the presence of RF or NRF. Due to RF or NRF interaction with dsDNA, the differential pulse voltammetry (DPV) peak currents of guanine and adenine decreased and the peak potentials shifted to more positive values with increasing drug concentration (RF or NRF). Binding constants (Kb) of complexes RF-dsDNA and NRF-dsDNA were calculated based on adenine and guanine oxidation signals. The Kb values for RF-dsDNA were 1.48 × 104 M-1/8.56 × 104 M-1, while for NRF-dsDNA were 2.51 × 104 M-1/1.78 × 103 M-1 (based on adenine or guanine oxidation signals, respectively). The values of Kb revealed intercalation mode of interaction with dsDNA for RF and mixed type of interaction (intercalation and electrostatic mode) for NRF. The estimated values of ΔG (Gibbs free energy) of the complex formation confirmed that drug-dsDNA interactions are spontaneous and favourable reactions.

Interactions of galeterone and its 3-keto-Δ4 metabolite (D4G) with one of the key enzymes of corticosteroid biosynthesis - steroid 21-monooxygenase (CYP21A2).

We have investigated interactions of galeterone and its pharmacologically active metabolite - 3-keto-Δ4-galeterone (D4G) - with one of the key enzymes of corticosteroid biosynthesis - steroid 21-monooxygenase (CYP21A2). It was shown by absorption spectroscopy that both compounds induce type I spectral changes of CYP21A2. Spectral dissociation constants (KS ) of complexes of CYP21A2 with galeterone or D4G were calculated as 3.1 ± 0.7 µm and 4.6 ± 0.4 µm, respectively. It was predicted by molecular docking that both ligands similarly bind to the active site of CYP21A2. We have revealed using reconstituted monooxygenase system that galeterone is a competitive inhibitor of CYP21A2 with the inhibition constant (Ki ) value of 12 ± 3 µm, while D4G at the concentrations of 10 and 25 µm does not inhibit the enzyme. Summarizing, based on the in vitro analyses we detected inhibition of CYP21A2 by galeterone and lack of the influence of D4G on this enzyme.

Rational Design of Amphiphilic Diblock Copolymer/MWCNT Surface Modifiers and Their Application for Direct Electrochemical Sensing of DNA.

We demonstrate the application of amphiphilic ionic poly(n-butylmethacrylate)-block- poly(2-(dimethylamino)ethyl methacrylate) diblock copolymers (PnBMA40-b-PDMAEMA40, PnBMA40-b-PDMAEMA120, PnBMA70-b-PDMAEMA120) for dispersing multiwalled carbon nanotubes (MWCNTs) in aqueous media, a subsequent efficient surface modification of screen-printed electrodes (SPEs), and the application of the modified SPEs for DNA electrochemistry. Stable and fine aqueous dispersions of MWCNTs were obtained with PnBMAx-b-PDMAEMAy diblock copolymers, regardless of the structure of the copolymer and the amount of MWCNTs in the dispersions. The effect of the diblock copolymer structure was important when the dispersions of MWCNTs were deposited as modifying layers on surfaces of SPEs, resulting in considerable increases of the electroactive surface areas and great acceleration of the electron transfer rate. The SPE/(PnBMAx-b-PDMAEMAy + MWCNT) constructs were further exploited for direct electrochemical oxidation of the guanine (G) and adenine (A) residues in a model salmon sperm double-stranded DNA (dsDNA). Two well-defined irreversible oxidation peaks were observed at about +600 and +900 mV, corresponding to the electrochemical oxidation of G and A residues, respectively. A multi-parametric optimization of dsDNA electrochemistry enables one to get the limits of detection (LOD) as low as 5 µg/mL (0.25 µM) and 1 µg/mL (0.05 µM) for G and A residues, respectively. The achieved sensitivity of DNA assay enables quantification of the A and G residues of dsDNA in the presence of human serum and DNA in isolated human leukocytes.