ИСТИНА

Deoxyribonucleic acids (DNA) are biompolymers which carry genetic information for protein production essential to all known forms of life. At present, nucleic acids are promising subjects for a ‘point-of-need’ testing as markers of environmental bacterial and viral contaminations or human deseases. DNA are known to be electoactive through their nitrogenous bases. However, the direct electrochemistry of nucleic acids suffers from high oxidation/reduction potentials and low values of registered currents. In voltammograms, the oxidation current is strongly affected by the length of a nucleic acid strand and the formation of double helix – nucleobases linked together by hydrogen bonds inside the helix become hardly accessible for electrode reactions. Therefore, direct electrochemistry of native double-stranded DNA (dsDNA) via oxidation of nitrogenous bases cannot provide detection sensitivity required for most practical applications. To overcome these problems, an exciting strategy of electrochemical sensing of DNA has been suggested by M. Hocek and M. Fojta with coworkers [1]. They inserted a palette of electrochemically active moieties into nucleic acid sequences by polymerase incorporation of chemically modified nucleotides. Following this way, we present two 2'-deoxyuridine-5'-triphosphates modified with 4-hydroxyphenyl groups attached through various linkers at the C5 position of the pyrimidine ring (dUTP-Y1 and dUTP-Y2) [2, 3]. The dUTP-Y were tested as bearers of electroactive ‘labels’ as well as proper substrates for polymerases used in polymerase chain reaction (PCR) and isothermal recombinase polymerase amplification (RPA) with the aim of electrochemical detection of dsDNA amplification products. dUTP-Y1 and dUTP-Y2 were successfully incorporated into various PCR- or RPA-generated dsDNA amplicons of 1–2 hundred base pairs long (at 80–100 % substitution of dTTP). The modified nucleotides and corresponding amplification products were studied by square wave voltammetry on carbon screen printed electrodes suitable for in situ analysis. Compared to dUTP, the oxidation of dUTP-Y took place at less positive potentials of 0.5–0.7 V, similar to free tyrosine amino acid. dsDNA fragments with modified nucleotides showed novel oxidation signals at micromolar concentrations, while no peak was observed for unmodified dsDNA at the same conditions. A strong effect of the structure of linker of 4-hydroxyphenyl group on the electrochemical and biochemical behavior of dUTP-Y was revealed. Finally, the developed approach was applied to the direct electrochemical detection of dsDNA fragments obtained as a result of PCR or RPA, using dUTP-Y1 as a substrate, of the causative agent of potato bacteriosis Dickeya solani. Therefore, the tested dUTP derivatives well complement the existing collection of electroactive ‘labeled’ nucleotides for direct electrochemical detection of nucleic acids. The main advantage of the developed dUTP-Y is a good compatibility of tyrosine side chain with polymerase enzymes including those used in isothermal amplification methods. This work was financially supported by the Russian Science Foundation, grant 19-14-00247. 1. M. Hocek, M. Fojta, Nucleobase modification as redox DNA labelling for electrochemical detection, Chem. Soc. Rev. 40 (2011) 5802. 2. E.V. Suprun, S.A. Khmeleva, G.R. Kutdusova, et al., Deoxyuridine triphosphates modified with tyrosine or tryptophan aromatic groups for direct electrochemical detection of double-stranded DNA, Electrochim. Acta 362 (2020) 137105. 3. E.V. Suprun, S.A. Khmeleva, G.R. Kutdusova, et al., Deoxyuridine triphosphates modified with tyrosine aromatic groups for direct electrochemical detection of double-stranded DNA products of isothermal recombinase polymerase amplification, Electrochem. Commun. 131 (2021) 107120.