ИСТИНА

Deoxyribonucleic acid (DNA) carries genetic information in all living things and serves as a reliable biomarker of bacterial or viral contamination. To selectively and sensitively detect DNA, various amplification approaches such as polymerase chain reaction (PCR) and isothermal amplification techniques, both producing double-stranded DNA (dsDNA) amplicons, have been developed. Yet, the formation of a double helix hinders the direct electrochemical oxidation of dsDNA through guanine or adenine residues which become inaccessible for reactions on the electrode surface [1]. The aim of this work was to combine the in vitro DNA amplification with electrochemical detection of the generated dsDNA amplicons by incorporating electroactive ‘labeled’ nucleotides with the enzymatic reaction. To solve the problem of dsDNA electroactivity, several derivatives of 2'-deoxyuridine-5'-triphosphate (dUTP) modified via various linkers by aromatic groups of tyrosine, tryptophan [2], 4-nitrotoluene, fluorescein, or rhodamine were studied. By the square wave voltammetry on carbon screen printed electrodes, the oxidation or reduction peaks of all modified dUTP were obtained, similar to their free ‘labels’. The tested derivatives of dUTP showed acceptable compatibility with PCR, when the electroactive ‘label’ was introduced into dsDNA amplicons with a length of 100‒200 base pairs. However, in some cases, dsDNA products were not obtained by using modified dUTP instead of dTTP, probably due to specific DNA sequences. Among nucleotides under study, only a few modified dUTP compounds revealed good compatibility with the broadly used isothermal recombinase polymerase amplification. Generated dsDNA fragments with modified nucleotides demonstrated electrochemical signals at micromolar concentrations while no peaks were observed for unmodified dsDNA under the same conditions. This work was financially supported by the Russian Science Foundation, grant 19-14-00247. 1. E. V. Suprun, G. R. Kutdusova, S. A. Khmeleva, S. P. Radko, Towards deeper understanding of DNA electrochemical oxidation on carbon electrodes. Electrochem. Commun. 2021, 124, 106947. 2. E. V. Suprun, S. A. Khmeleva, G. R. Kutdusova, K. G. Ptitsyn, V. E. Kuznetsova, S. A. Lapa, A. V. Chudinov, S. P. Radko, Deoxyuridine triphosphates modified with tyrosine aromatic groups for direct electrochemical detection of double-stranded DNA products of isothermal recombinase polymerase amplification, Electrochem. Commun. 2021, 131, 107120.