ИСТИНА

The detection of nucleic acids – macromolecules that store genetic information and enable protein production – is of a great interest for fundamental and applied biochemical studies. Nuclec acids are promising subjects for a point-of-use testing as markers of environmental bacterial and viral contaminations or human deseases. Deoxyribonucleic acids (DNA) are known to be electoactive through their nitrogenous bases: guanine, adenine, cytosine, and thymine [1]. However, the direct electrochemistry of nucleic acids suffers from high oxidation/reduction potentials and low values of registered currents. Guanine possessing the lowest electrooxidation potential among nucleobases oxidizes at 0.6–0.8 V (vs. Ag/AgCl), while 2'-deoxyguanosine-5'-triphosphate (dGTP) oxidizes at 0.9–1.0 V [1]. The oxidation current is strongly affected by the length of a nucleic acid strand [2] and the formation of double helix – nucleobases linked together by hydrogen bonds inside the helix become hardly accessible for electrode reactions [1]. To overcome these problems, an exciting strategy of electrochemical sensing of nucleic acids has been suggested by M. Hocek and M. Fojta with coworkers [3]. They inserted a palette of electrochemically active moieties into nucleic acid sequences by polymerase incorporation of chemically modified nucleotides. In this work, we present a set of 2'-deoxyuridine-5'-triphosphates (dUTP) modified with amino acid side chains attached through a linker at the C5 position of the pyrimidine ring which can be introduced into double stranded DNA (dsDNA) by polymerase chain reaction (PCR) [4, 5]. The modified nucleotides and corresponding PCR products were studied by square wave voltammetry on carbon screen printed electrodes suitable for in situ analysis. A strong effect of all additional functional groups on the electrochemical behavior of dUTP was revealed. Compared to the 2′-deoxythymidine-5′-triphosphate (a natural substrate for DNA polymerases), oxidation of dUTP derivatives took place at less positive potentials. In particular, 5-aminoallyl-dUTP derivatives modified with indole acetic, indole-3-propionic, indole-4-butyric, or 4-hydroxyphenylacetic acids demonstrated well-defined oxidation peaks at 0.5–0.7 V, similar to tryptophan and tyrosine amino acids, respectively. These signals were about 0.2–0.3 V less positive than the oxidation potential of dGTP. Moreover, PCR-generated dsDNA fragments (amplicons) with modified nucleotides showed novel oxidation signals at micromolar concentrations, while no peak was observed for unmodified dsDNA at the same conditions. 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 is a good compatibility of amino acid side chains with polymerase enzymes including those used in PCR. This work was financially supported by the Russian Science Foundation, grant 19-14-00247. 1. A. M. Oliveira-Brett, Electrochemical DNA Assays, in: P. N. Bartlett (Ed.), Bioelectrochemistry: Fundamentals, Experimental Techniques and Applications, John Wiley & Sons, 2008, pp. 411–442. 2. V. Brabec, J. Koudelka, Oxidation of deoxyribonucleic acid at carbon electrodes. The effect of the quality of the deoxyribonucleic acid sample, J. Electroanal. Chem. Interfacial Electrochem. 116 (1980) 793. 3. M. Hocek, M. Fojta, Nucleobase modification as redox DNA labelling for electrochemical detection, Chem. Soc. Rev. 40 (2011) 5802. 4. S. A. Lapa, K. S. Romashova, M. A. Spitsyn, et al., Preparation of Modified Combinatorial DNA Libraries via Emulsion PCR with Subsequent Strand Separation, Mol. Biol. 52 (2018) 854. 5. A. V. Chudinov, Y. Y. Kiseleva, V. E. Kuznetsov, et al., Enzymatic synthesis of high-modified DNA, Mol. Biol. 51 (2017) 534.