ИСТИНА

Direct electrochemical oxidation of DNA molecules may provide a reliable and cost-effective approach to quantify DNA amplification products (amplicons), with a great potential for coupling to isothermal amplification techniques [1] and for developing in situ assays. However, electrochemical behavior of the double-stranded DNA (dsDNA) amplicons is not fully understood. Many details of the relationship between nucleic acids electroactivity and their molecular structure are still unknown. It is generally accepted that DNA is oxidizable via its nitrogenous bases: guanine (Gua), adenine (Ade), cytosine, and thymine [2]. However, distinct oxidation signals of all nitrogenous bases are shown only for small mono- and oligonucleotides [3]. Large molecular weight DNA demonstrates oxidation peaks only for Gua and Ade residues at about 0.7–0.9 V and 0.9–1.2 V (vs. Ag/AgCl), respectively [4]. Moreover, a decrease of electrochemical signal with DNA molecular weight may be expected [4]. In this work, to get deeper insights into electrochemical behavior of DNA molecules, the electrooxidation of synthetic oligonucleotides of four to several dozen nucleotides long, their thermally stable duplexes, amplicons of hundreds base pairs long, generated by isothermal amplification technique or polymerase chain reaction, and samples of double- and single-stranded DNA (ssDNA) of natural origin was studied on carbon screen printed electrodes by square wave and cycling voltammetry within the potential range of 0.5–1.5 V (vs. Ag/AgCl) [5]. In voltammograms, synthetic DNA oligonucleotides exhibited the oxidation peaks at potentials of either around 0.85 V (Gua residues) or 1.15 V (Ade residues), or both, depending on their sequence. Oxidation signals of Gua and Ade residues exponentially decreased with the DNA oligonucleotide length. Similar to that, ssDNA of natural origin produced two distinct signals at approximately 0.75 V and 1.05 V, attributed to the oxidation of Gua and Ade residues, respectively. The oxidation reactions for both residues of ssDNA were found diffusion-controlled. In contrast to ssDNA, dsDNA molecules showed no sign of oxidation signal on carbon electrodes. In the thermally stable duplex DNA molecules even as short as those of 24 base pairs long, Gua and Ade restudies were shown to be inaccessible for oxidation on carbon electrodes. Therefore, the low molecular weight ssDNA fragments seem to be the main contributors to the overall electrooxidation currents generated via oxidation of Gua and Ade residues of DNA from a natural source. This work was financially supported by the Russian Science Foundation, grant 19-14-00247.