Deoxyuridine Triphosphates Modified with 4-Hydroxyphenyl or 4-Nitrophenyl Groups for Direct Electrochemical Detection of Double-Stranded DNA Amplification Productsтезисы доклада
Аннотация:Today, there is a growing trend toward the ‘point-of-need’ testing (PONT). DNA diagnostics of various infectious agents is widespread, and its adaptation to the PONT requirements can benefit from the development of methods of DNA electrochemical detection. DNA is known to be electoactive through their nitrogenous bases [1]. 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 DNA strand and the formation of double helix since nucleobases linked together inside the helix become hardly accessible for electrode reactions [2]. Therefore, direct electrochemistry of double-stranded DNA (dsDNA) via oxidation of nitrogenous bases cannot provide detection sensitivity required for most practical applications. As a way around, an atractive strategy of electrochemical sensing of DNA has been suggested via inserting a palette of electrochemically active moieties into DNA sequences by polymerase incorporation of chemically modified nucleotides [3]. Following this way, our group presents a set of novel modified nucleotides: two 2'-deoxyuridine-5'-triphosphates with 4-hydroxyphenyl groups (dUTP-Y1 and dUTP-Y2) [4, 5] and three 2'-deoxyuridine-5'-triphosphates with 4-nitrophenyl groups (dUTP-N1, dUTP-N2, and dUTP-N3), attached through various linkers at the C5 position of the pyrimidine ring (Fig. 1). In this study, the dUTP-Y and dUTP-N nucleotides 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-Y and dUTP-N nucleotides were successfully incorporated into various PCR- or RPA-generated dsDNA amplicons of 1–2 hundred base pairs long (at 80–100 % substitution of dTTP in the reaction mixture). The modified nucleotides and corresponding amplification products were detected by square wave voltammetry on carbon screen printed electrodes suitable for on-site 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. On the other hand, compared to dUTP, the reduction peaks of aromatic nitro groups of dUTP-N were registered at less negative potentials of –0.7 to –0.85 V, similar to free nitro aromatic compounds. dsDNA fragments with modified nucleotides showed novel oxidation/reduction signals at micromolar concentrations, while no peaks were observed for unmodified dsDNA at the same conditions. In addition, there was registered an effect of the structure of linker of 4-hydroxyphenyl or 4-nitrophenyl group on the electrochemical behavior of dUTP-Y or dUTP-N. The polymerase-driven incorporation of dUTP-Y or dUTP-N in PCR and RPA revealed that its efficiency can depend on the molecular structure of electroactive moiety, including the linker structure, and also on a template sequence. Therefore, the tested dUTP derivatives well complement the existing collection of electroactive ‘labeled’ nucleotides for direct electrochemical detection of DNA.
Acknowledgements
This work was financially supported by the Russian Science Foundation, grant 19-14-00247, https://rscf.ru/project/19-14-00247/.
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