Polymerase incorporation of Cy5 modified 2′-deoxyuridine-5′-triphosphate into double- and single-stranded DNA for direct electrochemical detection

Suprun, E.V.; Bibik, K.V.; Khmeleva, S.A.; Ptitsyn, K.G.; Kurbatov, L.K.; Radko, S.P.

Авторы: Suprun Elena V., Bibik Konstantin V., Khmeleva Svetlana A., Ptitsyn Konstantin G., Kurbatov Leonid K., Radko Sergey P.
Журнал: Journal of Electroanalytical Chemistry
Том: 962
Год издания: 2024
Издательство: Elsevier Sequoia
Местоположение издательства: Switzerland
Номер статьи: 118240
DOI: 10.1016/j.jelechem.2024.118240
Аннотация: The 2′-deoxyuridine-5′-triphosphate modified with Cy5 cyanine dye (dUTP-Cy5) was tested as a carrier of electroactive label and as a proper substrate for DNA polymerases used in polymerase chain reaction (PCR), recombinase polymerase amplification (RPA), rolling circle amplification (RCA), and loop-mediated amplification (LAMP) with the aim of electrochemical detection of amplification products. For this purpose, the electrochemical behavior of free Cy5-alkyne, as well as the Cy5 conjugated with dUTP was studied by cyclic (CV) and square wave (SWV) voltammetry on carbon screen printed electrodes. Both Cy5-alkyne and dUTP-Cy5 underwent pronounced oxidation at the potentials of 0.5–0.6 V (phosphate buffer, pH 7.4). The reduction peak of Cy5-alkyne and dUTP-Cy5 was registered between –0.8 V and –0.9 V. In contrast to the anodic signals of Cy5-alkyne and dUTP-Cy5, their cathodic peak was found insensitive to analyte concentration. The dUTP-Cy5 was further tested as a substrate for the incorporation of the Cy5 electroactive label into 210 or 206 base pair long double-stranded DNA (dsDNA) amplicons generated either by PCR or RPA, as well as dsDNA generated by LAMP, and RCA ultralong single-stranded DNA product. dUTP-Cy5 revealed rather good compatibility with PCR, RCA, and RPA, producing the full-size amplicons at 75 %, 50 %, and 25 % substitution of dTTP in the reaction mixture, respectively, though with a rather low reaction yields. Significant amount of Cy5 modified product was obtained by LAMP only at 12.5 % substitution of dTTP with dUTP-Cy5. No full-length Cy5 labeled amplicons were obtained at 100 % substitution of dTTP by dUTP-Cy5 for all amplification reaction under study. The fluorescent property of the tested modified nucleotide helped to reveal relation between the overall output of amplification reaction and an optimal molar ratio of modified dUTP to native dTTP in the reaction mixture. Finally, PCR generated modified dsDNA product (at 75 % substitution of dTTP by dUTP-Cy5, dsDNA-Cy5) and LAMP generated modified dsDNA product (at 6 % substitution) were detected by SWV at micromolar and sub-micromolar concentrations. The calibration curve for the PCR amplicon detection was linear in coordinates Ip, A vs. Log (c, M) within the 0.1–2 µM range. The limit of detection for dsDNA-Cy5 was estimated as 7 nM. Alongside with fluorescence DNA assays, the dUTP-Cy5 appears as a promising electroactive label to be employed in developing electrochemical approaches for detecting DNA amplification products.
Добавил в систему: Супрун Елена Владимировна

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Polymerase incorporation of Cy5 modified 2′-deoxyuridine-5′-triphosphate into double- and single-stranded DNA for direct electrochemical detectionстатья

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