Polymerase incorporation of fluorescein or rhodamine modified 2'-deoxyuridine-5'-triphosphates into double-stranded DNA for direct electrochemical detection

Elena V Suprun; Svetlana A Khmeleva; Konstantin V Bibik; Konstantin G Ptitsyn; Leonid K Kurbatov; Sergey P Radko

doi:10.1016/j.jpba.2023.115737

Polymerase incorporation of fluorescein or rhodamine modified 2'-deoxyuridine-5'-triphosphates into double-stranded DNA for direct electrochemical detection

J Pharm Biomed Anal. 2023 Nov 30:236:115737. doi: 10.1016/j.jpba.2023.115737. Epub 2023 Sep 19.

Authors

Elena V Suprun¹, Svetlana A Khmeleva², Konstantin V Bibik³, Konstantin G Ptitsyn², Leonid K Kurbatov², Sergey P Radko²

Affiliations

¹ Chemistry Faculty of M.V. Lomonosov Moscow State University, Lenin Hills, 1/3, Moscow 119991, Russia; Institute of Biomedical Chemistry, Pogodinskaya Street, 10/8, Moscow 119121, Russia. Electronic address: lenasuprun@mail.ru.
² Institute of Biomedical Chemistry, Pogodinskaya Street, 10/8, Moscow 119121, Russia.
³ Chemistry Faculty of M.V. Lomonosov Moscow State University, Lenin Hills, 1/3, Moscow 119991, Russia; Institute of Biomedical Chemistry, Pogodinskaya Street, 10/8, Moscow 119121, Russia.

PMID: 37774487
DOI: 10.1016/j.jpba.2023.115737

Abstract

The 2'-deoxyuridine-5'-triphosphates modified with fluorescein (dUTP-Fl) or rhodamine (dUTP-Rh) were tested as bearers of electroactive labels and as proper substrates for polymerases used in polymerase chain reaction (PCR) and isothermal recombinase polymerase amplification (RPA) with the aim of electrochemical detection of double-stranded DNA (dsDNA) amplification products. For this purpose, electrochemical behavior of free fluorescein and rhodamine as well as the modified nucleotides, dUTP-Fl and dUTP-Rh, was studied by cyclic (CV) and square wave (SWV) voltammetry on carbon screen printed electrodes. Both free fluorescein and dUTP-Fl underwent a two-step oxidation at the peak potentials (E_p) of 0.6-0.7 V and 0.8-0.9 V (phosphate buffer, pH 7.4). The reduction peaks of fluorescein and dUTP-Fl were registered between -0.9 V and -1 V, but they did not depend on concentration. The free rhodamine and dUTP-Rh have demonstrated the well-defined oxidation peaks at 0.8-0.9 V. In addition, the distinct reduction peaks at E_p between -0.8 V and -0.9 V were registered for both rhodamine and dUTP-Rh. The dUTP-Fl and dUTP-Rh were further tested as substrates to incorporate an electroactive label into 210 or 206 base pair long dsDNA amplicons generated either by PCR or RPA. Among two dUTP derivatives tested, dUTP-Fl revealed significantly better compatibility with PCR and RPA, producing the full-size amplicons at 50-90% substitution of dTTP in the reaction mixture. In the PCR, the best compromise between amplicon output and labeling was achieved at the dUTP-Fl : dTTP and dUTP-Rh : dTTP molar ratios of 70% : 30% and 20% : 80% in the PCR mixture, respectively, allowing the direct electrochemical detection of amplicons at micromolar concentrations. Alongside with fluorescence DNA assays, the fluorescein and rhodamine modified dUTP appear as promising electroactive labels to develop direct electrochemical DNA assays for detecting PCR and RPA products.

Keywords: Fluorescein; Modified nucleotide; Polymerase chain reaction; Recombinase polymerase amplification; Rhodamine; Voltammetric detection.

MeSH terms

DNA* / analysis
Deoxyuridine*
Fluorescein
Polymerase Chain Reaction
Rhodamines

Substances

Rhodamines
Fluorescein
DNA
Deoxyuridine