Investigation of the substrate properties of fluorescently labeled pyrimidine triphosphates in recombinase polymerase amplification

Objectives. To study the substrate properties of Cy5-labeled deoxynucleoside triphosphates of various natures (dU and dC) in the process of incorporation in the DNA chain during recombinase polymerase amplification (RPA).
Methods. The work used the real-time RPA method. The method of horizontal electrophoresis was used to control the quality of the amplification products obtained.
Results. The influence of the fluorophore structure and linker lengths on the substrate properties for deoxynucleoside triphosphates Cy5-dUTP and Cy5-dCTP was studied. The following values of the substrate efficiency parameters were determined: amplification efficiency (kinetic indicator), normalized product yield, and embedding coefficient.
Conclusions. Modified deoxynucleoside triphosphates (dNTP) with long linkers between the fluorophore and the nitrogenous base, as well as between the quaternary ammonium group and the second heterocycle of the fluorophore, showed greater substrate efficiency than fluorescently labeled dNTP with short linkers. The modified dU in each pair demonstrated greater substrate efficiency compared to the modified dC.

1. Boháčová S., Ludvíková L., Poštová Slavětínská L., Vaníková Z., Klán P., Hocek M. Protected 5-(hydroxymethyl)uracil nucleotides bearing visible-light photocleavable groups as building blocks for polymerase synthesis of photocaged DNA. Org. Biomol. Chem. 2018;16(9):1527–1535. https:// doi.org/10.1039/c8ob00160j

2. Dziuba D., Pohl R., Hocek M. Bodipy-labeled nucleoside triphosphates for polymerase synthesis of fluorescent DNA. Bioconjug. Chem. 2014;25(11):1984–1995. https://doi.org/10.1021/bc5003554

3. Wynne S.A., Pinheiro V.B., Holliger P., Leslie A.G.W. Structures of an Apo and a Binary Complex of an Evolved Archeal B Family DNA Polymerase Capable of Synthesising Highly Cy-Dye Labelled DNA. PLoS ONE. 2013;8(8):e70892. https://doi.org/10.1371/journal.pone.0070892

4. Ramsay N., Jemth A.S., Brown A., Crampton N., Dear P., Holliger P. CyDNA: Synthesis and Replication of Highly Cy-Dye Substituted DNA by an Evolved Polymerase. J. Am. Chem. Soc. 2010;132(14):5096–5104. https://doi.org/10.1021/ja909180c

5. Shershov V.E., Lapa S.A., Kuznetsova V.E., Spitsyn M.A., Guseinov T.O., Polyakov S.A., Stomahin A.A., Zasedatelev A.S., Chudinov A.V. Comparative Study of Novel Fluorescent Cyanine Nucleotides: Hybridization Analysis of Labeled PCR Products Using a Biochip. J. Fluoresc. 2017;27(6):2001–2016. https://doi.org/10.1007/s10895-017-2139-6

6. Bertocchi F., Delledonne A., Vargas-Nadal G., Terenziani F., Painelli A., Sissa C. Aggregates of Cyanine Dyes: When Molecular Vibrations and Electrostatic Screening Make the Difference. J. Phys. Chem. C. 2023;127(21):10185–10196. https://doi.org/10.1021/acs.jpcc.3c01253

7. Shershov V.E., Lapa S.A., Levashova A.I., et al. Synthesis of Fluorescent-Labeled Nucleotides for Labeling of Isothermal Amplification Products. Russ. J. Bioorg. Chem. 2023;49(5):1115–1158. https://doi.org/10.1134/S1068162023050242 [Original Russian Text: Shershov V.E., Lapa S.A., Levashova A.I., Shishkin I.Yu., Shtylev G.F., Shekalova E.Yu., Vasiliskov V.A., Zasedatelev A.S., Kuznetsova V.E., Chudinov A.V. Synthesis of Fluorescent-Labeled Nucleotides for Labeling of Isothermal Amplification Products. Bioorganicheskaya khimiya. 2023;49(6):649–656 (in Russ.). https://doi.org/10.31857/S0132342323050056 ]

8. Telser J., Cruickshank K.A., Morrison L.E., Netzel T.L. Synthesis and characterization of DNA oligomers and duplexes containing covalently attached molecular labels: comparison of biotin, fluorescein, and pyrene labels by thermodynamic and optical spectroscopic measurements. J. Am. Chem. Soc. 1989;111(18):6966–6976. https://doi.org/10.1021/ja00200a011

9. Ren X., El-Sagheer A.H., Brown T. Efficient enzymatic synthesis and dual-colour fluorescent labelling of DNA probes using long chain azido-dUTP and BCN dyes. Nucleic Acids Res. 2016;44(8):e79. https://doi.org/10.1093/nar/gkw028

10. Lapa S.A., Volkova O.S., Spitsyn M.A., et al. Amplification Efficiency and Substrate Properties of Fluorescently Labeled Deoxyuridine Triphosphates in PCR in the Presence of DNA Polymerases without 3’-5’ Exonuclease Activity. Russ. J. Bioorg. Chem. 2019;45(4):263–272 (in Russ.). https://doi.org/10.1134/S1068162019040046 [Original Russian Text: Lapa S.A., Volkova O.S., Spitsyn M.A., Shershov V.E., Kuznetsova V.E., Guseinov T.O., Zasedatelev A.S., Chudinov A.V. Amplification Efficiency and Substrate Properties of Fluorescently Labeled Deoxyuridine Triphosphates in PCR in the Presence of DNA Polymerases without 3’-5’ Exonuclease Activity. Bioorganicheskaya khimiya. 2019;45(4):392–402 (in Russ.). https://doi.org/10.1134/S0132342319040043 ]

11. Piepenburg O., Williams C.H., Stemple D.L., Armes N.A. DNA detection using recombination proteins. PLoS Biol. 2006;4(7):e204. https://doi.org/10.1371/journal.pbio.0040204

12. Bondareva O.S., Baturin A.A., Mironova A.V. Recombinase polymerase amplification: method’s characteristics and applications in diagnostics of infectious diseases. Zhurnal mikrobiologii, epidemiologii i immunobiologii = Journal of Microbiology, Epidemiology and Immunobiology. 2024;101(2):270–280 (in Russ.). https://doi.org/10.36233/0372-9311-470

13. Lobato I.M., O’Sullivan C.K. Recombinase polymerase amplification: Basics, applications and recent advances. Trends Analyt. Chem. 2018;98:19–35. https://doi.org/10.1016/j.trac.2017.10.015.

14. Lapa S.A., Miftakhov R.A., Klochikhina E.S., et al. Development of Multiplex RT-PCR with Immobilized Primers for Identification of Infectious Human Pneumonia Pathogens. Mol. Biol. 2021;55(6):828–838. https://doi.org/10.1134/S0026893321040063 [Original Russian Text: Lapa S.A., Miftakhov R.A., Klochikhina E.S., Ammour Yu.I., Blagodatskikh S.A., Shershov V.E., Zasedatelev A.S., Chudinov A.V. Development of Multiplex RT-PCR with Immobilized Primers for Identification of Infectious Human Pneumonia Pathogens. Molekulyarnaya biologiya. 2021;55(6):944–955 (in Russ.). https://doi.org/10.31857/S0026898421050062 ]

15. Ramakers C., Ruijter J.M., Deprez R.H., Moorman A.F. Assumption – free analysis of quantitative real-time polymerase chain reaction (PCR) data. Neurosci. Lett. 2003;339(1):62–66. https://doi.org/10.1016/s0304-3940(02)01423-4