Comparative Behavior Analysis of Cy5-Pyrimidine Nucleotides in the Rolling Circle Amplification

Cover Page

Cite item

Full Text

Open Access Open Access
Restricted Access Access granted
Restricted Access Subscription Access

Abstract

Two pairs of Cy-5-labeled dU and dC triphosphates with similar electroneutral fluorophore structures differing in the length of the hydrocarbon linker between the fluorophore and the nitrogenous base were synthesized. A comparative analysis of their substrate behavior in the rolling circle amplification (RCA) using Bst 3.0 DNA polymerase was carried out. It was found that nucleotides with a long linker between the fluorophore and pyrimidine base are more efficiently incorporated into the growing DNA chain, while nucleotides with a short linker inhibit RCA less. In each of the dU and dC pairs with similar fluorophores and linkers, fluorescently labeled uridine derivatives demonstrated a high embedding density. It was found that with simultaneous incorporation of labeled dU and dC, the inhibitory effect does not summarise. This gives grounds for a more careful study of various Cy5-dC variants in order to increase a sensitivity of the analysis with simultaneous introduction of labeled dU and dC.

Full Text

Restricted Access

About the authors

S. A. Lapa

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Author for correspondence.
Email: lapa@biochip.ru
Russian Federation, ul. Vavilova 32, Moscow, 119991

P. A. Chirkova

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: lapa@biochip.ru
Russian Federation, ul. Vavilova 32, Moscow, 119991

S. A. Surzhikov

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: lapa@biochip.ru
Russian Federation, ul. Vavilova 32, Moscow, 119991

V. E. Kuznetsova

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: lapa@biochip.ru
Russian Federation, ul. Vavilova 32, Moscow, 119991

V. E. Shershov

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: lapa@biochip.ru
Russian Federation, ul. Vavilova 32, Moscow, 119991

A. V. Chudinov

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: lapa@biochip.ru
Russian Federation, ul. Vavilova 32, Moscow, 119991

References

  1. Ali M.M., Li F., Zhang Z., Zhang K., Kang D.K., Ankrum J.A., Le X.C., Zhao W. // Chem. Soc. Rev. 2014. V. 43. P. 3324–3341. https://doi.org/10.1039/c3cs60439j
  2. Mori Y., Notomi T. // J. Infect. Chemother. 2009. V. 15. P. 62–69. https://doi.org/10.1007/s10156-009-0669-9
  3. Piepenburg O., Williams C.H., Stemple D.L., Armes N.A. // PLoS Biol. 2006. V. 4. P. e204. https://doi.org/10.1371/journal.pbio.0040204
  4. Чемисова О.С., Цырулина О.А., Трухачев А.Л., Носков А.К. // Журнал микробиологии, эпидемиологии и иммунобиологии. 2022. Т. 99. C. 126–138. https://doi.org/10.36233/0372-9311-176
  5. Lapa S.A., Volkova O.S., Spitsyn M.A., Shershov V.E., Kuznetsova V.E., Guseinov T.O., Zasedatelev A.S., Chudinov A.V. // Russ. J. Bioorg. Chem. 2019. V. 45. P. 263–272. https://doi.org/10.1134/S0132342319040043
  6. Lapa S.A., Guseinov T.O., Pavlov A.S., Shershov V.E., Kuznetsova V.E., Zasedatelev A.S., Chudinov A.V. // Russ. J. Bioorg. Chem. 2020. V. 46. P. 557–562. https://doi.org/10.31857/S0132342320040168
  7. Spitsyn M.A., Kuznetsova V.E., Shershov V.E., Emelyanova М.А., Guseinov T.O., Lapa S.A., Nasedkina T.V., Zasedatelev A.S., Chudinov A.V. // Dyes Pigments. 2017. V. 147. P. 199–210. https://doi.org/10.1016/j.dyepig.2017.07.052
  8. 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. // Russ. J. Bioorg. Chem. 2023. V. 49. P. 1151–1158. https://doi.org/10.1134/S1068162023050242
  9. Lapa S.A., Volkova O.S., Kuznetsova V.E., Zasedatelev A.S., Chudinov A.V. // Mol. Biol. 2022. V. 56. P. 115–123. https://doi.org/10.31857/S0026898422010050
  10. Lapa S.A., Miftakhov R.A., Klochikhina E.S., Ammur Y.I., Blagodatskikh S.A., Shershov V.E., Zasedatelev A.S., Chudinov A.V. // Mol. Biol. 2021. V. 55. P. 828–838. https://doi.org/10.1134/S0026893321040063

Supplementary files

Supplementary Files
Action
1. JATS XML
2. Fig. 1. Fluorophore and the linker that binds it to the 5-position of the nitrogenous base. B is the nitrogenous base. Other explanations are given in the text.

Download (97KB)
3. Fig. 2. (a) – RCA amplification kinetics using dUL and dCL as an example, both individually and in combination; (b) – electropherogram of RCA results in dual excitation/detection mode (“green” channel 530/585 nm and “red” channel 630/690 nm). L – double-stranded DNA length marker GeneRuler 50 bp (Thermo, Latvia).

Download (212KB)

Copyright (c) 2024 Russian Academy of Sciences