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

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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.

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作者简介

S. Lapa

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

编辑信件的主要联系方式.
Email: lapa@biochip.ru
俄罗斯联邦, ul. Vavilova 32, Moscow, 119991

P. Chirkova

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: lapa@biochip.ru
俄罗斯联邦, ul. Vavilova 32, Moscow, 119991

S. Surzhikov

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: lapa@biochip.ru
俄罗斯联邦, ul. Vavilova 32, Moscow, 119991

V. Kuznetsova

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: lapa@biochip.ru
俄罗斯联邦, ul. Vavilova 32, Moscow, 119991

V. Shershov

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: lapa@biochip.ru
俄罗斯联邦, ul. Vavilova 32, Moscow, 119991

A. Chudinov

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: lapa@biochip.ru
俄罗斯联邦, ul. Vavilova 32, Moscow, 119991

参考

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  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

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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.

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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).

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