Substrate efficiency of CY5-modified derivatives of deoxyuridine and deoxycytidine in the rolling circle amplification

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The kinetics of amplification and the features of individual and simultaneous incorporation of modified deoxynucleoside triphosphates in DNA during rolling circle amplification (RCA) have been studied. The study was carried out for six pairs of Sy5-labeled triphosphates of deoxyuridine (dU) and deoxycytidine (dC) previously synthesized with similar fluorescent substituents inside the pair. The effect of the linker length between the fluorophore and the pyrimidine base on the incorporation density was determined: nucleotides with a linker length of six carbon atoms are embedded in a growing DNA chain better than with three carbon atoms. It was found that the combined introduction of triphosphates into the reaction in an equivalent total concentration does not enhance the inhibitory effect, which gives grounds for a more detailed study of the simultaneous use of labeled dU and dC.

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作者简介

P. Chirkova

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: lapa@biochip.ru
俄罗斯联邦, Moscow

S. Surzhikov

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: lapa@biochip.ru
俄罗斯联邦, Moscow

V. Kuznetsova

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: lapa@biochip.ru
俄罗斯联邦, Moscow

V. Shershov

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: lapa@biochip.ru
俄罗斯联邦, Moscow

A. Chudinov

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: lapa@biochip.ru
俄罗斯联邦, Moscow

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, Moscow

参考

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  2. Ali M.M., Li F., Zhang Z., Zhang K., Kang D.K., Ankrum J.A., Le X.C., Zhao W. (2014) Rolling circle amplification: a versatile tool for chemical biology, materials science and medicine. Chem. Soc. Rev. 43, 3324–3341.
  3. Mujumdar R.B., Ernst L.A, Mujumdar S.R., Lewis C.J., Waggoner A.S. (1993) Cyanine dye labeling reagents: sulfoindocyanine succinimidyl esters. Bioconjug. Chem. 4, 105–111.
  4. Ren X., El-Sagheer A.H., Brown T. (2016) Efficient enzymatic synthesis and dual-colour fluorescent labelling of DNA probes using long chain azido-dUTP and BCN dyes. Nucl. Acids Res. 44, е79.
  5. Лапа С.А., Чиркова П.А., Суржиков С.А., Кузнецова В.Е., Шершов В.Е., Чудинов А.В. (2024) Сравнительный анализ поведения Cy5-пиримидиновых нуклеотидов в реакции амплификации по типу катящегося кольца. Биоорган. химия. 50, 1351–1356.
  6. Лапа С.А., Мифтахов Р.А., Клочихина Е.С., Аммур Ю.И., Благодатских С.А., Шершов В.Е., Заседателев А.С., Чудинов А.В. (2021) Разработка мультиплексной ОТ-ПЦР с иммобилизованными праймерами для идентификации возбудителей инфекционной пневмонии человека. Молекуляр. биология. 55, 944–955.
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  9. Шершов В.Е., Лапа С.А., Левашова А.И., Шишкин И.Ю., Штылев Г.Ф., Шекалова Е.Ю., Василисков В.А., Заседателев А.С., Кузнецова В.Е., Чудинов А.В. (2023) Синтез флуоресцентно-меченых нуклеотидов для маркирования продуктов изотермической амплификации. Биоорган. химия. 49, 649–656.
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2. Fig. 1. Structural formulas of nitrogenous bases and fluorophore. a — fluorescently labeled 5-allylamine-2ꞌ-deoxyuridine-5ꞌ-triphosphate (dUTP-Dye); b — fluorescently labeled 5-allylamine-2ꞌ-deoxycytidine-5ꞌ-triphosphate (dCTP-Dye); c — fluorescent dye (Dye). R1–4 are given in Table 1.

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3. Fig. 2. Kinetics of RCA amplification with individual and combined introduction of labeled deoxynucleoside triphosphates with equivalent total concentration (explanations in the text). For clarity, the curves in each group are normalized to the maximum value of the fluorescent signal.

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4. Fig. 3. Visualization of the degree of purification of reaction products from free fluorescently labeled nucleoside triphosphates in a dual-channel excitation/detection mode (“green” channel 530/585 nm and “red” channel 630/690 nm). a — Electropherogram of RCA products before purification; b — after purification. L — dsDNA length marker GeneRuler 50 bp; 1 — dUk; 2 — dCk; 3 — dUk+dCk; 4 — dU3; 5 — dC3; 6 — dU3+dC3; 7 — dU5; 8 — dC5; 9 — dU5+dC5.

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5. Fig. 4. Effect of linker length on the density of Cy5 nucleotide incorporation into RCA. a — RCA visualization in the “green” channel 530/585 nm; b — in the “red” channel 630/690 nm; c — in dual-channel excitation and detection mode. L — dsDNA length marker GeneRuler 50 bp; 1 — dUк; 2 — dCк; 3 — dUк + dCк; 4 — dU3; 5 — dC3; 6 — dU3 + dC3; 7 — dU5; 8 — dC5; 9 — dU5 + dC5.

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