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

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Abstract

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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About the authors

P. A. Chirkova

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: lapa@biochip.ru
Russian Federation, Moscow

S. A. Surzhikov

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: lapa@biochip.ru
Russian Federation, Moscow

V. E. Kuznetsova

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: lapa@biochip.ru
Russian Federation, Moscow

V. E. Shershov

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: lapa@biochip.ru
Russian Federation, Moscow

A. V. Chudinov

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: lapa@biochip.ru
Russian Federation, Moscow

S. A. Lapa

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Author for correspondence.
Email: lapa@biochip.ru
Russian Federation, Moscow

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Supplementary files

Supplementary Files
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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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