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

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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.

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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

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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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