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Study of the multiple incorporation of modified nucleotides into the growing DNA strand

https://doi.org/10.32362/2410-6593-2021-16-2-148-155

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Abstract

Objectives. This study investigated the substrate properties of the modified derivatives of triphosphates of purine and pyrimidine deoxynucleosides (5-propynyl-2’-deoxyuridine-5’-triphosphate, 5-propynyl2’-deoxycytidine-5’-triphosphate, 5-methyl-2’-deoxycytidine-5’-triphosphate, and N6-methyl-2’-deoxyadenosine-5’-triphosphate) during their simultaneous incorporation in enzymatic reactions (polymerase chain and primer extension reactions).

Methods. The real-time polymerase chain and primer extension reactions were used to study the substrate efficiency of modified deoxynucleotide triphosphates. Various pairwise combinations of modified derivatives were used; specially designed synthetic DNA fragments and libraries for the Systematic Evolution of Ligands by Exponential Enrichment technology were used as templates. Reactions were conducted using DNA polymerases: Taq, Vent (exo-), DeepVent (exo-), and KOD XL.

Results. In each case, a pair of compounds (modified dUTP + dCTP, dUTP + dATP, and dCTP + dATP) was selected to study the simultaneous incorporation into the growing DNA strand. The most effective combinations of nucleotides for simultaneous insertion were dU and dC, having 5-propynyl substitution. The Vent (exo-) DNA polymerase was found as the most effective for the modified substrates.

Conclusions. The selected compounds can be used for the enzymatic preparation of modified DNA, including aptamers with extended physicochemical properties. 

About the Authors

O. S. Volkova
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences
Russian Federation


A. V. Chudinov
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences
Russian Federation


S. A. Lapa
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences
Russian Federation


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Supplementary files

1. Fig. 3. PEX electrophoretic analysis with DNA polymerases and specially designed templates.
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2. This is to certify that the paper titled Study of the multiple incorporations of modified nucleotides into the growing DNA strand commissioned to us by Olga S. Volkova, Alexander V. Chudinov, and Sergey A. Lapa has been edited for English language, grammar, punctuation, and spelling by Enago, an editing brand of Crimson Interactive Consulting Co. Ltd.
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The substrate properties of the modified derivatives of purine and pyrimidine dNTPs were studied in enzymatic reactions: real-time PCR and PEX. In the experiments, we used four DNA polymerases of different families with no 3′–5′ correcting exonuclease activity. Both individual and joint pairwise insertions of unlike modified nucleotides into the growing DNA strand were conducted. The most effective nucleotide pairs found were 5-propynyl-2′-deoxyuridine-5′-triphosphate and 5-propynyl-2′-deoxycytidine-5′-triphosphate combined with the Vent DNA polymerase (exo-matric).

For citation:


Volkova O.S., Chudinov A.V., Lapa S.A. Study of the multiple incorporation of modified nucleotides into the growing DNA strand. Fine Chemical Technologies. 2021;16(2):148-155. https://doi.org/10.32362/2410-6593-2021-16-2-148-155

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ISSN 2410-6593 (Print)
ISSN 2686-7575 (Online)