Influence of Nucleotide Context on Non-Specific Amplification of DNA with Bst exo^– DNA Polymerase

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Ravil R. Garafutdinov^1,a*, Olga Yu. Kupova¹, and Assol R. Sakhabutdinova¹

¹Institute of Biochemistry and Genetics, Ufa Federal Research Center, Russian Academy of Sciences, 450054 Ufa, Russia

Received September 11, 2023; Revised November 30, 2023; Accepted December 2, 2023

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Isothermal nucleic acids amplification that requires DNA polymerases with strand-displacement activity gained more attention in the last two decades. Among the DNA polymerases with strand-displacement activity, Bst exo^– is the most widely used. However, it tends to carry out nonspecific DNA synthesis through multimerization. In this study, the effect of nucleotide sequence on the Bst exo^– binding with DNA and on the efficiency of multimerization initiation, are reported. Preference for binding of the “closed” form of Bst exo^– to the purine-rich DNA sequences, especially those containing dG at the 3′-end of the growing chain was revealed using molecular docking of the single-stranded trinucleotides (sst) and trinucleotide duplexes (dst). The data obtained in silico were confirmed in the experiments using oligonucleotide templates that differ in the structure of the 3′- and 5′-terminal motifs. It has been shown that templates with the oligopurine 3′-terminal fragment and oligopyrimidine 5′-terminal part contribute to the earlier start of multimerization. The results can be used for design of nucleotide sequences suitable for reliable isothermal amplification. To avoid multimerization, DNA templates and primers containing terminal dA and/or dG nucleotides should be excluded.

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KEY WORDS: Bst DNA polymerase, isothermal amplification, multimerization, specificity, molecular docking

DOI: 10.1134/S0006297924010036

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