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

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.

Effect of metal ions on isothermal amplification with Bst exo- DNA polymerase.

Over the last two decades, the isothermal amplification has become actively used for nucleic acids analysis. To perform isothermal techniques, DNA polymerases with strand-displacement activity are needed, and Bst exo- polymerase is one of the most widely used. However, Bst exo- is prone to non-specific DNA synthesis (e.g., DNA multimerization) occurring in the absence of the DNA target of interest. Here, we report on the activity of Bst exo- in the presence of Mg2+, Mn2+, Ca2+, Cd2+, Co2+, Cu2+, Ni2+ and Zn2+ in the model molecular systems which included amplification of circular and linear DNA templates; conditions providing effective and highly specific isothermal amplification were determined. It was found that amplification can proceed not only with Mg2+ but with Mn2+, Ca2+, Cd2+ and Cu2+ depending on the type of Bst exo- polymerase and the buffer. Manganese ions turned out to be the most suitable alternative cofactor, which prevents multimerization in some buffers. Molecular docking simulations showed the highest stability for the quaternary 'polymerase-DNA-triphosphate-cations' complexes containing Mg2+ and Mn2+, and the moderate one for complexes with Ca2+, Cd2+ and Cu2+. The frequency of nucleotide misincorporation increased in the following row: Mg2+ ≈ Mn2+ ≤ Cd2+ < Ca2+ â‰ª Cu2+.

Data on molecular docking simulations of quaternary complexes 'Bst exo- polymerase-DNA-dCTP-metal cations'.

This article reports data related to the research article entitled "Effect of metal ions on isothermal amplification with Bst exo- DNA polymerase" (R.R. Garafutdinov, A.R. Gilvanov, O.Y. Kupova, A.R. Sakhabutdinova, 2020) [1]. Here, the results of molecular simulations of the complexes of Bst exo- DNA polymerase with dCTP triphosphate, double-stranded DNA and divalent metal cations are presented. Energetic parameters, number and type of chemical bonds formed by dCTP with the environment are given.