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⁺ ≪ Cu²⁺.