The influence of nucleotide context on non-specific amplification of DNA with Bst exo- DNA polymerase

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Abstract

In recent years, nucleic acid amplification methods that proceed in isothermal mode and require the use of DNA polymerases with strand-displacement activity have become increasingly widespread. Among these, the most popular is the Bst exo- polymerase, but it tends to carry out nonspecific DNA synthesis through multimerization. This study shows the influence of the nucleotide sequence on the binding of Bst exo- with DNA and on the efficiency of multimerization initiation. On single-stranded trinucleotides (sst) and dinucleotide duplexes (dst), molecular docking revealed the preference for binding of the “closed” form of Bst exo- to purine-rich sequences, especially those containing dG at the 3′ end of the synthesized strand. The data obtained in silico were confirmed in experiments using oligonucleotide templates that differ in the structure of the 3′- and 5′-terminal motifs. It has been shown that templates with an oligopurine 3′-terminal fragment and an oligopyrimidine 5′-terminal part contribute to an earlier start of multimerization. The obtained data can be used at the stage of selecting optimal nucleotide sequences for isothermal amplification, ensuring more reliable results. Thus, to avoid multimerization, DNA templates and primers containing terminal dA and/or dG nucleotides should be excluded.

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About the authors

R. R. Garafutdinov

Ufa Federal Research Center, Russian Academy of Sciences

Email: garafutdinovr@gmail.com

Institute of Biochemistry and Genetics

Russian Federation, 450054 Ufa, Bashkortostan

O. Yu. Kupova

Ufa Federal Research Center, Russian Academy of Sciences

Email: garafutdinovr@gmail.com

Institute of Biochemistry and Genetics

Russian Federation, 450054 Ufa, Bashkortostan

A. R. Sakhabutdinova

Ufa Federal Research Center, Russian Academy of Sciences

Author for correspondence.
Email: garafutdinovr@gmail.com

Institute of Biochemistry and Genetics

Russian Federation, 450054 Ufa, Bashkortostan

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Supplementary files

Supplementary Files
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2. Fig. 1. Structure of model single-stranded trinucleotides (sst) and dinucleotide duplexes (dst)

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3. Fig. 2. Average docking score values ​​obtained when considering only nucleotides N2 and N3 (the dinucleotides corresponding to the strongest complexes are marked with an asterisk)

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4. Fig. 3. Diagram of ligand interaction using the example of the Bst exo- complex with dst (the trinucleotide 5'-CAC-3' is given as an example). The amino acids are presented as spheres: purple spheres are amino acids carrying positively charged groups, blue spheres are those containing polar groups, green spheres are hydrophobic, and red spheres are those carrying negatively charged groups; colored lines and arrows show chemical interactions between the amino acids and the environment: red arrows are ionic bonds (salt bridges, sb), blue arrows are hydrogen bonds (H), and no arrows are other weak interactions (+).

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5. Fig. 4. Scheme of the multimerization process: annealing and extension of primer R on DNA template I, leading to the formation of a double-stranded product of length 1L (step 1), formation of the pseudocyclic DNA structure Ini (step 2), elongation of chains in Ini, formation of a double-stranded product of length 2L (steps 3, 4 and 5b) or annealing and elongation of primer R (steps 3, 4 and 5a), formation of template II (step 5a), displacement of DNA template II and annealing of primer F on it (step 6), formation of full-length amplicons with tandem nucleotide sequences (steps i). Primer F and its complementary regions are shown in black, primer R and its complementary regions are shown in gray

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6. Fig. 5. Accumulation of multimerization products for templates with different primary structures of terminal fragments (data obtained using Taq buffer are shown). Insert: electrophoretic separation of multimers: 1 – template LM1, 2 – LM2, 3 – LM3, 4 – LM4, 5 – LM5, 6 – LM6, M – 50 bp DNA ladder marker

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7. Annex 1
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8. Annex 2
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9. Annex 3
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