Abstract
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.
Abbreviations
- dst:
-
double-stranded trinucleotide
- LID:
-
ligand interaction diagram
- LT:
-
linear template
- MM:
-
multimerization
- NA:
-
nucleic acids
- sb:
-
ionic bonds (salt bridges)
- sst:
-
single-stranded trinucleotide
References
Bodulev, O. L., and Sakharov, I. Y. (2020) Isothermal nucleic acid amplification techniques and their use in bioanalysis, Biochemistry (Moscow), 85, 147-166, https://doi.org/10.1134/S0006297920020030.
Soroka, M., Wasowicz, B., and Rymaszewska, A. (2021) Loop-mediated isothermal amplification (LAMP): the better sibling of PCR? Cells, 10, 1931, https://doi.org/10.3390/cells10081931.
Garafutdinov, R. R., Sakhabutdinova, A. R., Gilvanov, A. R., and Chemeris, A. V. (2021) Rolling circle amplification as a universal method for the analysis of a wide range of biological targets, Russ. J. Bioorg. Chem., 47, 1172-1189, https://doi.org/10.1134/S1068162021060078.
Kuznetsova, A. A., Fedorova, O. S., and Kuznetsov, N. A. (2022) Structural and molecular kinetic features of activities of DNA polymerases, Int. J. Mol. Sci., 23, 6373, https://doi.org/10.3390/ijms23126373.
Oscorbin, I., and Filipenko, M. (2023) Bst polymerase – a humble relative of Taq polymerase, Comput. Struct. Biotechnol. J., 21, 4519-4535, https://doi.org/10.1016/j.csbj.2023.09.008.
Hafner, G. J., Yang, I. C., Wolter, L. C., Stafford, M. R., and Giffard, P. M. (2001) Isothermal amplification and multimerization of DNA by Bst DNA polymerase, BioTechniques, 30, 852-886, https://doi.org/10.2144/01304rr03.
Garafutdinov, R. R., Gilvanov, A. R., and Sakhabutdinova, A. R. (2020) The influence of reaction conditions on DNA multimerization during isothermal amplification with Bst DNA polymerase, Appl. Biochem. Biotechnol., 190, 758-771, https://doi.org/10.1007/s12010-019-03127-6.
Wang, G., Ding, X., Hu, J., Wu, W., Sun, J., and Mu, Y. (2017) Unusual isothermal multimerization and amplification by the strand-displacing DNA polymerases with reverse transcription activities, Sci. Rep., 7, 13928, https://doi.org/10.1038/s41598-017-13324-0.
Sakhabutdinova, A. R., Kamalov, M. I., Salakhieva, D. V., Mavzyutov, A. R., and Garafutdinov, R. R. (2021) Inhibition of nonspecific polymerase activity using poly(aspartic) acid as a model anionic polyelectrolyte, Anal. Biochem., 628, 114267, https://doi.org/10.1016/j.ab.2021.114267.
Zyrina, N. V., and Antipova, V. N. (2021) Nonspecific Synthesis in the reactions of isothermal nucleic acid amplification, Biochemistry (Moscow), 86, 887-897, https://doi.org/10.1134/S0006297921070099.
Rolando, J. C., Jue, E., Barlow, J. T., and Ismagilov, R. F. (2020) Real-time kinetics and high-resolution melt curves in single-molecule digital LAMP to differentiate and study specific and non-specific amplification, Nucleic Acids Res., 48, e42, https://doi.org/10.1093/nar/gkaa099.
Reid, M. S., Le, X. C., and Zhang, H. (2018) Exponential isothermal amplification of nucleic acids and assays for proteins, cells, small molecules, and enzyme activities: an EXPAR example, Angew. Chem. Int. Ed. Engl., 57, 11856-11866, https://doi.org/10.1002/anie.201712217.
Tan, E., Erwin, B., Dames, S., Ferguson, T., Buechel, M., Irvine, B., Voelkerding, K., and Niemz, A. (2008) Specific versus nonspecific isothermal DNA amplification through thermophilic polymerase and nicking enzyme activities, Biochemistry, 47, 9987-9999, https://doi.org/10.1021/bi800746p.
Qian, J., Ferguson, T. M., Shinde, D. N., Ramírez-Borrero, A. J., Hintze, A., Adami, C., and Niemz, A. (2012) Sequence dependence of isothermal DNA amplification via EXPAR, Nucleic Acids Res., 40, e87, https://doi.org/10.1093/nar/gks230.
Garafutdinov, R. R., Sakhabutdinova, A. R., Kupryushkin, M. S., and Pyshnyi, D. V. (2020) Prevention of DNA multimerization during isothermal amplification with Bst exo–DNA polymerase, Biochimie, 168, 259-267, https://doi.org/10.1016/j.biochi.2019.11.013.
Sakhabutdinova, A. R., Mirsaeva, L. R., Oscorbin, I. P., Filipenko, M. L., and Garafutdinov, R. R. (2020) Elimination of DNA multimerization arising from isothermal amplification in the presence of Bst exo– DNA polymerase, Russ. J. Bioorg. Chem., 46, 52-59, https://doi.org/10.1134/S1068162020010082.
Garafutdinov, R. R., Gilvanov, A. R., Kupova, O. Y., and Sakhabutdinova, A. R. (2020) Effect of metal ions on isothermal amplification with Bst exo– DNA polymerase, Int. J. Biol. Macromol., 161, 1447-1455, https://doi.org/10.1016/j.ijbiomac.2020.08.028.
Schrödinger Suite, Small-Molecule Drug Discovery Suite 2016-4, Schrödinger, LLC, New York, 2016.
Garafutdinov, R. R., Kupova, O. Y., and Sakhabutdinova, A. R. (2020) Data on molecular docking simulations of quaternary complexes 'Bst exo– polymerase-DNA-dCTP-metal cations', Data-in-Brief, 33, 106549, https://doi.org/10.1016/j.dib.2020.106549.
Protein Preparation Wizard; Epik, Schrödinger, LLC, New York, NY, 2016; Impact, Schrödinger, LLC, New York, NY, 2016; Prime, Schrödinger, LLC, New York, NY, 2016.
Sastry, G. M., Adzhigirey, M., Day, T., Annabhimoju, R., and Sherman, W. (2013) Protein and ligand preparation: parameters, protocols, and influence on virtual screening enrichments, J. Comput. Aid. Mol. Des., 27, 221-234, https://doi.org/10.1007/s10822-013-9644-8.
Wu, E. Y., and Beese, L. S. (2011) The structure of a high fidelity DNA polymerase bound to a mismatched nucleotide reveals an “ajar” intermediate conformation in the nucleotide selection mechanism, J. Biol. Chem., 286, 19758-19767, https://doi.org/10.1074/jbc.M110.191130.
Knorre, D. G., Lavrik, O. I., and Nevinsky, G. A. (1988) Protein-nucleic acid interaction in reactions catalyzed with DNA polymerases, Biochimie, 70, 655-661, https://doi.org/10.1016/0300-9084(88)90250-7.
Kolocheva, T. I., Nevinsky, G. A., Levina, A. S., Khomov, V. V., and Lavrik, O. I. (1991) The mechanism of recognition of templates by DNA polymerases from pro- and eukaryotes as revealed by affinity modification data, J. Biomol. Struct. Dyn., 9, 169-186, https://doi.org/10.1080/07391102.1991.10507901.
Doronin, S. V., Nevinsky, G. A., Malygina, T. O., Podust, V. N., Khomov, V. V., and Lavrik, O. I. (1989) The efficiency of interaction of deoxyribonucleoside-5'-mono-, di- and triphosphates with the active center of E. coli DNA polymerase I Klenow fragment, FEBS Lett., 259, 83-85, https://doi.org/10.1016/0014-5793(89)81500-5.
Kolocheva, T. I., Nevinsky, G. A., Volchkova, V. A., Levina, A. S., Khomov, V. V., and Lavrik, O. I. (1989) DNA polymerase I (Klenow fragment): role of the structure and length of a template in enzyme recognition, FEBS Lett., 248, 97-100, https://doi.org/10.1016/0014-5793(89)80439-9.
Garafutdinov, R. R., Burkhanova, G. F., Maksimov, I. V., Sakhabutdinova, A. R. (2023) New method for microRNA detection based on multimerization, Anal. Biochem., 664, 115049, https://doi.org/10.1016/j.ab.2023.115049.
Sakhabutdinova, A. R., Chemeris, A. V., and Garafutdinov, R. R. (2023) Detection of specific RNA targets by multimerization, Biochemistry (Moscow), 88, 679-686, https://doi.org/10.1134/S0006297923050103.
Acknowledgments
The instruments and equipment of the regional Collective Use Centers “Agidel” and “Biomics” were used in the study.
Funding
This work was supported by the Russian Science Foundation (project no. 22-24-00235).
Author information
Authors and Affiliations
Contributions
R.R.G. planned experiments, supervised the study, and edited the manuscript. O.Yu.K. planned and performed in silico experiments, discussed the results. A.R.S. planned and performed experiments, discussed the results, and prepared the manuscript.
Corresponding author
Ethics declarations
This work does not contain any studies involving human and animal subjects. The authors of this work declare that they have no conflicts of interest.
Additional information
Publisher’s Note. Pleiades Publishing remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
Rights and permissions
About this article
Cite this article
Garafutdinov, R.R., Kupova, O.Y. & Sakhabutdinova, A.R. Influence of Nucleotide Context on Non-Specific Amplification of DNA with Bst exo– DNA Polymerase. Biochemistry Moscow 89, 53–64 (2024). https://doi.org/10.1134/S0006297924010036
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1134/S0006297924010036