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.

中文翻译：

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核苷酸背景对 Bst 外切 DNA 聚合酶 DNA 非特异性扩增的影响

摘要

需要具有链置换活性的 DNA 聚合酶的等温核酸扩增在过去二十年中获得了更多关注。在具有链置换活性的 DNA 聚合酶中，Bst exo ^–应用最广泛。然而，它倾向于通过多聚化进行非特异性DNA合成。在这项研究中，报告了核苷酸序列对 Bst exo 与 DNA 结合以及多聚化起始效率的^影响。通过单链三核苷酸 (sst) 的分子对接揭示了Bst exo 的“闭合”形式优先^与富含嘌呤的 DNA 序列结合，尤其是那些在生长链 3' 端含有 dG 的序列三核苷酸双链体（dst）。使用 3'- 和 5'- 末端基序结构不同的寡核苷酸模板在实验中证实了计算机中获得的数据。研究表明，具有寡嘌呤 3' 末端片段和寡嘧啶 5' 末端部分的模板有助于多聚化的较早开始。结果可用于设计适合可靠等温扩增的核苷酸序列。为了避免多聚化，应排除含有末端 dA 和/或 dG 核苷酸的 DNA 模板和引物。