Oscorbin Igor P, Belousova Ekaterina A, Boyarskikh Ulyana A, Zakabunin Aleksandr I, Khrapov Evgeny A, Filipenko Maksim L
Laboratory of Pharmacogenomics, Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Lavrentiev Avenue 8, Novosibirsk 630090, Russian Federation.
Laboratory of genomic technologies, Novosibirsk State University, Pirogova street 2, Novosibirsk 630090, Russian Federation.
Nucleic Acids Res. 2017 Sep 19;45(16):9595-9610. doi: 10.1093/nar/gkx645.
At the moment, one of the actual trends in medical diagnostics is a development of methods for practical applications such as point-of-care testing, POCT or research tools, for example, whole genome amplification, WGA. All the techniques are based on using of specific DNA polymerases having strand displacement activity, high synthetic processivity, fidelity and, most significantly, tolerance to contaminants, appearing from analysed biological samples or collected under purification procedures. Here, we have designed a set of fusion enzymes based on catalytic domain of DNA polymerase I from Geobacillus sp. 777 with DNA-binding domain of DNA ligase Pyrococcus abyssi and Sto7d protein from Sulfolobus tokodaii, analogue of Sso7d. Designed chimeric DNA polymerases DBD-Gss, Sto-Gss and Gss-Sto exhibited the same level of thermal stability, thermal transferase activity and fidelity as native Gss; however, the processivity was increased up to 3-fold, leading to about 4-fold of DNA product in WGA which is much more exiting. The attachment of DNA-binding proteins enhanced the inhibitor tolerance of chimeric polymerases in loop-mediated isothermal amplification to several of the most common DNA sample contaminants-urea and whole blood, heparin, ethylenediaminetetraacetic acid, NaCl, ethanol. Therefore, chimeric Bst-like Gss-polymerase will be promising tool for both WGA and POCT due to increased processivity and inhibitor tolerance.
目前，医学诊断领域的一个实际趋势是开发用于实际应用的方法，如即时检测（POCT）或研究工具，例如全基因组扩增（WGA）。所有这些技术都基于使用具有链置换活性、高合成持续性、保真度且最重要的是对来自分析生物样品或在纯化过程中收集的污染物具有耐受性的特定DNA聚合酶。在此，我们基于嗜热栖热放线菌DNA聚合酶I的催化结构域与深渊热球菌DNA连接酶的DNA结合结构域以及来自嗜热栖热放线菌的Sto7d蛋白（Sso7d的类似物）设计了一组融合酶。设计的嵌合DNA聚合酶DBD-Gss、Sto-Gss和Gss-Sto表现出与天然Gss相同水平的热稳定性、热转移酶活性和保真度；然而，持续性提高了3倍，导致WGA中的DNA产物增加了约4倍，这更令人兴奋。DNA结合蛋白的附着增强了嵌合聚合酶在环介导等温扩增中对几种最常见DNA样品污染物（尿素、全血、肝素、乙二胺四乙酸、氯化钠、乙醇）的耐受性。因此，由于持续性增加和抑制剂耐受性增强，嵌合Bst样Gss聚合酶将成为WGA和POCT的有前途的工具。
