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Molecules 2010, 15(11), 8229-8240; doi:10.3390/molecules15118229

Study on Suitability of KOD DNA Polymerase for Enzymatic Production of Artificial Nucleic Acids Using Base/Sugar Modified Nucleoside Triphosphates

1
Graduate School of Engineering, Gunma University, 1-5-1 Tenjin-cho, Kiryu, Gunma 376-8515, Japan
2
Tsuruga Institute of Biotechnology, Toyobo Co., Ltd., 10-24 Toyo-cho, Tsuruga, Fukui 914-0047, Japan
3
Graduate School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871, Japan
*
Author to whom correspondence should be addressed.
Received: 30 September 2010 / Revised: 6 November 2010 / Accepted: 10 November 2010 / Published: 12 November 2010
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Abstract

Recently, KOD and its related DNA polymerases have been used for preparing various modified nucleic acids, including not only base-modified nucleic acids, but also sugar-modified ones, such as bridged/locked nucleic acid (BNA/LNA) which would be promising candidates for nucleic acid drugs. However, thus far, reasons for the effectiveness of KOD DNA polymerase for such purposes have not been clearly elucidated. Therefore, using mutated KOD DNA polymerases, we studied here their catalytic properties upon enzymatic incorporation of nucleotide analogues with base/sugar modifications. Experimental data indicate that their characteristic kinetic properties enabled incorporation of various modified nucleotides. Among those KOD mutants, one achieved efficient successive incorporation of bridged nucleotides with a 2′-ONHCH2CH2-4′ linkage. In this study, the characteristic kinetic properties of KOD DNA polymerase for modified nucleoside triphosphates were shown, and the effectiveness of genetic engineering in improvement of the enzyme for modified nucleotide polymerization has been demonstrated.
Keywords: KOD DNA polymerase; artificial nucleic acids; modified nucleoside triphosphates KOD DNA polymerase; artificial nucleic acids; modified nucleoside triphosphates
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MDPI and ACS Style

Kuwahara, M.; Takano, Y.; Kasahara, Y.; Nara, H.; Ozaki, H.; Sawai, H.; Sugiyama, A.; Obika, S. Study on Suitability of KOD DNA Polymerase for Enzymatic Production of Artificial Nucleic Acids Using Base/Sugar Modified Nucleoside Triphosphates. Molecules 2010, 15, 8229-8240.

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