title: Consecutive incorporation of functionalized nucleotides with amphiphilic side chains by novel KOD polymerase mutant.
abstract: Recently, 7-substituted 7-deazapurine nucleoside triphosphates and 5-substituted pyrimidine nucleoside triphosphates (dN(am)TPs) were synthesized to extend enzymatically using commercially available polymerase. However, extension was limited when we attempted to incorporate the substrates consecutively. To address this, we have produced a mutant polymerase that can efficiently accept the modified nucleotide with amphiphilic groups as substrates. Here we show that the KOD polymerase mutant, KOD exo(-)/A485L, had the ability to incorporate dN(am)TP continuously over 50nt, indicating that the mutant is sufficient for generating functional nucleic acid molecules.
authors.affiliation: Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan; National Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN), 7-6-8 Saito-Asagi, Ibaraki, Osaka 567-0085, Japan.
authors.forename: Hidekazu
authors.initials: H
authors.lastname: Hoshino
authors.affiliation: Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan; National Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN), 7-6-8 Saito-Asagi, Ibaraki, Osaka 567-0085, Japan.
authors.forename: Yuuya
authors.initials: Y
authors.lastname: Kasahara
authors.affiliation: Graduate School of Science and Technology, Gunma University, 1-5-1 Tenjin-cho, Kiryu, Gunma 376-8515, Japan.
authors.forename: Hiroto
authors.initials: H
authors.lastname: Fujita
authors.affiliation: Graduate School of Science and Technology, Gunma University, 1-5-1 Tenjin-cho, Kiryu, Gunma 376-8515, Japan. Electronic address: mkuwa@gunma-u.ac.jp.
authors.forename: Masayasu
authors.initials: M
authors.lastname: Kuwahara
authors.affiliation: Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan; National Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN), 7-6-8 Saito-Asagi, Ibaraki, Osaka 567-0085, Japan.
authors.forename: Kunihiko
authors.initials: K
authors.lastname: Morihiro
authors.affiliation: National Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN), 7-6-8 Saito-Asagi, Ibaraki, Osaka 567-0085, Japan.
authors.forename: Shin-Ichi
authors.initials: SI
authors.lastname: Tsunoda
authors.affiliation: Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan; National Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN), 7-6-8 Saito-Asagi, Ibaraki, Osaka 567-0085, Japan. Electronic address: obika@phs.osaka-u.ac.jp.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Obika
mesh_terms: D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics; D009838:Oligodeoxyribonucleotides / Q000737:chemistry* / Q000235:genetics; D017354:Point Mutation; D011092:Polyethylene Glycols / Q000737:chemistry; D011685:Purine Nucleotides / Q000737:chemistry* / Q000235:genetics; D011742:Pyrimidine Nucleotides / Q000737:chemistry* / Q000235:genetics; D013696:Temperature
chemical_list: D009838:Oligodeoxyribonucleotides; D011685:Purine Nucleotides; D011742:Pyrimidine Nucleotides; D011092:Polyethylene Glycols; D004259:DNA-Directed DNA Polymerase
keywords: DNA polymerase mutant; High-density incorporation; KOD polymerase; Modified nucleoside triphosphate; Primer extension