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

Masayasu Kuwahara,Satoshi Obika,Yuuki Takano,Akio Sugiyama,Hiroaki Ozaki,Yuuya Kasahara,Hiroaki Sawai,Hiroki Nara

doi:10.3390/molecules15118229

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.

Highlights

To diversify the ability and enhance the nuclease resistance of functional nucleic acids like ribozymes and aptamers [1,2,3,4], enzymatic production of various modified nucleic acids has been investigated [5,6,7,8,9,10,11,12,13,14,15]
Four KOD mutants (KOD1–3, 8) and Vent(exo-) DNA polymerase were used as the enzyme, and thymidine analogues 1 and 2, thymidine-5′-triphosphate (TTP) and 2′-deoxycytidine-5′-triphospate, were used as the substrates
KOD mutants and Vent(exo-) DNA polymerase. This catalytic property of KOD mutants would be favourable for enzymatic production of artificial nucleic acids, that is, large Vmax values of KOD

Summary

Introduction

To diversify the ability and enhance the nuclease resistance of functional nucleic acids like ribozymes and aptamers [1,2,3,4], enzymatic production of various modified nucleic acids has been investigated [5,6,7,8,9,10,11,12,13,14,15]. A series of experimental results from our laboratories indicated that KOD Dash DNA polymerase was more tolerant than any of the other aforementioned polymerases, towards base-modification, and sugar-modification, and would be excellent for this purpose [17,18,19,20]. This is further supported by recent reports [21, 22] regarding enzymatic production of bridged/locked nucleic acid (BNA [23, 24]/LNA [25]) using KOD DNA polymerase. Biolabs) were used as reference materials; Vent(exo-) is a genetically engineered form of the native DNA polymerase, which belongs to family B, from Thermococcus litoralis [31, 32]

Results and Discussion

Synthesis of modified nucleoside triphosphate analogs and their intermediates

Preparation of mutated KOD DNA polymerases

Conclusions