Nucleotide Modification and Polymerase Engineering for Creating a Novel Class of Artificial Nucleic Acid Aptamers

Abstract

Enzymatic transcription and reverse transcription of artificial nucleic acids would be an important technique to allow the application of artificial nucleic acids to random screening methods of nucleic acids such as systematic evolution of ligands by exponential enrichment (SELEX), Non-SELEX selection, one-step selection etc. These random screening methods would be able to identify various functional nucleic acids such as aptamers and ribozymes with functions similar to antibodies and enzymes, which could be useful not only as research tools for molecular biology but also as diagnostic agents, therapeutic drugs etc. 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 and phosphate-modified ones. 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/ phosphate modifications. Experimental data indicate that their characteristic kinetic properties enabled recognition of various modified nucleotides. Among those KOD mutants, one achieved efficient successive incorporation of bridged nucleotides with a 2′-ONHCH₂CH₂-4′ linkage, which would be promising candidates for nucleic acid drugs. 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.