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

Enzymatic transcription and reverse transcrip- tion 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 system- atic 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 diag- nostic agents, therapeutic drugs etc. Recently, KOD and its re- lated DNA polymerases have been used for preparing various modified nucleic acids, including not only base-modified nu- cleic 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 poly- merases, we studied here their catalytic properties upon enzy- matic 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 nucleo- tides with a 2�c -ONHCH2CH2-4�c linkage, which would be prom- ising candidates for nucleic acid drugs. In this study, the char- acteristic kinetic properties of KOD DNA polymerase for modified nucleoside triphosphates were shown, and the effec- tiveness of genetic engineering in improvement of the enzyme for modified nucleotide polymerization has been demon- strated.