An unnatural nucleobase, pyridin-2-one (denoted as y), can be specifically incorporated into RNA opposite 2-amino-6-(dimethylamino)purine (denoted as x) in DNA templates by T7 RNA polymerase. For the easy preparation of DNA templates containing x, protecting groups for the 2-amino group of x were examined. Although the general N2 protecting groups, such as isobutyryl and acetyl, resisted removal by the usual treatment with concentrated ammonia at 55 degreesC for 6 h, the N2-phenoxyacetyl group was successfully removed from the protected oligomers. This DNA synthesis would be a useful method for preparation of DNA templates toward enzymatic incorporation of unnatural nucleosides into RNA at desired positions.