An unnatural base pair for incorporating amino acid analogs into proteins

Ichiro Hirao, Takashi Ohtsuki, Tsuyoshi Fujiwara, Tsuneo Mitsui, Tomoko Yokogawa, Taeko Okuni, Hiroshi Nakayama, Koji Takio, Takashi Yabuki, Takanori Kigawa, Koichiro Kodama, Takashi Yokogawa, Kazuya Nishikawa, Shigeyuki Yokoyama

Research output: Contribution to journalArticle

177 Citations (Scopus)


An unnatural base pair of 2-amino-6-(2-thienyl)purine (denoted by s) and pyridin-2-one (denoted by y) was developed to expand the genetic code. The ribonucleoside triphosphate of y was site-specifically incorporated into RNA, opposite s in a template, by T7 RNA polymerase. This transcription was coupled with translation in an Escherichia coli cell-free system. The yAG codon in the transcribed ras mRNA was recognized by the CUs anticodon of a yeast tyrosine transfer RNA (tRNA) variant, which had been enzymatically aminoacylated with an unnatural amino acid, 3-chlorotyrosine. Site-specific incorporation of 3-chlorotyrosine into the Ras protein was demonstrated by liquid chromatography-mass spectrometry (LC-MS) analysis of the products. This coupled transcription-translation system will permit the efficient synthesis of proteins with a tyrosine analog at the desired position.

Original languageEnglish
Pages (from-to)177-182
Number of pages6
JournalNature Biotechnology
Issue number2
Publication statusPublished - 2002
Externally publishedYes


ASJC Scopus subject areas

  • Microbiology

Cite this

Hirao, I., Ohtsuki, T., Fujiwara, T., Mitsui, T., Yokogawa, T., Okuni, T., Nakayama, H., Takio, K., Yabuki, T., Kigawa, T., Kodama, K., Yokogawa, T., Nishikawa, K., & Yokoyama, S. (2002). An unnatural base pair for incorporating amino acid analogs into proteins. Nature Biotechnology, 20(2), 177-182.