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An unnatural base pair for incorporating amino acid analogs into proteins

Abstract

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.

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Figure 1
Figure 2: T7 transcription employing s·y pairing.
Figure 3: The transcription–translation system involving the s·y base pair.
Figure 4: Preparation of aminoacylated tRNACUs.
Figure 5: Incorporation of ClTyr into the Ras protein.

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Acknowledgements

We thank T. Masaki, of Ibaraki University, for providing the Achromobacter Protease I, and R. Ishitani, The University of Tokyo, for helpful discussion.

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Correspondence to Ichiro Hirao or Shigeyuki Yokoyama.

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Hirao, I., Ohtsuki, T., Fujiwara, T. et al. An unnatural base pair for incorporating amino acid analogs into proteins. Nat Biotechnol 20, 177–182 (2002). https://doi.org/10.1038/nbt0202-177

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