The ability to introduce unnatural amino acids (UAAs) with novel chemical, physical and biological properties into proteins adds a new dimension to study the structure and function of proteins. In order to site-specifically incorporate UAAs into proteins in vivo, one unique 'blank' codon designated for UAAs and an orthogonal aminoacyl-tRNA synthetase (aaRS) /tRNA pair are required. However there are very few 'blank' codons, which dramatically limits the efficiency and diversity of UAA incorporation. Till now, only three stop codons and few frame-shift quadruplets, such as AGGA, were reported to be used for UAAs. In order to provide more blank codons, we reassigned rare sense codons to unnatural amino acids. We created new UAA-RS/tRNA pairs from TyrRS/tRNA to recode two sense codons (AGG or AGG) from arginine to UAAs. Also, multiple UAAs were incorporated into one protein simultaneously in response to a sense codon and the amber codon UAG by using the new UAA-RS/tRNA pair and PylRS/tRNA_CUA. To overcome the difficulties during sense codon reassignment, we adopted the following strategies. First, a sensitive probe was created for the detection of a single amino acid mutation due to the success of codon reassignment. Second, new UAA-RS/tRNA pairs were evolved to effectively and orthogonally recode the sense codon. Third, the UAA incorporation conditions were optimized to maximize the UAA incorporation efficiency and to minimize the toxicity caused by global suppressing of the sense codon. Mass spectrometry results showed several UAAs were introduced into proteins in response to AGG or AGA at a single position by using the newly evolved UAA-RS/tRNA pair, and the arginine suppression rates were over 90%. Further, multiple UAAs were simultaneously incorporated into proteins in responding to AGG and UAG, respectively. Double UAA incorporation rates were about 80% or above. In addition, fluorescent dye tagging, and PEGylation were used to confirm the successful incorporation of target UAAs. In this work, for the first time the resource of 'blank' codons was successfully expanded to sense codons. The same strategies can be applied to reassign many other sense codons which contain 61 potential candidate codons for 20 canonical amino acids.

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