Expanding the genetic code of Caenorhabditis elegans by incorporating unnatural amino acids

The genetic code specifies 20 common amino acids and is largely preserved in both single and multicellular organisms. Unnatural amino acids have been genetically incorporated into proteins by using engineered tRNA/ aminoacyl-tRNA synthetase pairs in single cells, enabling new research capabilities and precision inaccessible with common amino acids. Here I show that unnatural amino acids can be genetically incorporated into the coding region of proteins in Caenorhabditis elegans, a multicellular model organism extensively used to study basic biology and human diseases. I established that Escherichia coli tyrosyl and leucyl amber suppressor tRNA/synthetase pairs can be evolved to incorporate different unnatural amino acids in response to the amber stop codon (UAG) into various proteins in worms. To accurately report unnatural amino acid incorporation, I found that it is crucial to integrate the UAG-containing reporter gene into the genome rather than to express it on an extrachromosomal array, where expression can be variable. Unnatural amino acid incorporation is greatly affected by bioavailability, which can be improved by delivering in a dipeptide form. Incorporation levels also depend on dosage, exposure time, and tRNA copy number. Unnatural amino acid incorporation can be improved by stabilizing UAG-containing mRNA by RNAi knockdown of smg-1. I have generated stable transgenic worms capable of genetically encoding unnatural amino acids, enabling exploitation of new functional groups to address complex biological problems in a metazoan. I anticipate my strategies will be generally applicable to other multicellular organisms