Expanding the Genetic Code of Caenorhabditis elegans Using Bacterial Aminoacyl-tRNA Synthetase/tRNA Pairs

Abstract

The genetic code specifies 20 common amino acids and is largely preserved in both single and multicellular organisms. Unnatural amino acids (Uaas) have been genetically incorporated into proteins by using engineered orthogonal tRNA/aminoacyltRNA synthetase (RS) pairs, enabling new research capabilities and precision inaccessible with common amino acids. We show here that Escherichia coli tyrosyl and leucyl amber suppressor tRNA/RS pairs can be evolved to incorporate different Uaas in response to the amber stop codon UAG into various proteins in Caenorhabditis elegans. To accurately report Uaa incorporation in worms, we found that it is crucial to integrate the UAG-containing reporter gene into the genome rather than to express it on an extrachromosomal array from which variable expression can lead to reporter activation independent of the amber-suppressing tRNA/RS. Synthesizing a Uaa in a dipeptide drives Uaa uptake and bioavailability. Uaa incorporation has dosage, temporal, tRNA copy, and temperature dependencies similar to endogenous amber suppression. Uaa incorporation efficiency was improved by impairing the nonsense-mediated mRNA decay pathway through knockdown of smg-1. We have generated stable transgenic worms capable of genetically encoding Uaas, enabling Uaa exploitation to address complex biological problems within a metazoan. We anticipate our strategies will be generally extendable to other multicellular organisms.

Selective chemical protein modification

“…Yet, limitations of the technology remain: most importantly these include the need for more widespread access to the required plasmids and the global applicability towards all protein systems. However, recent impressive advances in the field such as amber suppression in living animals 197,198 , and significant increases in suppression efficiency by knockout of the gene for RF1 in E. coli 199 are beginning to open up the possibility of amber-stop codon suppression becoming an indispensable tool for the incorporation of unnatural 'tags' into proteins to undertake site-selective modification. The creation of an 'amber-free' E. coli variant is also farsighted in this regard 200 .…”

Playing with the Molecules of Life

“…24, 42, 43 These latter pairs are especially advantageous as they allow aaRSs to be evolved in E. coli prior to transfer of the machinery to more diverse eukaryotic hosts. Utilizing these orthogonal pairs, ncAAs have been encoded in B. cereus, 89 P. pastoris, 90 C. elegans, 91, 92 D. melanogaster, 93 A. thaliana, 94 Zebrafish embryos, 95 and the mouse. 96–99 More recently, it has been demonstrated that a native Trp aaRS/tRNA pair in E. coli can be functionally replaced with a counterpart from yeast, and the liberated Trp pair can be used to encode ncAAs in bacteria.…”

Engineering the Genetic Code in Cells and Animals: Biological Considerations and Impacts

“…We showed that this method can be used to express different tRNAs in mammalian cells, including ${\mathrm{tRNA}}_{\mathrm{CUA}}^{\mathrm{Tyr}}$ and ${\mathrm{tRNA}}_{\mathrm{CUA}}^{\mathrm{leu}}$ from E. coli 18 and ${\mathrm{tRNA}}_{\mathrm{CUA}}^{\mathrm{pyl}}$ from Methanosarcina . 22 In addition, we applied this approach successfully in various cells and animals, including mammalian cell lines, 18 primary neurons, 18 stem cells, 19 C. elegans , 23 mouse, 24,25 and zebrafish. 25 Others have later applied this approach in plant 26 and fruit fly.…”

“…18 In combination with the efficient tRNA expression discussed above, the transfer strategy has enabled various Uaas to be genetically encoded in mammalian cells, 18 primary neurons, 18 stem cells, 19 and animals. 23-25 …”