Angew. Chem. Int. Ed. Engl. doi:10.1002/anie.201308584

Translational recoding to incorporate non-natural or otherwise modified amino acids has generally focused on stop codons as the modifiable triplet. However, recent studies have shown that incorporation of selenocysteine (Sec) is possible at low levels at the sense codons UUA (leucine) and UGG (tryptophan). To investigate whether these low levels were due to competition with the 20 canonical amino acids generally or whether other codons might be more amenable to replacement, Bröcker et al. systematically tested each of the 64 codons to see whether Sec could be inserted and to what extent. Both formate dehydrogenase and thioredoxin reductase naturally include and are functionally dependent on Sec, meaning that activity assays provide a straightforward and quantitative proxy for Sec incorporation. Surprisingly, in formate dehydrogenase, 58 codons could be translated with Sec, yielding activities ranging from 12% to 100% of the parent sequence. The fully active codon replacements included all three stop codons as well as 15 sense codons for which the proteins not only were active but also were expressed at much higher levels than those using stop codons. The most active of these, UAC, showed the same behavior in recoding thioredoxin reductase, yielding wild-type activity and better yields than the most commonly used stop codon, UGA. These data raise new questions about translational fidelity and create new opportunities for protein engineering.