Scheme of 2Nal incorporation into multiple UUG Leu codons based on forced ambiguity of the Leu codons.

E. coli leucyl-tRNA synthetase (LeuRS) charges Leu into its cognate tRNA^Leu_s containing UAA anticodon (E. coli tRNA^Leu_UAA) and CAA anticodon (E. coli tRNA^Leu_CAA). Leu charged into E. coli tRNA^Leu_UAA is incorporated into multiple UUA Leu codon sites of a target protein. The yPheRS_naph charges 2Nal into ytRNA^Phe containing CAA anticodon (ytRNA^Phe_CAA). Then, 2Nal is incorporated into multiple UUG Leu codons. According to the (extended) wobble rules, ytRNA^Phe_CAA and E. coli tRNA^Leu_UAA do not recognize UUA and UUG, respectively. UUG Leu codons can also be recognized by Leu-charged E. coli tRNA^Leu_CAA resulting in partial occupancy of UUG codons by Leu.