Genetic code expansion via integration of redundant amino acid assignment by finely tuning tRNA pools

In all translation systems, the genetic code assigns codons to amino acids as building blocks of polypeptides, defining their chemical, structural and physiological properties. The canonical genetic code, however, utilizes only 20 proteinogenic amino acids redundantly encoded in 61 codons. In order to expand the building block repertoire, this redundancy was reduced by tuning composition of the transfer RNA (tRNA) mixture in vitro. Depletion of particular tRNAs from the total tRNA mixture or its reconstitution with in vitro-transcribed tRNA_SNNs (S = C or G, N = U, C, A or G) divided a codon box to encode two amino acids, expanding the repertoire to 23. The expanded genetic codes may benefit analysis of cellular regulatory pathways and drug screening.