Removing redundancy of the NCN codons in vitro for maximal sense codon reassignment

Expanding the genetic code affords exciting opportunities for synthetic biology, studies of protein function, and creation of diverse peptide libraries by mRNA display. Maximal expansion with the standard 64 codon code requires breaking the degeneracy of the 61 sense codons which encode for only 20 amino acids. In E. coli these 61 codons are decoded by 46 different tRNAs. Moreover, many codons are decoded by multiple tRNAs, further complicating efforts to break this redundancy. The overlapping decoding patterns of the 11 tRNAs in E. coli which read the 16 codons that encode serine, proline, threonine, and alanine codons exemplify this difficulty. Here we tackle this challenge by first outlining a general process to evaluate codons for their potential for reassignment. We then use this knowledge to assign these 16 codons to 10 different amino acids, more than doubling their encoding potential. Our work highlights the expanded potential of sense codon reassignment and points the way to a dramatically expanded code containing more than 30 monomers.