Automated prototyping of genetic codes

The standard genetic code uses 64 codons to encode 20 canonical amino acids across domains of life. New-to-nature genetic codes enable new chemistries, therapeutics and ecosystem engineering, but recoding the genome of an organism is exceptionally challenging^1-6. Here we describe automated genetic tRNA expansion (AGENTEX) for multiplexed robotic prototyping of genetic codes in cell-free translation systems. Two Watson-Crick interactions in the ribosomal large subunit (LSU) mediate recognition of the 3' CCA end of tRNAs, preventing tRNAs with alternative 3' sequences from being accommodated during translation^7,8. Building on these interactions, we investigated the extent to which non-CCA-3' tRNAs (otRNAs) would be aminoacylated by natural aminoacyl tRNA synthetases (aaRSs), allowing pools of otRNAs to specify unique genetic codes using ribosomes with altered LSU. We developed multiplexed and automated methods to read aminoacylation in libraries of synthetic tRNAs. We discovered that the tRNA 3' end shows remarkable flexibility to mutation, allowing aminoacylation of most otRNAs by all Escherichia coli aaRSs. Building on our discovery, we developed cell-free translation systems enabling compressed genetic codes of 34 aaRSs for 34 codons. Using AGENTEX, we evaluated two genetic codes alongside the standard genetic code, with non-standard amino acid incorporation and reassignment of up to three codons. Our findings have implications for the design of radically new translation systems, the synthesis of biopolymers with several instances of non-standard monomers, and understanding of possible past and future genetic codes.