De novo synthesis of a recoded genome with expanded decoding capacity
We further plan to expand the number of reassigned codons by a factor of four using quadruplet decoding (Figure 3, step vi). When fully utilized, this can create up to 44 distinct quadruplet codons based on the initial 11 freed-up triplet codons to dramatically expand the number of unnatural amino acids that can be incorporated simultaneously.
Figure 3. Expanding decoding capacity through removing and reassigning decoding redundancy.
Step i -identifies target codons for removal (e.g. serine codon TCG and TCA, and stop codon TAG; in grey).
Step ii-identifies the best synonymous replacements by testing defined recoding schemes. For example, TAA is identified as a viable replacement to TAG.
Step iii- systematically recodes the target codons to the identified synonymous replacements in the de novo synthesized genome across all genomic positions.
Step iv- removes the redundant tRNAs (Ser tRNACGA and Ser tRNAUGA, in grey) and release factor 1 (RF1, in grey) to free-up the decoding capacity of removed codons TCG, TCA, and TAG.
Step v- introduces evolved synthetase/tRNA pairs (in blue) to encode unnatural amino acids (u.a.a., blue star) quantitatively in response to reassigned triplet codons TCG, TCA, and TAG. Through the five defined steps, the previous serine codon TCG and TCA, and stop codon TAG in the wildtype genome have been reassigned to solely encode unnatural amino acids in the synthetic genome.
Step vi expands the reassigned decoding capacities by a factor of four by introducing 12 quadruplet codons and corresponding quadruplet decoding evolved synthetase/tRNA pairs (in orange).