Directed Evolution of an Orthogonal Quadruplet Codon-based Genetic Code

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Strategies to expand the genetic code with non-canonical amino acids provide an unparalleled ability to site-specifically endowproteins with new functionalities. This has enabled a multitude of strategies to study protein function, structure, interactions, andlocalisation by, for example, encoding biophysical probes or photocaged residues. Although orthogonal aminoacyl-tRNA synthetase/tRNA pairs have been generated for the incorporation of >150 structurally diverse amino acids, the number of distinct amino acidsthat can be added to the genetic code simultaneously is restricted by a lack of free codons. Recently, the synthesis of an E. coligenome wherein three codons were recoded to synonyms has enabled straightforward access to a 23-amino acid genetic code. Ipropose to drastically expand the utility of this E. coli strain by splitting the three blank triplet codons into twelve quadruplet codonsto be decoded by quadruplet tRNAs. Significantly, this approach eliminates the competition of quadruplet tRNAs with endogenoustRNAs and removes the risk of toxic frameshifting of the proteome. I will establish a directed evolution strategy to evolve highly activeand specific quadruplet tRNAs. Subsequently, I will combine sets of mutually orthogonal aminoacyl-tRNA synthetase/quadruplettRNA pairs in the recoded E. coli strain, establishing an up-to-32-amino acid genetic code. Next, I will utilise this condensedquadruplet codon-based genetic code to evolve an orthogonal ribosome for improved quadruplet but suppressed triplet codondecoding, creating an orthogonal quadruplet codon genetic code. DEQode will thus unlock the potential of quadruplet codondecoding, provide unprecedented capacity to encode multiple non-canonical amino acids into proteins simultaneously, and generatethe first exclusively-quadruplet orthogonal genetic code. This multidisciplinary project will utilise my protein chemistry expertisewhile providing me with extensive synthetic biology training.