Directed Evolution of an Orthogonal Quadruplet Codon-based Genetic Code
Lead Research Organisation:
MRC Laboratory of Molecular Biology
Department Name: Protein and Nucleic Acid Chemistry
Abstract
Strategies to expand the genetic code with non-canonical amino acids provide an unparalleled ability to site-specifically endow
proteins with new functionalities. This has enabled a multitude of strategies to study protein function, structure, interactions, and
localisation 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 acids
that can be added to the genetic code simultaneously is restricted by a lack of free codons. Recently, the synthesis of an E. coli
genome wherein three codons were recoded to synonyms has enabled straightforward access to a 23-amino acid genetic code. I
propose to drastically expand the utility of this E. coli strain by splitting the three blank triplet codons into twelve quadruplet codons
to be decoded by quadruplet tRNAs. Significantly, this approach eliminates the competition of quadruplet tRNAs with endogenous
tRNAs and removes the risk of toxic frameshifting of the proteome. I will establish a directed evolution strategy to evolve highly active
and specific quadruplet tRNAs. Subsequently, I will combine sets of mutually orthogonal aminoacyl-tRNA synthetase/quadruplet
tRNA pairs in the recoded E. coli strain, establishing an up-to-32-amino acid genetic code. Next, I will utilise this condensed
quadruplet codon-based genetic code to evolve an orthogonal ribosome for improved quadruplet but suppressed triplet codon
decoding, creating an orthogonal quadruplet codon genetic code. DEQode will thus unlock the potential of quadruplet codon
decoding, provide unprecedented capacity to encode multiple non-canonical amino acids into proteins simultaneously, and generate
the first exclusively-quadruplet orthogonal genetic code. This multidisciplinary project will utilise my protein chemistry expertise
while providing me with extensive synthetic biology training.
proteins with new functionalities. This has enabled a multitude of strategies to study protein function, structure, interactions, and
localisation 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 acids
that can be added to the genetic code simultaneously is restricted by a lack of free codons. Recently, the synthesis of an E. coli
genome wherein three codons were recoded to synonyms has enabled straightforward access to a 23-amino acid genetic code. I
propose to drastically expand the utility of this E. coli strain by splitting the three blank triplet codons into twelve quadruplet codons
to be decoded by quadruplet tRNAs. Significantly, this approach eliminates the competition of quadruplet tRNAs with endogenous
tRNAs and removes the risk of toxic frameshifting of the proteome. I will establish a directed evolution strategy to evolve highly active
and specific quadruplet tRNAs. Subsequently, I will combine sets of mutually orthogonal aminoacyl-tRNA synthetase/quadruplet
tRNA pairs in the recoded E. coli strain, establishing an up-to-32-amino acid genetic code. Next, I will utilise this condensed
quadruplet codon-based genetic code to evolve an orthogonal ribosome for improved quadruplet but suppressed triplet codon
decoding, creating an orthogonal quadruplet codon genetic code. DEQode will thus unlock the potential of quadruplet codon
decoding, provide unprecedented capacity to encode multiple non-canonical amino acids into proteins simultaneously, and generate
the first exclusively-quadruplet orthogonal genetic code. This multidisciplinary project will utilise my protein chemistry expertise
while providing me with extensive synthetic biology training.
People |
ORCID iD |
| Jason Chin (Principal Investigator) |