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Reprogramming the genetic code

Abstract

The encoded biosynthesis of proteins provides the ultimate paradigm for high-fidelity synthesis of long polymers of defined sequence and composition, but it is limited to polymerizing the canonical amino acids. Recent advances have built on genetic code expansion — which commonly permits the cellular incorporation of one type of non-canonical amino acid into a protein — to enable the encoded incorporation of several distinct non-canonical amino acids. Developments include strategies to read quadruplet codons, use non-natural DNA base pairs, synthesize completely recoded genomes and create orthogonal translational components with reprogrammed specificities. These advances may enable the genetically encoded synthesis of non-canonical biopolymers and provide a platform for transforming the discovery and evolution of new materials and therapeutics.

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Fig. 1: Reprogramming the genetic code for the encoded cellular synthesis of non-canonical biopolymers.
Fig. 2: Genetic code expansion.
Fig. 3: Replication, transcription and decoding of non-natural base pairs.
Fig. 4: Synthesis of an E. coli genome with synonymous codon compression.
Fig. 5: Engineered mutually orthogonal aaRS/tRNA pairs for incorporating three distinct ncAAs.
Fig. 6: In vivo evolution of new intrinsic polymerization function in an orthogonal stapled ribosome.

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Acknowledgements

The authors thank R. Mehl, D. Cervettini, W. Robertson, C. Morgan, A. Beattie and J. Fredens for discussions and comments regarding this Review.

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Correspondence to Jason W. Chin.

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Glossary

Aminoacylated

A tRNA is aminoacylated when an amino acid is attached to its 3′ end by an aminoacyl-tRNA synthetase. The carboxylate of the amino acid is attached to either the 2′-hydroxyl or the 3′-hydroxyl of the ribose, on the terminal A of the tRNA’s conserved 3′ CCA sequence, via an ester bond. The process is described as ‘aminoacylation’.

Aminoacyl-tRNA synthetases

(aaRSs). Enzymes that catalyse the aminoacylation of a specific amino acid onto its cognate tRNA. Each aaRS recognizes the correct tRNAs, among a pool of structurally similar tRNA molecules, on the basis of specific nucleotide sequences in the tRNA known as identity elements.

Genetic code expansion

The process of incorporating a non-canonical amino acid into a protein in cells, commonly in response to an amber stop codon.

Non-canonical amino acids

(ncAAs). Amino acids beyond the canonical 20 amino acids plus pyrrolysine and selenocystine. ncAAs are not naturally co-translationally incorporated into proteins during ribosomal synthesis, are normally synthesized chemically in the laboratory and can have a myriad of different side chain structures and backbone configurations.

Genetic code reprogramming

An effort to convert protein translation into a system for non-canonical biopolymer synthesis, including strategies to provide codons beyond stop codons for incorporation of non-canonical amino acids, for creating mutually orthogonal aminoacyl-tRNA synthetase/tRNA pairs and for expanding the chemical scope of the ribosome.

Orthogonal ribosomes

A ribosome in which the 3′ end of the 16S ribosomal RNA has been altered to efficiently and specifically read an mRNA with an orthogonal ribosome-binding site in Escherichia coli.

Orthogonal aaRS/tRNA pairs

An aminoacyl-tRNA synthetase (aaRS) and its cognate tRNA are described as an orthogonal pair if the synthetase does not function with endogenous tRNAs and the tRNA does not function with endogenous synthetases.

Directed evolution

The process of generating libraries of genes or mutations in genes, and selecting for combinations of genes or mutations that confer a particular phenotype.

Amber suppressor

A tRNA that decodes the amber stop codon to direct the incorporation of an amino acid.

Extended anticodon tRNAs

tRNAs in which a nucleotide is inserted into the anticodon to extend it from three bases to four bases.

Mutually orthogonal aaRS/tRNA pairs

Orthogonal aminoacyl-tRNA synthetase (aaRS)/tRNA pairs are mutually orthogonal to each other if the aaRS from each pair does not aminoacylate the tRNA from the other pair.

Episome

Extrachromosomal DNA that is capable of replicating independently of the host chromosome.

Synonymous codon compression

Substitution of a fixed set of target codons by other synonyms (defined or variable) such that the target codons are never added to the genome. Genome-wide synonymous codon compression removes every known occurrence of a target codon from the genome and thereby compresses the number of codons used to encode an amino acid.

Codon reassignment

Synonymous codon compression, followed by the removal of all tRNAs that read the deleted codon may allow the codon to be reassigned using orthogonal aminoacyl-tRNA synthetase/tRNA pairs.

Conjugation

A natural mechanism of horizontal DNA transfer between bacteria via a donor cell-encoded pilus.

Epistasis

Mutations in genetic sequences within an organism are not always independent. The effect of perturbations in one genetic element on the function of another genetic element defines epistasis between the elements.

Recoding

The substitution of codons by their synonyms in the genome. Recoding may or may not remove codons.

Bacterial artificial chromosome

A single-copy episome, with an origin of replication and segregation machinery derived from a natural fertility plasmid involved in natural conjugation. Bacterial artificial chromosomes can support the propagation of large megabase-scale DNA sequences.

Peptidyltransferase centre

Region of the ribosome in the large subunit that contains the catalytic activity required for formation of peptide bonds during protein synthesis.

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de la Torre, D., Chin, J.W. Reprogramming the genetic code. Nat Rev Genet 22, 169–184 (2021). https://doi.org/10.1038/s41576-020-00307-7

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