Expanded genetic code

An expanded genetic code refers to an artificially modified genetic code in which one or more specific codons have been allocated to encode an amino acid which is not among the twenty/twenty-two found in nature.[1]

Contents

Background

The translation is catalysed by ribosomes. Transfer RNAs (tRNA) are used as keys to decode the RNA into its equivalent polypeptide. The tRNA recognises a specific three nucleotide codon thanks to a complementary sequence called the anticodon on one of its loops. Each three nucleotide codon is translated into one amino acid. There is at least one tRNA for any codon. If there are more than one, they code for the same amino acid. Many tRNAs are compatible with several codons. The encoding of a codon to its amino acid is a result of the aminoacyl tRNA synthetase which adds the aminoacyl group to its allocated tRNA. The aminoacyl tRNA synthetase often does not recognise the anticodon, but another part of the tRNA, meaning that if the anticodon were to be mutated the encoding of that amino acid would change to a new codon.

Introduction

For successful translation of a novel amino acid, the codon to which the amino acid is reassigned must be free or unfavoured and the novel tRNA and synthetase set (called the orthogonal set when including the codon) must not crosstalk with the endogenous tRNA and synthetase sets, while still being functionally compatible with the ribosome and other components of the translation apparatus. The tRNA synthetase pair is taken from a distant organism, generally from a different domain, and the active site of the synthetase is modified to accept the non-natural amino acid.

The possibility of reassigning codons was realized by Normanly et al. in 1990 when a viable mutant strain of E. coli read through the amber (stop) codon.[2] As a result the amber codon became the choice codon to be assigned a novel amino acid. Later, in the Schultz lab the tRNATyr/tyrosyl-tRNA synthetase (TyrRS) from Methanococcus jannaschii was used to introduce a tyrosine instead of STOP, the default value of the amber codon.[3] As mentioned, this was possible because of the differences between the endogenous bacterial synthases and the orthologous archeal synthase which do not recognise each other.

Directed evolution

This orthologous set can then be mutated and screened through directed evolution to accept a different, even novel, amino acid. Mutations to the plasmid containing the pair can be introduced by error-prone PCR or through degenerate primers for the synthetase's active site. Selection involves multiple rounds of a two-step process, where the plasmid is transferred into cells expressing chloramphenicol acetyl transferase with a premature amber codon. In the presence of toxic chloramphenicol and the non-natural amino acid, the surviving cells will have overridden the amber codon using the orthogonal tRNA aminoacylated with either the standard amino acids or the non-natural one. To remove the former, the plasmid is inserted into cells with a barnase gene (toxic) with a premature amber codon but without the non-natural amino acid, removing all the orthogonal synthases which do not specifically recognize the non-natural amino acid.[4] In addition to the recoding of the tRNA to a different codon, they can be mutated to recognize a four base codon, allowing additional free coding options.[5] The non natural amino acid, as a result, introduces diverse physicochemical and biological properties in order to be used as a tool to explore protein structure and function or to create novel or enhanced protein for practical purposes.

Diversity

The orthogonal pairs of synthase and tRNA which work for one organism may not work for another as the synthase may mis-aminoacylate endogenous tRNAs or the tRNA be mis-aminoacylated itself by an endogenous synthase. As a result the sets created to date differ between organisms.

orthogonal sets in E. coli

orthogonal sets in yeast

orthogonal sets in mammalian cells

Protein studies

With an expanded genetic code, the unnatural amino acid can be genetically directed to any chosen site in the protein of interest. The high efficiency and fidelity allows a better control of the placement of the modification compared to modifying the protein post-translationally, which generally will target all amino acids of the same type, such as the thiol group of cysteine and the -amino group of lysine.[15] Also, an expanded genetic code allows modifications to be carried out in vivo. The ability to site-specifically direct lab-synthesized chemical moieties into proteins allows many types of studies which would otherwise be extremely difficult.

An example of the possible application for this method is the biomedical where "chemical warheads" can be added to protein which target specific cellular components.[16]

See also

References

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