expanded genetic code


  • The ability to site-specifically direct lab-synthesized chemical moieties into proteins allows many types of studies that would otherwise be extremely difficult, such as:
    • Probing protein structure and function: By using amino acids with slightly different size such as O-methyltyrosine or dansylalanine instead of tyrosine, and by inserting genetically coded reporter moieties (color-changing and/or spin-active)
    into selected protein sites, chemical information about the protein’s structure and function can be measured.

  • In 2002, they developed an unnatural base pair between and pyridine-2-one (y) that functions in vitro in transcription and translation for the site-specific incorporation
    of non-standard amino acids into proteins.

  • In May 2019, researchers, in a milestone effort, reported the creation of a new synthetic (possibly artificial) form of viable life, a variant of the bacteria Escherichia
    coli, by reducing the natural number of 64 codons in the bacterial genome to 61 codons (eliminating two out of the six codons coding for serine and one out of three stop codons) – of which 59 used to encode 20 amino acids.

  • [63] Applications With an expanded genetic code, the unnatural amino acid can be genetically directed to any chosen site in the protein of interest.

  • Non-standard amino acids The first element of the system is the amino acid that is added to the genetic code of a certain strain of organism.

  • • Changing the mode of action of a protein: One can start with the gene for a protein that binds a certain sequence of DNA and, by inserting a chemically active amino acid
    into the binding site, convert it to a protein that cuts the DNA rather than binding it.

  • [82][87][88][89] Related methods Selective pressure incorporation (SPI) method for production of alloproteins[edit] There have been many studies that have produced protein
    with non-standard amino acids, but they do not alter the genetic code.

  • Even accounting for a variety of stop codons, more than 200 different amino acids could potentially be encoded this way.

  • [33] Recent developments in genetic code engineering also showed that quadruplet codon could be used to encode non-standard amino acids under experimental conditions.

  • [4] In general, the introduction of new functional unnatural amino acids into proteins of living cells breaks the universality of the genetic language, which ideally leads
    to alternative life forms.

  • An expanded genetic code is an artificially modified genetic code in which one or more specific codons have been re-allocated to encode an amino acid that is not among the
    22 common naturally-encoded proteinogenic amino acids.

  • [28] This allowed an experiment to be done with this strain to make it “addicted” to the amino acid biphenylalanine by evolving several key enzymes to require it structurally,
    therefore putting its expanded genetic code under positive selection.

  • • Probing the role of post-translational modifications in protein structure and function: By using amino acids that mimic post-translational modifications such as phosphoserine,
    biologically active protein can be obtained, and the site-specific nature of the amino acid incorporation can lead to information on how the position, density, and distribution of protein phosphorylation effect protein function.

  • In the second case, a biosynthetic pathway needs to be engineered, for example, an E. coli strain that biosynthesizes a novel amino acid (p-aminophenylalanine) from basic
    carbon sources and includes it in its genetic code.

  • [10] Due to technical details (easier chemical synthesis of NSAAs, less crosstalk and easier evolution of the aminoacyl-tRNA synthase), the NSAAs are generally larger than
    standard amino acids and most often have a phenylalanine core but with a large variety of different substituents.

  • [10] A library of compounds is usually tested for use in incorporation of the new amino acid, but this is not always necessary, for example, various transport systems can
    handle unnatural amino acids with apolar side-chains.

  • [7] In order to incorporate a novel amino acid into the genetic code several changes are required.

  • [42] 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.

  • [8] A feature exploited in the expansion of the genetic code is the fact that the aminoacyl tRNA synthetase often does not recognize 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.

  • [86] In May 2014, researchers announced that they had successfully introduced two new artificial nucleotides into bacterial DNA, and by including individual artificial nucleotides
    in the culture media, were able to induce amplification of the plasmids containing the artificial nucleotides by a factor of (24 doublings); they did not create mRNA or proteins able to use the artificial nucleotides.

  • The high efficiency and fidelity of this process allows a better control of the placement of the modification compared to modifying the protein post-translationally, which,
    in general, will target all amino acids of the same type, such as the thiol group of cysteine and the amino group of lysine.

  • Current methodology uses only one non-standard amino acid at the time, whereas ideally multiple could be used.

  • 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.

  • These protein, called alloprotein, are made by incubating cells with an unnatural amino acid in the absence of a similar coded amino acid in order for the former to be incorporated
    into protein in place of the latter, for example L-2-aminohexanoic acid (Ahx) for methionine (Met).

  • [75] The first genetically recoded organism was created by a collaboration between George Church’s and Farren Isaacs’ labs, when the wild type E.coli MG1655 was recoded in
    such a way that all 321 known stop codons (UAG) were substituted with synonymous UAA codons and release factor 1 was knocked out in order to eliminate the interaction with the exogenous stop codon and improve unnatural protein synthesis.

  • [94] The objective of expanding the genetic code is more radical as it does not replace an amino acid, but it adds one or more to the code.

  • In 2017 a mouse engineered with an extended genetic code that can produce proteins with unnatural amino acids was reported.

  • The successful incorporation of a third base pair into a living micro-organism is a significant breakthrough toward the goal of greatly expanding the number of amino acids
    which can be encoded by DNA, thereby expanding the potential for living organisms to produce novel proteins.

  • 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 syntheses that do not specifically recognize the non-natural amino acid.

  • [3][2] In addition to the elimination of the usage of rare codons, the specificity of the system needs to be increased as many tRNA recognise several codons[74] Expanded genetic
    alphabet[edit] Main article: Unnatural base pair Another approach is to expand the number of nucleobases to increase the coding capacity.

  • [109] Chemical synthesis[edit] Main article: Peptide synthesis There are several techniques to produce peptides chemically, generally it is by solid-phase protection chemistry.

  • First, for successful translation of a novel amino acid, the codon to which the novel amino acid is assigned cannot already code for one of the 20 natural amino acids.

  • [32] Four base (quadruplet) codons[edit] While triplet codons are the basis of the genetic code in nature, programmed +1 frameshift is a natural process that allows the use
    of a four-nucleotide sequence (quadruplet codon) to encode an amino acid.

  • [69] • Selective destruction of selected cellular components: using an expanded genetic code, unnatural, destructive chemical moieties (sometimes called “chemical warheads”)
    can be incorporated into proteins that target specific cellular components.

  • [97] Moreover, many biological phenomena, such as protein folding and stability, are based on synergistic effects at many positions in the protein sequence.

  • This means that any (protected) amino acid can be added into the nascent sequence.

  • Orthogonal ribosomes ideally use different mRNA transcripts than their natural counterparts and ultimately should draw on a separate pool of tRNA as well.

  • Most often, a library of mutant synthetases is screened for one which charges the tRNA with the desired amino acid.

  • [111] This unnatural base pair has been demonstrated previously,[112][113] but this is the first report of transcription and translation of proteins using an unnatural base

  • The orthologous set of synthetase and tRNA can be mutated and screened through directed evolution to charge the tRNA with a different, even novel, amino acid.

  • However, by co-mutating the binding nucleotides in such a way, that they can still base pair, the translational fidelity can be conserved.

  • Thus far, this system has only been shown to work in an in-vitro translation setting where the aminoacylation of the orthogonal tRNA was achieved using so called “flexizymes”.

  • [73] Moreover, there has been development in software that allows combination of orthogonal ribosomes and unnatural tRNA/RS pairs in order to improve protein yield and fidelity.

  • [91] In protein crystallography, for example, the addition of selenomethionine to the media of a culture of a methionine-auxotrophic strain results in proteins containing
    selenomethionine as opposed to methionine (viz.

  • [70] • Producing better protein: the evolution of T7 bacteriophages on a non-evolving E. coli strain that encoded 3-iodotyrosine on the amber codon, resulted in a population
    fitter than wild-type thanks to the presence of iodotyrosine in its proteome[71] • Probing protein localization and protein-protein interaction in bacteria.

  • [14][15][16] Availability of the non-standard amino acid requires that the organism either import it from the medium or biosynthesize it.

  • [58] The 16S rRNA was mutated in such a way that it bound the release factor RF1 less strongly than the natural ribosome does.

  • [82][83] In 2014 the same team from the Scripps Research Institute reported that they synthesized a stretch of circular DNA known as a plasmid containing natural T-A and C-G
    base pairs along with the best-performing UBP Romesberg’s laboratory had designed, and inserted it into cells of the common bacterium E. coli that successfully replicated the unnatural base pairs through multiple generations.

  • [92] Another example is that photoleucine and photomethionine are added instead of leucine and methionine to cross-label protein.

  • [37] This stems from the fact that the interaction between engineered tRNAs with ternary complexes or other translation components is not as favorable and strong as with cell
    endogenous translation elements.

  • [98] In this context, the SPI method generates recombinant protein variants or alloproteins directly by substitution of natural amino acids with unnatural counterparts.

  • This ribosome did not eliminate the problem of lowered cell fitness caused by suppressed stop codons in natural proteins.

  • Thus far, this optimized 16S rRNA was combined with natural large-subunits to form orthogonal ribosomes.

  • [108] in vitro synthesis[edit] Main article: mRNA display The genetic code expansion described above is in vivo.

  • Subsequently, the group evolved the orthologonal tRNA/synthase pair to utilise the non-standard amino acid O-methyltyrosine.

  • Selection involves multiple rounds of a two-step process, where the plasmid is transferred into cells expressing chloramphenicol acetyl transferase with a premature amber

  • In fact, the group of Jason Chin has recently broken the record for a genetically recoded E.coli strain that can simultaneously incorporate up to 4 unnatural amino acids.

  • However through the improved specificity it raised the yields of correctly synthesized target protein significantly (from percent for one amber codon to be suppressed and
    from for two amber codons).

  • [73] Recoded synthetic genome[edit] One way to achieve the encoding of multiple unnatural amino acids is by synthesising a rewritten genome.

  • For example, global proteome-wide substitutions of natural amino acids with fluorinated analogs have been attempted in E. coli[95] and B.

  • [6] There is at least one tRNA for any codon, and sometimes multiple codons code for the same amino acid.

  • The genetic code has a non-random layout that shows tell-tale signs of various phases of primordial evolution, however, it has since frozen into place and is near-universally


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