Expanded genetic code
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.[1]
![](../I/Orthogonal_pair_to_extend_code.svg.png.webp)
The key prerequisites to expand the genetic code are:
- the non-standard amino acid to encode,
- an unused codon to adopt,
- a tRNA that recognises this codon, and
- a tRNA synthetase that recognises only that tRNA and only the non-standard amino acid.
Expanding the genetic code is an area of research of synthetic biology, an applied biological discipline whose goal is to engineer living systems for useful purposes. The genetic code expansion enriches the repertoire of useful tools available to science.
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.[2][3]
Introduction
It is noteworthy that the genetic code for all organisms is basically the same, so that all living beings use the same ’genetic language’.[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.[5] Proteins are produced thanks to the translational system molecules, which decode the RNA messages into a string of amino acids. The translation of genetic information contained in messenger RNA (mRNA) into a protein is catalysed by ribosomes. Transfer RNAs (tRNA) are used as keys to decode the mRNA into its encoded polypeptide. The tRNA recognizes a specific three nucleotide codon in the mRNA with a complementary sequence called the anticodon on one of its loops. Each three-nucleotide codon is translated into one of twenty naturally occurring amino acids.[6] There is at least one tRNA for any codon, and sometimes multiple codons code for the same amino acid. Many tRNAs are compatible with several codons. An enzyme called an aminoacyl tRNA synthetase covalently attaches the amino acid to the appropriate tRNA.[7] Most cells have a different synthetase for each amino acid (20 or more synthetases). On the other hand, some bacteria have fewer than 20 aminoacyl tRNA synthetases, and introduce the "missing" amino acid(s) by modification of a structurally related amino acid by an aminotransferase enzyme.[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. In the ribosome, the information in mRNA is translated into a specific amino acid when the mRNA codon matches with the complementary anticodon of a tRNA, and the attached amino acid is added onto a growing polypeptide chain. When it is released from the ribosome, the polypeptide chain folds into a functioning protein.[7]
In order to incorporate a novel amino acid into the genetic code several changes are required. 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. Usually a nonsense codon (stop codon) or a four-base codon are used.[6] Second, a novel pair of tRNA and aminoacyl tRNA synthetase are required, these are called the orthogonal set. The orthogonal set 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 active site of the synthetase is modified to accept only the novel amino acid. Most often, a library of mutant synthetases is screened for one which charges the tRNA with the desired amino acid. The synthetase is also modified to recognize only the orthogonal tRNA.[6] The tRNA synthetase pair is often engineered in other bacteria or eukaryotic cells.[9]
In this area of research, the 20 encoded proteinogenic amino acids are referred to as standard amino acids, or alternatively as natural or canonical amino acids, while the added amino acids are called non-standard amino acids (NSAAs), or unnatural amino acids (uAAs; term not used in papers dealing with natural non-proteinogenic amino acids, such as phosphoserine), or non-canonical amino acids.
Non-standard amino acids
![](../I/Tyr_based_non_natural_aa.svg.png.webp)
The first element of the system is the amino acid that is added to the genetic code of a certain strain of organism.
Over 71 different NSAAs have been added to different strains of E. coli, yeast or mammalian cells.[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. These allow a large repertoire of new functions, such as labeling (see figure), as a fluorescent reporter (e.g. dansylalanine)[11] or to produce translational proteins in E. coli with Eukaryotic post-translational modifications (e.g. phosphoserine, phosphothreonine, and phosphotyrosine).[10][12]
Unnatural amino acids incorporated into proteins include heavy atom-containing amino acids to facilitate certain x-ray crystallographic studies; amino acids with novel steric/packing and electronic properties; photocrosslinking amino acids which can be used to probe protein-protein interactions in vitro or in vivo; keto, acetylene, azide, and boronate-containing amino acids which can be used to selectively introduce a large number of biophysical probes, tags, and novel chemical functional groups into proteins in vitro or in vivo; redox active amino acids to probe and modulate electron transfer; photocaged and photoisomerizable amino acids to photoregulate biological processes; metal binding amino acids for catalysis and metal ion sensing; amino acids that contain fluorescent or infra-red active side chains to probe protein structure and dynamics; α-hydroxy acids and D-amino acids as probes of backbone conformation and hydrogen bonding interactions; and sulfated amino acids and mimetics of phosphorylated amino acids as probes of post-translational modifications.[13][14][15]
Availability of the non-standard amino acid requires that the organism either import it from the medium or biosynthesize it. In the first case, the unnatural amino acid is first synthesized chemically in its optically pure L-form.[16] It is then added to the growth medium of the cell.[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. 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.[15][17][18] Another example: the production of phosphoserine, a natural metabolite, and consequently required alteration of its pathway flux to increase its production.[12]
Codon assignment
Another element of the system is a codon to allocate to the new amino acid.
A major problem for the genetic code expansion is that there are no free codons. 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 conserved.[19] Nevertheless, some codons are rarer than others. In fact, in E. coli (and all organisms) the codon usage is not equal, but presents several rare codons (see table), the rarest being the amber stop codon (UAG).
Codon | Amino acid | Abundance (%) |
---|---|---|
UUU | Phe (F) | 1.9 |
UUC | Phe (F) | 1.8 |
UUA | Leu (L) | 1.0 |
UUG | Leu (L) | 1.1 |
CUU | Leu (L) | 1.0 |
CUC | Leu (L) | 0.9 |
CUA | Leu (L) | 0.3 |
CUG | Leu (L) | 5.2 |
AUU | Ile (I) | 2.7 |
AUC | Ile (I) | 2.7 |
AUA | Ile (I) | 0.4 |
AUG | Met (M) | 2.6 |
GUU | Val (V) | 2.0 |
GUC | Val (V) | 1.4 |
GUA | Val (V) | 1.2 |
GUG | Val (V) | 2.4 |
UCU | Ser (S) | 1.1 |
UCC | Ser (S) | 1.0 |
UCA | Ser (S) | 0.7 |
UCG | Ser (S) | 0.8 |
CCU | Pro (P) | 0.7 |
CCC | Pro (P) | 0.4 |
CCA | Pro (P) | 0.8 |
CCG | Pro (P) | 2.4 |
ACU | Thr (T) | 1.2 |
ACC | Thr (T) | 2.4 |
ACA | Thr (T) | 0.1 |
ACG | Thr (T) | 1.3 |
GCU | Ala (A) | 1.8 |
GCC | Ala (A) | 2.3 |
GCA | Ala (A) | 0.1 |
GCG | Ala (A) | 3.2 |
UAU | Tyr (Y) | 1.6 |
UAC | Tyr (Y) | 1.4 |
UAA | Stop | 0.2 |
UAG | Stop | 0.03 |
CAU | His (H) | 1.2 |
CAC | His (H) | 1.1 |
CAA | Gln (Q) | 1.3 |
CAG | Gln (Q) | 2.9 |
AAU | Asn (N) | 1.6 |
AAC | Asn (N) | 2.6 |
AAG | Lys (K) | 3.8 |
AAA | Lys (K) | 1.2 |
GAU | Asp (D) | 3.3 |
GAC | Asp (D) | 2.3 |
GAA | Glu (E) | 4.4 |
GAG | Glu (E) | 1.9 |
UGU | Cys (C) | 0.4 |
UGC | Cys (C) | 0.6 |
UGA | Stop | 0.1 |
UGG | Trp (W) | 1.4 |
CGU | Arg (R) | 2.4 |
CGC | Arg (R) | 2.2 |
CGA | Arg (R) | 0.3 |
CGG | Arg (R) | 0.5 |
AGU | Ser (S) | 0.7 |
AGC | Ser (S) | 1.5 |
AGA | Ser (S) | 0.2 |
AGG | Ser (S) | 0.2 |
GGU | Gly (G) | 2.8 |
GGC | Gly (G) | 3.0 |
GGC | Gly (G) | 0.7 |
GGA | Gly (G) | 0.9 |
Amber codon suppression
The possibility of reassigning codons was realized by Normanly et al. in 1990, when a viable mutant strain of E. coli read through the UAG ("amber") stop codon.[21] This was possible thanks to the rarity of this codon and the fact that release factor 1 alone makes the amber codon terminate translation. Later, in the Schultz lab, the tRNATyr/tyrosyl-tRNA synthetase (TyrRS) from Methanococcus jannaschii, an archaebacterium,[6] was used to introduce a tyrosine instead of STOP, the default value of the amber codon.[22] This was possible because of the differences between the endogenous bacterial synthases and the orthologous archaeal synthase, which do not recognize each other. Subsequently, the group evolved the orthologonal tRNA/synthase pair to utilise the non-standard amino acid O-methyltyrosine.[23] This was followed by the larger naphthylalanine[24] and the photocrosslinking benzoylphenylalanine,[25] which proved the potential utility of the system.
The amber codon is the least used codon in Escherichia coli, but hijacking it results in a substantial loss of fitness. One study in fact found that there were at least 83 peptides majorly affected by the readthrough[26] Additionally, the labelling was incomplete. As a consequence, several strains have been made to reduce the fitness cost, including the removal of all amber codons from the genome. In most E. coli K-12 strains (viz. Escherichia coli (molecular biology) for strain pedigrees) there are 314 UAG stop codons. Consequently, a gargantuan amount of work has gone into the replacement of these. One approach pioneered by the group of Prof. George Church from Harvard, was dubbed MAGE in CAGE: this relied on a multiplex transformation and subsequent strain recombination to remove all UAG codons—the latter part presented a halting point in a first paper,[27] but was overcome. This resulted in the E. coli strain C321.ΔA, which lacks all UAG codons and RF1.[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.[29]
Rare sense codon reassignment
In addition to the amber codon, rare sense codons have also been considered for use. The AGG codon codes for arginine, but a strain has been successfully modified to make it code for 6-N-allyloxycarbonyl-lysine.[30] Another candidate is the AUA codon, which is unusual in that its respective tRNA has to differentiate against AUG that codes for methionine (primordially, isoleucine, hence its location). In order to do this, the AUA tRNA has a special base, lysidine. The deletion of the synthase (tilS) was possible thanks to the replacement of the native tRNA with that of Mycoplasma mobile (no lysidine). The reduced fitness is a first step towards pressuring the strain to loose all instances of AUA, allowing it to be used for genetic code expansion.[31]
Four base codons
Other approaches include the addition of extra base pairing or the use of orthologous ribosomes that accept in addition to the regular triplet genetic code, tRNAs with quadruple code.[32] This allowed the simultaneous usage of two unnatural amino acids, p-azidophenylalanine (pAzF) and N6-[(2-propynyloxy)carbonyl]lysine (CAK), which cross-link with each other by Huisgen cycloaddition.[33]
tRNA/synthetase pair
Another key element is the tRNA/synthetase pair.
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. 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 that do not specifically recognize the non-natural amino acid.[6] 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.[34] 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.
![](../I/Selection_for_nonnatural_aa.svg.png.webp)
Orthogonal sets in model organisms
The orthogonal pairs of synthetase and tRNA that work for one organism may not work for another, as the synthetase may mis-aminoacylate endogenous tRNAs or the tRNA be mis-aminoacylated itself by an endogenous synthetase. As a result, the sets created to date differ between organisms.
Pair | Source | E. coli | Yeast | Mammals | Notes and references |
---|---|---|---|---|---|
tRNATyr-TyrRS | Methanococcus jannaschii | Yes | No | No | |
tRNALys–LysRS | Pyrococcus horikoshii | Yes | No | No | [35] |
tRNAGlu–GluRS | Pyrococcus horikoshii | Yes | No | No | [36] |
tRNALeu–LeuRS | tRNA: mutant Halobacterium sp. RS: Methanobacterium thermoautotrophicum | Yes | No | No | [37] |
tRNAAmber-PylRS | Methanosarcina barkeri and Methanosarcina mazei | Yes | Yes | Yes | [38] |
tRNAAmber-3-iodotyrosyl-RS | RS: variant Methanocaldococcus jannaschii aaRS | Yes | No | No | [39] |
tRNATyr/Amber-TyrRS | Escherichia coli | No | Yes | No | Reported in 2003,[40] mentioned in 2014 LeuRS[41] |
tRNAiMet-GlnRS | tRNA: human RS: Escherichia coli | No | Yes | No | Switched to Amber codon.[42] |
tRNAifMet-TyrRS | tRNA: Escherichia coli RS: S. cerevisiae | Yes | Yes | No | Switched to Amber codon.[42] |
tRNALeu/Amber-LeuRS | Escherichia coli | No | Yes | Yes | Reported in 2004 and mutated for 2-Aminooctanoic acid, o-methyl tyrosine, and o-nitrobenzyl cysteine.[41] Evolved in yeast for 4,5-dimethoxy-2-nitrobenzyl serine,[43] tested in mice with photosensitive 4,5-dimethoxy-2-nitrobenzyl-cysteine.[44] |
tRNATyr-TyrRS | Bacillus stearothermophilus | No | No | Yes | [9] |
tRNATrp-TrpRS | Bacillus subtilis, RS modified | No | No | Yes | New AA is 5-OH Trp.[45] |
In 2017 a mouse engineered with an extended genetic code that can produce proteins with unnatural amino acids was reported.[46]
Orthogonal ribosomes
Similarly to orthogonal tRNAs and aminoacyl tRNA synthetases (aaRSs), orthogonal ribosomes have been engineered to work in parallel to the natural ribosomes. Orthogonal ribosomes ideally use different mRNA transcripts than their natural counterparts and ultimately should draw on a separate pool of tRNA as well. This should alleviate some of the loss of fitness which currently still arises from techniques such as Amber codon suppression. Additionally, orthogonal ribosomes can be mutated and optimized for particular tasks, like the recognition of quadruplet codons. Such an optimization is not possible, or highly disadvantageous for natural ribosomes.
o-Ribosome
In 2005 three sets of ribosomes were published, which did not recognize natural mRNA, but instead translated a separate pool of orthogonal mRNA (o-mRNA).[47] This was achieved by changing the recognition sequence of the mRNA, the Shine-Dalgarno sequence, and the corresponding recognition sequence in the 16S rRNA of ribosomes, the so-called Anti-Shine-Darlgarno-Sequence. This way the base pairing, which is usually lost if either sequence is mutated, stays available. However the mutations in the 16S rRNA were not limited to the obviously base-pairing nucleotides of the classical Anti-Shine-Darlgarno sequence.
Ribo-X
In 2007 the group of Jason W. Chin presented an orthogonal ribosome, which was optimized for Amber codon suppression.[48] The 16S rRNA was mutated in such a way that it bound the release factor RF1 less strongly than the natural ribosome does. This ribosome did not eliminate the problem of lowered cell fitness caused by suppressed stop codons in natural proteins. However through the improved specificity it raised the yields of correctly synthesized target protein significantly (from ~20% to >60% percent for one amber codon to be suppressed and form <1% to >20% for two amber codons).
Ribo-Q
In 2010 the group of Jason W. Chin presented a further optimized version of the orthogonal ribosome. The Ribo-Q is a 16S rRNA optimized to recognize tRNAs, which have quadruplet anti-codons to recognize quadruplet codons, instead of the natural triplet codons.[33] With this approach the number of possible codons rises from 64 to 256. Even accounting for a variety of stop codons, more than 200 different amino acids could potentially be encoded this way.
Ribosome stapling
The orthogonal ribosomes described above all focus on optimizing the 16S rRNA. Thus far, this optimized 16S rRNA was combined with natural large-subunits to form orthogonal ribosomes. If the 23S rRNA, the main RNA-component of the large ribosomal subunit, is to be optimized as well, it had to be assured, that there was no crosstalk in the assembly of orthogonal and natural ribosomes (see figureX B). To ensure that optimized 23S rRNA would only form into ribosomes with the optimized 16S rRNA, the two rRNAs were combined into one transcript.[49] By inserting the sequence for the 23S rRNA into a loop-region of the 16S rRNA sequence, both subunits still adopt functioning folds. Since the two rRNAs are linked and thus in constant proximity, they preferably bind each other, not other free floating ribosomal subunits.
Engineered peptidyl transferase center
In 2014 it was shown that by altering the peptidyl transferase center of the 23S rRNA, ribosomes could be created which draw on orthogonal pools of tRNA.[50] The 3’ end of tRNAs is universally conserved to be CCA. The two cytidines base pair with two guanines the 23S rRNA to bind the tRNA to the ribosome. This interaction is required for translational fidelity. However, by co-mutating the binding nucleotides in such a way, that they can still base pair, the translational fidelity can be conserved. The 3’-end of the tRNA is mutated from CCA to CGA, while two cytidine nucleotides in the ribosomes A- and P-sites are mutated to guanidine. This leads to ribosomes which do not accept naturally occurring tRNAs as substrates and to tRNAs, which cannot be used as substrate by natural ribosomes.
To use such tRNAs effectively, they would have to be aminoacylated by specific, orthogonal aaRSs. Most naturally occurring aaRSs recognize the 3’-end of their corresponding tRNA.[51][52] aaRSs for these 3’-mutated tRNAs are not available yet. 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”. Flexizymes are ribozymes with tRNA-amino-aclylation activity.[53]
Applications
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 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.[54] 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 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.
- 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.[55][56][57][58]
- Identifying and regulating protein activity: By using photocaged aminoacids, protein function can be "switched" on or off by illuminating the 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.
- Improving immunogenicity and overcoming self-tolerance: By replacing strategically chosen tyrosines with p-nitro phenylalanine, a tolerated self-protein can be made immunogenic.[59]
- 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.[60]
- 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[61]
Future
The expansion of the genetic code is still in its infancy. Current methodology uses only one non-standard amino acid at the time, whereas ideally multiple could be used.
Recoded synthetic genome
One way to achieve the encoding of multiple unnatural amino acids is by synthesising a rewritten genome.[62] In 2010, at the cost of $40 million an organism, Mycoplasma laboratorium, was constructed that was controlled by a synthetic, but not recoded, genome.[63] In 2019, Eschericia coli Syn61 was created, with a 4 megabase recoded genome consisting of only 61 codons instead of the natural 64.[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[62]
Expanded genetic alphabet
Another approach is to expand the number of nucleobases to increase the coding capacity.
An unnatural base pair (UBP) is a designed subunit (or nucleobase) of DNA which is created in a laboratory and does not occur in nature. A demonstration of UBPs were achieved in vitro by Ichiro Hirao's group at RIKEN institute in Japan. In 2002, they developed an unnatural base pair between 2-amino-8-(2-thienyl)purine (s) 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.[64] In 2006, they created 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds) and pyrrole-2-carbaldehyde (Pa) as a third base pair for replication and transcription.[65] Afterward, Ds and 4-[3-(6-aminohexanamido)-1-propynyl]-2-nitropyrrole (Px) was discovered as a high fidelity pair in PCR amplification.[66][67] In 2013, they applied the Ds-Px pair to DNA aptamer generation by in vitro selection (SELEX) and demonstrated the genetic alphabet expansion significantly augment DNA aptamer affinities to target proteins.[68]
In 2012, a group of American scientists led by Floyd Romesberg, a chemical biologist at the Scripps Research Institute in San Diego, California, published that his team designed an unnatural base pair (UBP).[69] The two new artificial nucleotides or Unnatural Base Pair (UBP) were named "d5SICS" and "dNaM." More technically, these artificial nucleotides bearing hydrophobic nucleobases, feature two fused aromatic rings that form a (d5SICS–dNaM) complex or base pair in DNA.[70][71] 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.[72] This is the first known example of a living organism passing along an expanded genetic code to subsequent generations.[70][73] This was in part achieved by the addition of a supportive algal gene that expresses a nucleotide triphosphate transporter which efficiently imports the triphosphates of both d5SICSTP and dNaMTP into E. coli bacteria.[70] Then, the natural bacterial replication pathways use them to accurately replicate the plasmid containing d5SICS–dNaM.
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.[72] The artificial strings of DNA do not encode for anything yet, but scientists speculate they could be designed to manufacture new proteins which could have industrial or pharmaceutical uses.[74]
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 passage the bacteria 24 times; they did not create mRNA or proteins able to use the artificial nucleotides.[70][75][76][77]
Related methods
Selective pressure incorporation (SPI) method for production of alloproteins
There have been many studies that have produced protein with non-standard amino acids, but they do not alter the genetic code. 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).[78]
These studies rely on the natural promiscuous activity of the aminoacyl tRNA synthetase to add to its target tRNA an unnatural amino acid (i.e. analog) similar to the natural substrate, for example methionyl-tRNA synthase's mistaking isoleucine for methionine.[79] 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. Multi-wavelength anomalous dispersion for reason).[80] Another example is that photoleucine and photomethionine are added instead of leucine and methionine to cross-label protein.[81] Similarly, some tellurium-tolerant fungi can incorporate tellurocysteine and telluromethionine into their protein instead of cysteine and methionine.[82] 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. On the other hand, proteome-wide replacements are most efficiently performed by global amino acid substitutions. For example, global proteome-wide substitutions of natural amino acids with fluorinated analogs have been attempted in E. coli[83] and B. subtilis.[84] A complete tryptophan substitution with thienopyrrole-alanine in response to 20899 UGG codons in E. coli was reported in 2015 by Budisa and Söll.[85] Moreover, many biological phenomena, such as protein folding and stability, are based on synergistic effects at many positions in the protein sequence.[86]
In this context, the SPI method generates recombinant protein variants or alloproteins directly by substitution of natural amino acids with unnatural counterparts.[87] An amino acid auxotrophic expression host is supplemented with an amino acid analog during target protein expression.[88] This approach avoids the pitfalls of suppression-based methods[89] and it is superior to it in terms of efficiency, reproducibility and an extremely simple experimental setup.[90] Numerous studies demonstrated how global substitution of canonical amino acids with various isosteric analogs caused minimal structural perturbations but dramatic changes in thermodynamic,[91] folding,[92] aggregation[93] spectral properties[94][95] and enzymatic activity.[96]
in vitro synthesis
The genetic code expansion described above is in vivo. An alternative is the change of coding in vitro translation experiments. This requires the depletion of all tRNAs and the selective reintroduction of certain aminoacylated-tRNAs, some chemically aminoacylated.[97]
Chemical synthesis
There are several techniques to produce peptides chemically, generally it is by solid-phase protection chemistry. This means that any (protected) amino acid can be added into the nascent sequence.
In November 2017, a team from the Scripps Research Institute reported having constructed a semi-synthetic E. coli bacteria genome using six different nucleic acids (versus four found in nature). The two extra 'letters' form a third, unnatural base pair. The resulting organisms were able to thrive and synthesize proteins using "unnatural amino acids".[98][99] The unnatural base pair used is dNaM–dTPT3.[99] This unnatural base pair has been demonstrated previously,[100][101] but this is the first report of transcription and translation of proteins using an unnatural base pair.
See also
References
- Xie J, Schultz PG (December 2005). "Adding amino acids to the genetic repertoire". Current Opinion in Chemical Biology. 9 (6): 548–54. doi:10.1016/j.cbpa.2005.10.011. PMID 16260173.
- Zimmer C (15 May 2019). "Scientists Created Bacteria With a Synthetic Genome. Is This Artificial Life? - In a milestone for synthetic biology, colonies of E. coli thrive with DNA constructed from scratch by humans, not nature". The New York Times. Retrieved 16 May 2019.
- Fredens J, Wang K, de la Torre D, Funke LF, Robertson WE, Christova Y, et al. (May 2019). "Total synthesis of Escherichia coli with a recoded genome". Nature. 569 (7757): 514–518. Bibcode:2019Natur.569..514F. doi:10.1038/s41586-019-1192-5. PMC 7039709. PMID 31092918.
- Kubyshkin V, Acevedo-Rocha CG, Budisa N (February 2018). "On universal coding events in protein biogenesis". Bio Systems. 164: 16–25. doi:10.1016/j.biosystems.2017.10.004. PMID 29030023.
- Kubyshkin V, Budisa N (August 2017). "Synthetic alienation of microbial organisms by using genetic code engineering: Why and how?". Biotechnology Journal. 12 (8): 1600097. doi:10.1002/biot.201600097. PMID 28671771.
- Wang L, Brock A, Herberich B, Schultz PG (April 2001). "Expanding the genetic code of Escherichia coli". Science. 292 (5516): 498–500. Bibcode:2001Sci...292..498W. doi:10.1126/science.1060077. PMID 11313494. S2CID 6702011.
- Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P (2008). Molecular Biology of the Cell (5th ed.). New York: Garland Science. ISBN 978-0-8153-4105-5.
- Woese CR, Olsen GJ, Ibba M, Söll D (March 2000). "Aminoacyl-tRNA synthetases, the genetic code, and the evolutionary process". Microbiology and Molecular Biology Reviews. 64 (1): 202–36. doi:10.1128/mmbr.64.1.202-236.2000. PMC 98992. PMID 10704480.
- Sakamoto K, Hayashi A, Sakamoto A, Kiga D, Nakayama H, Soma A, et al. (November 2002). "Site-specific incorporation of an unnatural amino acid into proteins in mammalian cells". Nucleic Acids Research. 30 (21): 4692–9. doi:10.1093/nar/gkf589. PMC 135798. PMID 12409460.
- Liu CC, Schultz PG (2010). "Adding new chemistries to the genetic code". Annual Review of Biochemistry. 79: 413–44. doi:10.1146/annurev.biochem.052308.105824. PMID 20307192.
- Summerer D, Chen S, Wu N, Deiters A, Chin JW, Schultz PG (June 2006). "A genetically encoded fluorescent amino acid". Proceedings of the National Academy of Sciences of the United States of America. 103 (26): 9785–9. Bibcode:2006PNAS..103.9785S. doi:10.1073/pnas.0603965103. PMC 1502531. PMID 16785423.
- Steinfeld JB, Aerni HR, Rogulina S, Liu Y, Rinehart J (May 2014). "Expanded cellular amino acid pools containing phosphoserine, phosphothreonine, and phosphotyrosine". ACS Chemical Biology. 9 (5): 1104–12. doi:10.1021/cb5000532. PMC 4027946. PMID 24646179.
- Wang L, Xie J, Schultz PG (2006). "Expanding the genetic code". Annual Review of Biophysics and Biomolecular Structure. 35: 225–49. doi:10.1146/annurev.biophys.35.101105.121507. PMID 16689635.
- Young TS, Schultz PG (April 2010). "Beyond the canonical 20 amino acids: expanding the genetic lexicon". The Journal of Biological Chemistry. 285 (15): 11039–44. doi:10.1074/jbc.R109.091306. PMC 2856976. PMID 20147747.
- "The Peter G. Schultz Laboratory". Schultz.scripps.edu. Retrieved 2015-05-05.
- Cardillo G, Gentilucci L, Tolomelli A (March 2006). "Unusual amino acids: synthesis and introduction into naturally occurring peptides and biologically active analogues". Mini Reviews in Medicinal Chemistry. 6 (3): 293–304. doi:10.2174/138955706776073394. PMID 16515468.
- Journal of the American Chemical Society. 2003 Jan 29;125(4):935-9. Generation of a bacterium with a 21 amino acid genetic code. Mehl RA, Anderson JC, Santoro SW, Wang L, Martin AB, King DS, Horn DM, Schultz PG.
- "context :: 21-amino-acid bacteria: expanding the genetic code". Straddle3.net. Retrieved 2015-05-05.
- Koonin EV, Novozhilov AS (February 2009). "Origin and evolution of the genetic code: the universal enigma". IUBMB Life. 61 (2): 99–111. arXiv:0807.4749. doi:10.1002/iub.146. PMC 3293468. PMID 19117371.
- Maloy SR, Valley Joseph Stewart VJ, Taylor RK (1996). Genetic analysis of pathogenic bacteria : a laboratory manual. New York: Cold Spring Harbor Laboratory. ISBN 978-0-87969-453-1.
- Normanly J, Kleina LG, Masson JM, Abelson J, Miller JH (June 1990). "Construction of Escherichia coli amber suppressor tRNA genes. III. Determination of tRNA specificity". Journal of Molecular Biology. 213 (4): 719–26. doi:10.1016/S0022-2836(05)80258-X. PMID 2141650.
- Wang L, Magliery TJ, Liu DR, Schultz PG (2000). "A new functional suppressor tRNA/aminoacyl-tRNA synthetase pair for the in vivo incorporation of unnatural amino acids into proteins" (PDF). J. Am. Chem. Soc. 122 (20): 5010–5011. doi:10.1021/ja000595y.
- Wang L, Brock A, Herberich B, Schultz PG (April 2001). "Expanding the genetic code of Escherichia coli". Science. 292 (5516): 498–500. Bibcode:2001Sci...292..498W. doi:10.1126/science.1060077. PMID 11313494. S2CID 6702011.
- Wang L, Brock A, Schultz PG (March 2002). "Adding L-3-(2-Naphthyl)alanine to the genetic code of E. coli". Journal of the American Chemical Society. 124 (9): 1836–7. doi:10.1021/ja012307j. PMID 11866580.
- Chin JW, Martin AB, King DS, Wang L, Schultz PG (August 2002). "Addition of a photocrosslinking amino acid to the genetic code of Escherichiacoli". Proceedings of the National Academy of Sciences of the United States of America. 99 (17): 11020–4. Bibcode:2002PNAS...9911020C. doi:10.1073/pnas.172226299. PMC 123203. PMID 12154230.
- Aerni HR, Shifman MA, Rogulina S, O'Donoghue P, Rinehart J (January 2015). "Revealing the amino acid composition of proteins within an expanded genetic code". Nucleic Acids Research. 43 (2): e8. doi:10.1093/nar/gku1087. PMC 4333366. PMID 25378305.
- Isaacs FJ, Carr PA, Wang HH, Lajoie MJ, Sterling B, Kraal L, et al. (July 2011). "Precise manipulation of chromosomes in vivo enables genome-wide codon replacement". Science. 333 (6040): 348–53. Bibcode:2011Sci...333..348I. doi:10.1126/science.1205822. PMC 5472332. PMID 21764749.
- Lajoie MJ, Rovner AJ, Goodman DB, Aerni HR, Haimovich AD, Kuznetsov G, et al. (October 2013). "Genomically recoded organisms expand biological functions". Science. 342 (6156): 357–60. Bibcode:2013Sci...342..357L. doi:10.1126/science.1241459. PMC 4924538. PMID 24136966.
- Mandell DJ, Lajoie MJ, Mee MT, Takeuchi R, Kuznetsov G, Norville JE, et al. (February 2015). "Biocontainment of genetically modified organisms by synthetic protein design". Nature. 518 (7537): 55–60. Bibcode:2015Natur.518...55M. doi:10.1038/nature14121. PMC 4422498. PMID 25607366.
- Zeng Y, Wang W, Liu WR (August 2014). "Towards reassigning the rare AGG codon in Escherichia coli". ChemBioChem. 15 (12): 1750–4. doi:10.1002/cbic.201400075. PMC 4167342. PMID 25044341.
- Bohlke N, Budisa N (February 2014). "Sense codon emancipation for proteome-wide incorporation of noncanonical amino acids: rare isoleucine codon AUA as a target for genetic code expansion". FEMS Microbiology Letters. 351 (2): 133–44. doi:10.1111/1574-6968.12371. PMC 4237120. PMID 24433543.
- Hoesl MG, Budisa N (October 2012). "Recent advances in genetic code engineering in Escherichia coli". Current Opinion in Biotechnology. 23 (5): 751–7. doi:10.1016/j.copbio.2011.12.027. PMID 22237016.
- Neumann H, Wang K, Davis L, Garcia-Alai M, Chin JW (March 2010). "Encoding multiple unnatural amino acids via evolution of a quadruplet-decoding ribosome" (PDF). Nature. 464 (7287): 441–4. Bibcode:2010Natur.464..441N. doi:10.1038/nature08817. PMID 20154731. S2CID 4390989.
- Watanabe T, Muranaka N, Hohsaka T (March 2008). "Four-base codon-mediated saturation mutagenesis in a cell-free translation system". Journal of Bioscience and Bioengineering. 105 (3): 211–5. doi:10.1263/jbb.105.211. PMID 18397770.
- Anderson JC, Wu N, Santoro SW, Lakshman V, King DS, Schultz PG (May 2004). "An expanded genetic code with a functional quadruplet codon". Proceedings of the National Academy of Sciences of the United States of America. 101 (20): 7566–71. Bibcode:2004PNAS..101.7566A. doi:10.1073/pnas.0401517101. PMC 419646. PMID 15138302.
- Santoro SW, Anderson JC, Lakshman V, Schultz PG (December 2003). "An archaebacteria-derived glutamyl-tRNA synthetase and tRNA pair for unnatural amino acid mutagenesis of proteins in Escherichia coli". Nucleic Acids Research. 31 (23): 6700–9. doi:10.1093/nar/gkg903. PMC 290271. PMID 14627803.
- Anderson JC, Schultz PG (August 2003). "Adaptation of an orthogonal archaeal leucyl-tRNA and synthetase pair for four-base, amber, and opal suppression". Biochemistry. 42 (32): 9598–608. doi:10.1021/bi034550w. PMID 12911301.
- Hancock SM, Uprety R, Deiters A, Chin JW (October 2010). "Expanding the genetic code of yeast for incorporation of diverse unnatural amino acids via a pyrrolysyl-tRNA synthetase/tRNA pair". Journal of the American Chemical Society. 132 (42): 14819–24. doi:10.1021/ja104609m. PMC 2956376. PMID 20925334.
- Minaba M, Kato Y (March 2014). "High-yield, zero-leakage expression system with a translational switch using site-specific unnatural amino Acid incorporation". Applied and Environmental Microbiology. 80 (5): 1718–25. doi:10.1128/AEM.03417-13. PMC 3957627. PMID 24375139.
- Chin JW, Cropp TA, Anderson JC, Mukherji M, Zhang Z, Schultz PG (August 2003). "An expanded eukaryotic genetic code". Science. 301 (5635): 964–7. Bibcode:2003Sci...301..964C. doi:10.1126/science.1084772. PMID 12920298. S2CID 2376187.
- Wu N, Deiters A, Cropp TA, King D, Schultz PG (November 2004). "A genetically encoded photocaged amino acid". Journal of the American Chemical Society. 126 (44): 14306–7. doi:10.1021/ja040175z. PMID 15521721.
- Kowal AK, Kohrer C, RajBhandary UL (February 2001). "Twenty-first aminoacyl-tRNA synthetase-suppressor tRNA pairs for possible use in site-specific incorporation of amino acid analogues into proteins in eukaryotes and in eubacteria". Proceedings of the National Academy of Sciences of the United States of America. 98 (5): 2268–73. Bibcode:2001PNAS...98.2268K. doi:10.1073/pnas.031488298. PMC 30127. PMID 11226228.
- Lemke EA, Summerer D, Geierstanger BH, Brittain SM, Schultz PG (December 2007). "Control of protein phosphorylation with a genetically encoded photocaged amino acid". Nature Chemical Biology. 3 (12): 769–72. doi:10.1038/nchembio.2007.44. PMID 17965709.
- Kang JY, Kawaguchi D, Coin I, Xiang Z, O'Leary DD, Slesinger PA, Wang L (October 2013). "In vivo expression of a light-activatable potassium channel using unnatural amino acids". Neuron. 80 (2): 358–70. doi:10.1016/j.neuron.2013.08.016. PMC 3815458. PMID 24139041.
- Zhang Z, Alfonta L, Tian F, Bursulaya B, Uryu S, King DS, Schultz PG (June 2004). "Selective incorporation of 5-hydroxytryptophan into proteins in mammalian cells". Proceedings of the National Academy of Sciences of the United States of America. 101 (24): 8882–7. Bibcode:2004PNAS..101.8882Z. doi:10.1073/pnas.0307029101. PMC 428441. PMID 15187228.
- Han S, Yang A, Lee S, Lee HW, Park CB, Park HS (February 2017). "Expanding the genetic code of Mus musculus". Nature Communications. 8: 14568. Bibcode:2017NatCo...814568H. doi:10.1038/ncomms14568. PMC 5321798. PMID 28220771.
- Rackham O, Chin JW (August 2005). "A network of orthogonal ribosome x mRNA pairs". Nature Chemical Biology. 1 (3): 159–66. doi:10.1038/nchembio719. PMID 16408021. S2CID 37181098.
- Wang K, Neumann H, Peak-Chew SY, Chin JW (July 2007). "Evolved orthogonal ribosomes enhance the efficiency of synthetic genetic code expansion" (PDF). Nature Biotechnology. 25 (7): 770–7. doi:10.1038/nbt1314. PMID 17592474. S2CID 19683574.
- Fried SD, Schmied WH, Uttamapinant C, Chin JW (October 2015). "Ribosome Subunit Stapling for Orthogonal Translation in E. coli". Angewandte Chemie. 54 (43): 12791–4. doi:10.1002/anie.201506311. PMC 4678508. PMID 26465656.
- Terasaka N, Hayashi G, Katoh T, Suga H (July 2014). "An orthogonal ribosome-tRNA pair via engineering of the peptidyl transferase center". Nature Chemical Biology. 10 (7): 555–7. doi:10.1038/nchembio.1549. PMID 24907900.
- Cavarelli J, Moras D (January 1993). "Recognition of tRNAs by aminoacyl-tRNA synthetases". FASEB Journal. 7 (1): 79–86. doi:10.1096/fasebj.7.1.8422978. PMID 8422978. S2CID 46222849.
- Schimmel PR, Söll D (1979). "Aminoacyl-tRNA synthetases: general features and recognition of transfer RNAs". Annual Review of Biochemistry. 48: 601–48. doi:10.1146/annurev.bi.48.070179.003125. PMID 382994.
- Ohuchi M, Murakami H, Suga H (October 2007). "The flexizyme system: a highly flexible tRNA aminoacylation tool for the translation apparatus". Current Opinion in Chemical Biology. 11 (5): 537–42. doi:10.1016/j.cbpa.2007.08.011. PMID 17884697.
- Wang Q, Parrish AR, Wang L (March 2009). "Expanding the genetic code for biological studies". Chemistry & Biology. 16 (3): 323–36. doi:10.1016/j.chembiol.2009.03.001. PMC 2696486. PMID 19318213.
- Park HS, Hohn MJ, Umehara T, Guo LT, Osborne EM, Benner J, et al. (August 2011). "Expanding the genetic code of Escherichia coli with phosphoserine". Science. 333 (6046): 1151–4. Bibcode:2011Sci...333.1151P. doi:10.1126/science.1207203. PMC 5547737. PMID 21868676.
- Oza JP, Aerni HR, Pirman NL, Barber KW, Ter Haar CM, Rogulina S, et al. (September 2015). "Robust production of recombinant phosphoproteins using cell-free protein synthesis". Nature Communications. 6: 8168. Bibcode:2015NatCo...6.8168O. doi:10.1038/ncomms9168. PMC 4566161. PMID 26350765.
- Pirman NL, Barber KW, Aerni HR, Ma NJ, Haimovich AD, Rogulina S, et al. (September 2015). "A flexible codon in genomically recoded Escherichia coli permits programmable protein phosphorylation". Nature Communications. 6: 8130. Bibcode:2015NatCo...6.8130P. doi:10.1038/ncomms9130. PMC 4566969. PMID 26350500.
- Rogerson DT, Sachdeva A, Wang K, Haq T, Kazlauskaite A, Hancock SM, et al. (July 2015). "Efficient genetic encoding of phosphoserine and its nonhydrolyzable analog". Nature Chemical Biology. 11 (7): 496–503. doi:10.1038/nchembio.1823. PMC 4830402. PMID 26030730.
- Gauba V, Grünewald J, Gorney V, Deaton LM, Kang M, Bursulaya B, et al. (August 2011). "Loss of CD4 T-cell-dependent tolerance to proteins with modified amino acids". Proceedings of the National Academy of Sciences of the United States of America. 108 (31): 12821–6. Bibcode:2011PNAS..10812821G. doi:10.1073/pnas.1110042108. PMC 3150954. PMID 21768354.
- Liu CC, Mack AV, Brustad EM, Mills JH, Groff D, Smider VV, Schultz PG (July 2009). "Evolution of proteins with genetically encoded "chemical warheads"". Journal of the American Chemical Society. 131 (28): 9616–7. doi:10.1021/ja902985e. PMC 2745334. PMID 19555063.
- Hammerling MJ, Ellefson JW, Boutz DR, Marcotte EM, Ellington AD, Barrick JE (March 2014). "Bacteriophages use an expanded genetic code on evolutionary paths to higher fitness". Nature Chemical Biology. 10 (3): 178–80. doi:10.1038/nchembio.1450. PMC 3932624. PMID 24487692.
- Krishnakumar R, Ling J (January 2014). "Experimental challenges of sense codon reassignment: an innovative approach to genetic code expansion". FEBS Letters. 588 (3): 383–8. doi:10.1016/j.febslet.2013.11.039. PMID 24333334. S2CID 10152595.
- Gibson DG, Glass JI, Lartigue C, Noskov VN, Chuang RY, Algire MA, et al. (July 2010). "Creation of a bacterial cell controlled by a chemically synthesized genome". Science. 329 (5987): 52–6. Bibcode:2010Sci...329...52G. doi:10.1126/science.1190719. PMID 20488990.
- Hirao I, Ohtsuki T, Fujiwara T, Mitsui T, Yokogawa T, Okuni T, et al. (February 2002). "An unnatural base pair for incorporating amino acid analogs into proteins". Nature Biotechnology. 20 (2): 177–82. doi:10.1038/nbt0202-177. PMID 11821864. S2CID 22055476.
- Hirao I, Kimoto M, Mitsui T, Fujiwara T, Kawai R, Sato A, et al. (September 2006). "An unnatural hydrophobic base pair system: site-specific incorporation of nucleotide analogs into DNA and RNA". Nature Methods. 3 (9): 729–35. doi:10.1038/nmeth915. PMID 16929319. S2CID 6494156.
- Kimoto M, Kawai R, Mitsui T, Yokoyama S, Hirao I (February 2009). "An unnatural base pair system for efficient PCR amplification and functionalization of DNA molecules". Nucleic Acids Research. 37 (2): e14. doi:10.1093/nar/gkn956. PMC 2632903. PMID 19073696.
- Yamashige R, Kimoto M, Takezawa Y, Sato A, Mitsui T, Yokoyama S, Hirao I (March 2012). "Highly specific unnatural base pair systems as a third base pair for PCR amplification". Nucleic Acids Research. 40 (6): 2793–806. doi:10.1093/nar/gkr1068. PMC 3315302. PMID 22121213.
- Kimoto M, Yamashige R, Matsunaga K, Yokoyama S, Hirao I (May 2013). "Generation of high-affinity DNA aptamers using an expanded genetic alphabet". Nature Biotechnology. 31 (5): 453–7. doi:10.1038/nbt.2556. PMID 23563318. S2CID 23329867.
- Malyshev DA, Dhami K, Quach HT, Lavergne T, Ordoukhanian P, Torkamani A, Romesberg FE (July 2012). "Efficient and sequence-independent replication of DNA containing a third base pair establishes a functional six-letter genetic alphabet". Proceedings of the National Academy of Sciences of the United States of America. 109 (30): 12005–10. Bibcode:2012PNAS..10912005M. doi:10.1073/pnas.1205176109. PMC 3409741. PMID 22773812.
- Malyshev DA, Dhami K, Lavergne T, Chen T, Dai N, Foster JM, et al. (May 2014). "A semi-synthetic organism with an expanded genetic alphabet". Nature. 509 (7500): 385–8. Bibcode:2014Natur.509..385M. doi:10.1038/nature13314. PMC 4058825. PMID 24805238.
- Callaway E (May 7, 2014). "Scientists Create First Living Organism With 'Artificial' DNA". Nature News. Huffington Post. Retrieved 8 May 2014.
- Fikes BJ (May 8, 2014). "Life engineered with expanded genetic code". San Diego Union Tribune. Archived from the original on 9 May 2014. Retrieved 8 May 2014.
- Sample I (May 7, 2014). "First life forms to pass on artificial DNA engineered by US scientists". The Guardian. Retrieved 8 May 2014.
- Pollack A (May 7, 2014). "Scientists Add Letters to DNA's Alphabet, Raising Hope and Fear". The New York Times. Retrieved 8 May 2014.
- Pollack A (May 7, 2014). "Researchers Report Breakthrough in Creating Artificial Genetic Code". The New York Times. Retrieved May 7, 2014.
- Callaway E (May 7, 2014). "First life with 'alien' DNA". Nature. doi:10.1038/nature.2014.15179. S2CID 86967999. Retrieved May 7, 2014.
- Amos J (8 May 2014). "Semi-synthetic bug extends 'life's alphabet'". BBC News. Retrieved 2014-05-09.
- Koide H, Yokoyama S, Kawai G, Ha JM, Oka T, Kawai S, et al. (September 1988). "Biosynthesis of a protein containing a nonprotein amino acid by Escherichia coli: L-2-aminohexanoic acid at position 21 in human epidermal growth factor". Proceedings of the National Academy of Sciences of the United States of America. 85 (17): 6237–41. Bibcode:1988PNAS...85.6237K. doi:10.1073/pnas.85.17.6237. PMC 281944. PMID 3045813.
- Ferla MP, Patrick WM (August 2014). "Bacterial methionine biosynthesis". Microbiology. 160 (Pt 8): 1571–1584. doi:10.1099/mic.0.077826-0. PMID 24939187.
- Doublié S (2007). "Production of Selenomethionyl Proteins in Prokaryotic and Eukaryotic Expression Systems". Macromolecular Crystallography Protocols. Methods in Molecular Biology. 363. pp. 91–108. doi:10.1007/978-1-59745-209-0_5. ISBN 978-1-58829-292-6. PMID 17272838.
- Suchanek M, Radzikowska A, Thiele C (April 2005). "Photo-leucine and photo-methionine allow identification of protein-protein interactions in living cells". Nature Methods. 2 (4): 261–7. doi:10.1038/NMETH752. PMID 15782218.
- Ramadan SE, Razak AA, Ragab AM, el-Meleigy M (June 1989). "Incorporation of tellurium into amino acids and proteins in a tellurium-tolerant fungi". Biological Trace Element Research. 20 (3): 225–32. doi:10.1007/BF02917437. PMID 2484755. S2CID 9439946.
- Bacher JM, Ellington AD (September 2001). "Selection and characterization of Escherichia coli variants capable of growth on an otherwise toxic tryptophan analogue". Journal of Bacteriology. 183 (18): 5414–25. doi:10.1128/jb.183.18.5414-5425.2001. PMC 95426. PMID 11514527.
- Wong JT (October 1983). "Membership mutation of the genetic code: loss of fitness by tryptophan". Proceedings of the National Academy of Sciences of the United States of America. 80 (20): 6303–6. Bibcode:1983PNAS...80.6303W. doi:10.1073/pnas.80.20.6303. PMC 394285. PMID 6413975.
- Hoesl MG, Oehm S, Durkin P, Darmon E, Peil L, Aerni HR, et al. (August 2015). "Chemical Evolution of a Bacterial Proteome". Angewandte Chemie. 54 (34): 10030–4. doi:10.1002/anie.201502868. PMC 4782924. PMID 26136259. NIHMSID: NIHMS711205
- Moroder L, Budisa N (April 2010). "Synthetic biology of protein folding". ChemPhysChem. 11 (6): 1181–7. doi:10.1002/cphc.201000035. PMID 20391526.
- Budisa N (December 2004). "Prolegomena to future experimental efforts on genetic code engineering by expanding its amino acid repertoire". Angewandte Chemie. 43 (47): 6426–63. doi:10.1002/anie.200300646. PMID 15578784.
- Link AJ, Mock ML, Tirrell DA (December 2003). "Non-canonical amino acids in protein engineering". Current Opinion in Biotechnology. 14 (6): 603–9. doi:10.1016/j.copbio.2003.10.011. PMID 14662389.
- Nehring S, Budisa N, Wiltschi B (2012). "Performance analysis of orthogonal pairs designed for an expanded eukaryotic genetic code". PLOS ONE. 7 (4): e31992. Bibcode:2012PLoSO...731992N. doi:10.1371/journal.pone.0031992. PMC 3320878. PMID 22493661.
- Agostini F, Völler JS, Koksch B, Acevedo-Rocha CG, Kubyshkin V, Budisa N (August 2017). "Biocatalysis with Unnatural Amino Acids: Enzymology Meets Xenobiology". Angewandte Chemie. 56 (33): 9680–9703. doi:10.1002/anie.201610129. PMID 28085996.
- Rubini M, Lepthien S, Golbik R, Budisa N (July 2006). "Aminotryptophan-containing barstar: structure--function tradeoff in protein design and engineering with an expanded genetic code". Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics. 1764 (7): 1147–58. doi:10.1016/j.bbapap.2006.04.012. PMID 16782415.
- Steiner T, Hess P, Bae JH, Wiltschi B, Moroder L, Budisa N (February 2008). "Synthetic biology of proteins: tuning GFPs folding and stability with fluoroproline". PLOS ONE. 3 (2): e1680. Bibcode:2008PLoSO...3.1680S. doi:10.1371/journal.pone.0001680. PMC 2243022. PMID 18301757.
- Wolschner C, Giese A, Kretzschmar HA, Huber R, Moroder L, Budisa N (May 2009). "Design of anti- and pro-aggregation variants to assess the effects of methionine oxidation in human prion protein". Proceedings of the National Academy of Sciences of the United States of America. 106 (19): 7756–61. Bibcode:2009PNAS..106.7756W. doi:10.1073/pnas.0902688106. PMC 2674404. PMID 19416900.
- Lepthien S, Hoesl MG, Merkel L, Budisa N (October 2008). "Azatryptophans endow proteins with intrinsic blue fluorescence". Proceedings of the National Academy of Sciences of the United States of America. 105 (42): 16095–100. Bibcode:2008PNAS..10516095L. doi:10.1073/pnas.0802804105. PMC 2571030. PMID 18854410.
- Bae JH, Rubini M, Jung G, Wiegand G, Seifert MH, Azim MK, et al. (May 2003). "Expansion of the genetic code enables design of a novel "gold" class of green fluorescent proteins". Journal of Molecular Biology. 328 (5): 1071–81. doi:10.1016/s0022-2836(03)00364-4. PMID 12729742.
- Hoesl MG, Acevedo-Rocha CG, Nehring S, Royter M, Wolschner C, Wiltschi B, Budisa N, Antranikian G (2011). "Lipase Congeners Designed by Genetic Code Engineering". ChemCatChem. 3 (1): 213–221. doi:10.1002/cctc.201000253. ISSN 1867-3880. S2CID 86352672.
- Hong SH, Kwon YC, Jewett MC (2014). "Non-standard amino acid incorporation into proteins using Escherichia coli cell-free protein synthesis". Frontiers in Chemistry. 2: 34. Bibcode:2014FrCh....2...34H. doi:10.3389/fchem.2014.00034. PMC 4050362. PMID 24959531.
- 'Unnatural' microbe can make proteins. BBC News. 29 November 2017.
- Zhang Y, Ptacin JL, Fischer EC, Aerni HR, Caffaro CE, San Jose K, et al. (November 2017). "A semi-synthetic organism that stores and retrieves increased genetic information". Nature. 551 (7682): 644–647. Bibcode:2017Natur.551..644Z. doi:10.1038/nature24659. PMC 5796663. PMID 29189780.
- Howgego J (February 2014). "On stranger nucleotides". Chemistry World.
- Li L, Degardin M, Lavergne T, Malyshev DA, Dhami K, Ordoukhanian P, Romesberg FE (January 2014). "Natural-like replication of an unnatural base pair for the expansion of the genetic alphabet and biotechnology applications". Journal of the American Chemical Society. 136 (3): 826–9. doi:10.1021/ja408814g. PMC 3979842. PMID 24152106.