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
    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.

  • 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
    codon.

  • 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
    conserved.

 

Works Cited

[‘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.
2. ^ Jump up to:a b 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.
3. ^ Jump up to:a b 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.
4. ^ 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.
5. ^ 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.
6. ^ Jump up to:a b c d e f 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.
7. ^ Jump up to:a b 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.
8. ^ 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.
9. ^ Jump up to:a b 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.
10. ^ Jump up to:a b c 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.
11. ^ 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.
12. ^ Jump up to:a b 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.
13. ^ Furter R (February 1998). “Expansion of the genetic code:
site-directed p-fluoro-phenylalanine incorporation in Escherichia coli”. Protein Science. 7 (2): 419–26. doi:10.1002/pro.5560070223. PMC 2143905. PMID 9521119.
14. ^ 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.
15. ^ 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.
16. ^ Jump up to:a b “The Peter G. Schultz Laboratory”. Schultz.scripps.edu. Archived from the original on 2018-07-12. Retrieved 2015-05-05.
17. ^ 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.
18. ^ Mehl RA, Anderson JC, Santoro SW, Wang L, Martin AB, King DS, Horn DM, Schultz PG (January 2003). “Generation of a bacterium with a 21 amino acid genetic code”. Journal of the American Chemical Society. 125 (4): 935–9. doi:10.1021/ja0284153.
PMID 12537491.
19. ^ “context :: 21-amino-acid bacteria: expanding the genetic code”. Straddle3.net. Retrieved 2015-05-05.
20. ^ 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.
21. ^ 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.
22. ^ 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.
23. ^ 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.
24. ^ 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.
25. ^ 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.
26. ^ 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.
27. ^ 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.
28. ^ Jump up to:a b 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.
29. ^ 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.
30. ^ 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.
31. ^ 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.
32. ^ Robertson, Wesley E.; Funke,
Louise F. H.; de la Torre, Daniel; Fredens, Julius; Elliott, Thomas S.; Spinck, Martin; Christova, Yonka; Cervettini, Daniele; Böge, Franz L.; Liu, Kim C.; Buse, Salvador; Maslen, Sarah; Salmond, George P. C.; Chin, Jason W. (4 June 2021). “Sense
codon reassignment enables viral resistance and encoded polymer synthesis”. Science. 372 (6546): 1057–1062. doi:10.1126/science.abg3029. PMC 7611380.
33. ^ Atkins, J. F.; Bjoerk, G. R. “A gripping tale of ribosomal frameshifting: extragenic suppressors
of frameshift mutations spotlight P-site realignment.” Microbiol. Mol. Biol. Rev. 2009, 73, 178-210.
34. ^ Anderson, J. C.; Wu, N.; Santoro, S. W.; Lakshman, V.; King, D. S.; Schultz, P. G. “An expanded genetic code with a functional quadruplet
codon.” Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 7566-7571.
35. ^ Neumann, H.; Wang, K.; Davis, L.; Garcia-Alai, M.; Chin, J. W. “Encoding multiple unnatural amino acids via evolution of a quadruplet-decoding ribosome.” Nature 2010, 464, 441-444.
36. ^
Niu, W.; Schultz, P. G.; Guo, J. “An expanded genetic code in mammalian cells with a functional quadruplet codon.” ACS Chem. Biol. 2013, 8, 1640-1645.
37. ^ Jump up to:a b c 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”. Nature. 464 (7287): 441–4. Bibcode:2010Natur.464..441N. doi:10.1038/nature08817. PMID 20154731. S2CID 4390989.
38. ^ Hong S, Sunita S, Maehigashi T, Hoffer ED, Dunkle
JA, Dunham CM (October 2018). “Mechanism of tRNA-mediated +1 ribosomal frameshifting”. Proceedings of the National Academy of Sciences of the United States of America. 115 (44): 11226–11231. doi:10.1073/pnas.1809319115. PMC 6217423. PMID 30262649.
39. ^
Niu, W., Schultz, P. G., and Guo, J. (2013) An expanded genetic code in mammalian cells with a functional quadruplet codon. ACS Chem Biol 8, 1640-1645.
40. ^ DeBenedictis EA, Carver GD, Chung CZ, Söll D, Badran AH (September 2021). “Multiplex suppression
of four quadruplet codons via tRNA directed evolution”. Nature Communications. 12 (1): 5706. doi:10.1038/s41467-021-25948-y. PMC 8481270. PMID 34588441.
41. ^ Chen, Y., Wan, Y., Wang, N., Yuan, Z., Niu, W., Li, Q., and Guo, J. (2018) Controlling
the Replication of a Genomically Recoded HIV-1 with a Functional Quadruplet Codon in Mammalian Cells. ACS Synth. Biol. 7, 1612-1617.
42. ^ 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.
43. ^ 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.
44. ^ 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.
45. ^
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.
46. ^ 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.
47. ^ 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.
48. ^ 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.
49. ^ Jump up to:a b 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.
50. ^ Jump up to:a b 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.
51. ^ 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.
52. ^ Palei S, Buchmuller B, Wolffgramm
J, Muñoz-Lopez Á, Jung S, Czodrowski P, Summerer D (April 2020). “Light-Activatable TET-Dioxygenases Reveal Dynamics of 5-Methylcytosine Oxidation and Transcriptome Reorganization”. Journal of the American Chemical Society. 142 (16): 7289–7294. doi:10.1021/jacs.0c01193.
PMID 32286069. S2CID 215757172.
53. ^ 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.
54. ^ Wolffgramm J, Buchmuller B, Palei S, Muñoz-López Á, Kanne J, Janning P, et al. (June 2021). “Light-Activation of DNA-Methyltransferases”. Angewandte Chemie. 60 (24): 13507–13512. doi:10.1002/anie.202103945. PMC
8251764. PMID 33826797.
55. ^ 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.
56. ^ 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.
57. ^ 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.
58. ^ 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.
59. ^ 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.
60. ^ 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.
61. ^
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.
62. ^ 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.
63. ^ 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.
64. ^ 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.
65. ^ 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.
66. ^ 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.
67. ^ 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.
68. ^ 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.
69. ^
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.
70. ^ 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.
71. ^ 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.
72. ^ Kipper K, Lundius EG, Ćurić V, Nikić I, Wiessler M, Lemke EA, Elf J (February 2017). “Application
of Noncanonical Amino Acids for Protein Labeling in a Genomically Recoded Escherichia coli”. ACS Synthetic Biology. 6 (2): 233–255. doi:10.1021/acssynbio.6b00138. PMID 27775882.
73. ^ Jump up to:a b Dunkelmann DL, Oehm SB, Beattie AT, Chin JW (August
2021). “A 68-codon genetic code to incorporate four distinct non-canonical amino acids enabled by automated orthogonal mRNA design”. Nature Chemistry. 13 (11): 1110–1117. doi:10.1038/s41557-021-00764-5. PMC 7612796. PMID 34426682. S2CID 237271721.
74. ^
Jump up to:a b 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.
75. ^
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.
76. ^ 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.
77. ^ 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.
78. ^ 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.
79. ^ 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.
80. ^ 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.
81. ^ 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.
82. ^ Jump up to:a b c d 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.
83. ^ Callaway E (May 7, 2014). “Scientists Create First Living Organism With ‘Artificial’ DNA”. Nature News. Huffington Post. Retrieved 8 May 2014.
84. ^ Jump up to:a b 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.
85. ^ Sample I (May 7, 2014). “First life forms to pass on artificial DNA engineered by US scientists”. The Guardian.
Retrieved 8 May 2014.
86. ^ Pollack A (May 7, 2014). “Scientists Add Letters to DNA’s Alphabet, Raising Hope and Fear”. The New York Times. Retrieved 8 May 2014.
87. ^ Pollack A (May 7, 2014). “Researchers Report Breakthrough in Creating Artificial
Genetic Code”. The New York Times. Retrieved May 7, 2014.
88. ^ Callaway E (May 7, 2014). “First life with ‘alien’ DNA”. Nature. doi:10.1038/nature.2014.15179. S2CID 86967999. Retrieved May 7, 2014.
89. ^ Amos J (8 May 2014). “Semi-synthetic
bug extends ‘life’s alphabet'”. BBC News. Retrieved 2014-05-09.
90. ^ 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.
91. ^ Ferla
MP, Patrick WM (August 2014). “Bacterial methionine biosynthesis”. Microbiology. 160 (Pt 8): 1571–1584. doi:10.1099/mic.0.077826-0. PMID 24939187.
92. ^ Doublié S (2007). “Production of Selenomethionyl Proteins in Prokaryotic and Eukaryotic Expression
Systems”. Macromolecular Crystallography Protocols. Methods in Molecular Biology. Vol. 363. pp. 91–108. doi:10.1007/978-1-59745-209-0_5. ISBN 978-1-58829-292-6. PMID 17272838.
93. ^ 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.
94. ^ 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.
95. ^ 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.
96. ^ 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.
97. ^ 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
98. ^ Moroder
L, Budisa N (April 2010). “Synthetic biology of protein folding”. ChemPhysChem. 11 (6): 1181–7. doi:10.1002/cphc.201000035. PMID 20391526.
99. ^ 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.
100. ^ 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.
101. ^ 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.
102. ^ 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.
103. ^ 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.
104. ^ 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.
105. ^ 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.
106. ^ 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.
107. ^ 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.
108. ^ 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.
109. ^ 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.
110. ^ ‘Unnatural’ microbe can make proteins. BBC News. 29
November 2017.
111. ^ Jump up to:a b 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.
112. ^ Howgego J (February 2014). “On stranger nucleotides”. Chemistry World.
113. ^ 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.
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