Applications of genetic code expansion technology in eukaryotes

Abstract

Unnatural amino acids (UAAs) have gained significant attention in protein engineering and drug development owing to their ability to introduce new chemical functionalities to proteins. In eukaryotes, genetic code expansion (GCE) enables the incorporation of UAAs and facilitates posttranscriptional modification (PTM), which is not feasible in prokaryotic systems. GCE is also a powerful tool for cell or animal imaging, the monitoring of protein interactions in target cells, drug development, and switch regulation. Therefore, there is keen interest in utilizing GCE in eukaryotic systems. This review provides an overview of the application of GCE in eukaryotic systems and discusses current challenges that need to be addressed.

Key words： genetic code expansion; unnatural amino acid; eukaryotes; basic research; therapeutic applications

References

1 Adams MD, Celniker SE, Holt RA et al. The genome sequence of Drosophila melanogaster. Science (New York, N.Y.) 2000; 287:2185–95.

2 Adumeau P, Sharma SK, Brent C et al. Site-specifically labeled immunoconjugates for molecular imaging—part 1: cysteine residues and glycans. Mol Imaging Biol 2016; 18:1–17.

3 Aemissegger A, Hilvert D. Synthesis and application of an azobenzene amino acid as a light-switchable turn element in polypeptides. Nat Protocols 2007; 2:161–7.

4 Agard NJ, Baskin JM, Prescher JA et al. A comparative study of bioorthogonal reactions with azides. ACS Chem Biol 2006; 1:644–8.

5 Agostini F, Völler J-S, Koksch B et al. Biocatalysis with unnatural amino acids: enzymology meets xenobiology. Angew Chem Int Ed Engl 2017; 56:9680–703.

6 Alexpandi R, Prasanth MI, Ravi AV et al. Protective effect of neglected plant Diplocyclos palmatus on quorum sensing mediated infection of Serratia marcescens and UV-A induced photoaging in model Caenorhabditis elegans. J Photochem Photobiol B 2019; 201:111637.

7 Alouane A, Labruère R, Le Saux T et al. Self-immolative spacers: kinetic aspects, structure–property relationships, and applications. Angew Chem Int Ed Engl 2015; 54:7492–509.

8 Arbely E, Torres-Kolbus J, Deiters A et al. Photocontrol of tyrosine phosphorylation in mammalian cells via genetic encoding of photocaged tyrosine. J Am Chem Soc 2012; 134:11912–5.

9 Asiful Islam M, Alam F, Kamal MA et al. Therapeutic suppression of nonsense mutation: an emerging target in multiple diseases and thrombotic disorders. Curr Pharm Des 2017; 23:1598–609.

10 Axup JY, Bajjuri KM, Ritland M et al. Synthesis of site-specific antibody-drug conjugates using unnatural amino acids. Proc Natl Acad Sci USA 2012; 109:16101–6.

11 Baillie TA. Targeted covalent inhibitors for drug design. Angew Chem Int Ed Engl 2016; 55:13408–21.

12 Barber KW, Rinehart J. The ABCs of PTMs. Nat Chem Biol 2018; 14:188–92.

13 Beatty KE, Liu JC, Xie F et al. Fluorescence visualization of newly synthesized proteins in mammalian cells. Angew Chem Int Ed Engl 2006; 45:7364–7.

14 Beharry AA, Woolley GA. Azobenzene photoswitches for biomolecules. Chem Soc Rev 2011; 40:4422–37.

15 Beránek V, Reinkemeier CD, Zhang MS et al. Genetically encoded protein phosphorylation in mammalian cells. Cell Chem Biol 2018; 25:1067–1074.e5.

16 Bianco A, Townsley FM, Greiss S et al. Expanding the genetic code of Drosophila melanogaster. Nat Chem Biol 2012; 8:748–50.

17 Blaskovich MAT. Unusual amino acids in medicinal chemistry. J Med Chem 2016; 59:10807–36.

18 Bose M, Groff D, Xie J et al. The incorporation of a photoisomerizable amino acid into proteins in E. coli. J Am Chem Soc 2006; 128:388–9.

19 Brown W, Deiters A. Light-activation of Cre recombinase in zebrafish embryos through genetic code expansion. Methods Enzymol 2019; 624:265–81.

20 Brown W, Galpin JD, Rosenblum C et al. Chemically Acylated tRNAs are functional in zebrafish embryos. J Am Chem Soc 2023a; 145:2414–20.

21 Brown W, Wesalo J, Tsang M et al. Engineering small molecule switches of protein function in zebrafish embryos. J Am Chem Soc 2023b; 145:2395–403.

22 Burnett K, Edsinger E, Albrecht DR. Rapid and gentle hydrogel encapsulation of living organisms enables long-term microscopy over multiple hours. Commun Biol 2018; 1:73.

23 Cao Y, Axup JY, Ma JSY et al. Multiformat T-cell-engaging bispecific antibodies targeting human breast cancers. Angew Chem Int Ed Engl 2015; 54:7022–7.

24 Cao Y, Rodgers DT, Du J et al. Design of switchable chimeric antigen receptor T cells targeting breast cancer. Angew Chem Int Ed Engl 2016; 55:7520–4.

25 Cao YJ, Wang X, Wang Z et al. Switchable CAR-T cells out-performed traditional antibody-redirected therapeutics targeting breast cancers. ACS Synth Biol 2021; 10:1176–83.

26 Chang H, Han M, Huang W et al. Light-induced protein translocation by genetically encoded unnatural amino acid in Caenorhabditis elegans. Protein Cell 2013; 4:883–6.

27 Chang S-W, Liu W-C, Liao K-Y et al. Phosphorylation of HPV-16 E2 at serine 243 enables binding to Brd4 and mitotic chromosomes. PLoS One 2014; 9:e110882.

28 Chatterjee A, Guo J, Lee HS et al. A genetically encoded fluorescent probe in mammalian cells. J Am Chem Soc 2013a; 135:12540–3.

29 Chatterjee A, Xiao H, Bollong M et al. Efficient viral delivery system for unnatural amino acid mutagenesis in mammalian cells. Proc Natl Acad Sci USA 2013b; 110:11803–8.

30 Chemla Y, Ozer E, Algov I et al. Context effects of genetic code expansion by stop codon suppression. Curr Opin Chem Biol 2018; 46:146–55.

31 Chen J, Tsai Y-H. Applications of genetic code expansion in studying protein post-translational modification. J Mol Biol 2022; 434:167424.

32 Chen S, Schultz PG, Brock A. An improved system for the generation and analysis of mutant proteins containing unnatural amino acids in Saccharomyces cerevisiae. J Mol Biol 2007; 371:112–22.

33 Chen Y, Ma J, Lu W et al. Heritable expansion of the genetic code in mouse and zebrafish. Cell Res 2017; 27:294–7.

34 Chen Y, Loredo A, Gordon A et al. A noncanonical amino acid-based relay system for site-specific protein labeling. Chem Commun (Cambridge, England) 2018; 54:7187–90.

35 Chen Y, Wu K-L, Tang J et al. Addition of isocyanide-containing amino acids to the genetic code for protein labeling and activation. ACS Chem Biol 2019; 14:2793–9.

36 Chen L, Zhu C, Guo H et al. Epitope-directed antibody selection by site-specific photocrosslinking. Sci Adv 2020; 6:eaaz7825.

37 Chen Y, Jin S, Zhang M et al. Unleashing the potential of noncanonical amino acid biosynthesis to create cells with precision tyrosine sulfation. Nat Commun 2022a; 13:5434.

38 Chen C, Yu G, Huang Y et al. Genetic-code-expanded cellbased therapy for treating diabetes in mice. Nat Chem Biol 2022b; 18:47–55.

39 Chen Y, Liu W-Q, Zheng X et al. Cell-free biosynthesis of lysine-derived unnatural amino acids with chloro, alkene, and alkyne groups. ACS Synth Biol 2023; 12:1349–57.

40 Chin JW. Modular approaches to expanding the functions of living matter. Nat Chem Biol 2006; 2:304–11.

41 Chin JW, Martin AB, King DS et al. Addition of a photocrosslinking amino acid to the genetic code of Escherichia coli. Proc Natl Acad Sci USA 2002; 99:11020–4.

42 Chin JW, Cropp TA, Anderson JC et al. An expanded eukaryotic genetic code. Science (New York, N.Y.) 2003; 301:964–7.

43 Choi B-K, Bobrowicz P, Davidson RC et al. Use of combinatorial genetic libraries to humanize N-linked glycosylation in the yeast Pichia pastoris. Proc Natl Acad Sci USA 2003; 100:5022–7.

44 Chou C, Deiters A. Light-activated gene editing with a photocaged zinc-finger nuclease. Angew Chem Int Ed Engl 2011; 50:6839–42.

45 Chou C, Young DD, Deiters A. Photocaged t7 RNA polymerase for the light activation of transcription and gene function in pro- and eukaryotic cells. Chembiochem 2010; 11:972–7.

46 Cigler M, Müller TG, Horn-Ghetko D et al. Proximitytriggered covalent stabilization of low-affinity protein complexes in vitro and in vivo. Angew Chem Int Ed Engl 2017; 56:15737–41.

47 Coin I. Application of non-canonical crosslinking amino acids to study protein-protein interactions in live cells. Curr Opin Chem Biol 2018; 46:156–63.

48 Coin I, Perrin MH, Vale WW et al. Photo-cross-linkers incorporated into G-protein-coupled receptors in mammalian cells: a ligand comparison. Angew Chem Int Ed Engl 2011; 50:8077–81.

49 Courtney T, Deiters A. Recent advances in the optical control of protein function through genetic code expansion. Curr Opin Chem Biol 2018; 46:99–107.

50 Courtney TM, Deiters A. Optical control of protein phosphatase function. Nat Commun 2019; 10:4384.

51 Crnković A, Vargas-Rodriguez O, Söll D. Plasticity and constraints of tRNA aminoacylation define directed evolution of aminoacyl-tRNA synthetases. Int J Mol Sci 2019; 20:2294.

52 Darby N, Creighton TE. Disulfide bonds in protein folding and stability. Methods Mol Biol (Clifton, N.J.) 1995; 40:219–52.

53 Davis L, Radman I, Goutou A et al. Precise optical control of gene expression in C. elegans using improved genetic code expansion and Cre recombinase. ELife 2021; 10:e67075.

54 Dieterich DC, Hodas JJL, Gouzer G et al. In situ visualization and dynamics of newly synthesized proteins in rat hippocampal neurons. Nat Neurosci 2010; 13:897–905.

55 Dirksen A, Hackeng TM, Dawson PE. Nucleophilic catalysis of oxime ligation. Angew Chem Int Ed Engl 2006; 45:7581–4.

56 Edwards WF, Young DD, Deiters A. Light-activated Cre recombinase as a tool for the spatial and temporal control of gene function in mammalian cells. ACS Chem Biol 2009; 4:441–5.

57 Elia N. Using unnatural amino acids to selectively label proteins for cellular imaging: a cell biologist viewpoint. FEBS J 2021; 288:1107–17.

58 Elliott TS, Townsley FM, Bianco A et al. Proteome labeling and protein identification in specific tissues and at specific developmental stages in an animal. Nat Biotechnol 2014; 32:465–72.

59 Elsässer SJ, Ernst RJ, Walker OS et al. Genetic code expansion in stable cell lines enables encoded chromatin modification. Nat Methods 2016; 13:158–64.

60 Engelke H, Chou C, Uprety R et al. Control of protein function through optochemical translocation. ACS Synth Biol 2014; 3:731–6.

61 Ernst RJ, Krogager TP, Maywood ES et al. Genetic code expansion in the mouse brain. Nat Chem Biol 2016; 12:776–8.

62 Esvelt KM, Carlson JC, Liu DR. A system for the continuous directed evolution of biomolecules. Nature 2011; 472:499–503.

63 Fang Y, Judkins JC, Boyd SJ et al. Studies on the stability and stabilization of trans-Cyclooctenes through radical inhibition and silver (I) metal complexation. Tetrahedron 2019; 75:4307–17.

64 Fickel TE, Gilvarg C. Transport of impermeant substances in E. coli by way of oligopeptide permease. Nat New Biol 1973; 241:161–3.

65 Fok JA, Mayer C. Genetic-code-expansion strategies for vaccine development. Chembiochem 2020; 21:3291–300.

66 Foster H, Popplewell L, Dickson G. Genetic therapeutic approaches for Duchenne muscular dystrophy. Hum Gene Ther 2012; 23:676–87.

67 Fottner M, Brunner A-D, Bittl V et al. Site-specific ubiquitylation and SUMOylation using genetic-code expansion and sortase. Nat Chem Biol 2019; 15:276–84.

68 Friedrich M, Aigner A. Therapeutic siRNA: state-of-the-art and future perspectives. BioDrugs 2022; 36:549–71.

69 Frøkjaer-Jensen C, Davis MW, Hopkins CE et al. Single-copy insertion of transgenes in Caenorhabditis elegans. Nat Genet 2008; 40:1375–83.

70 Furman JL, Kang M, Choi S et al. A genetically encoded aza- Michael acceptor for covalent cross-linking of protein -receptor complexes. J Am Chem Soc 2014; 136:8411–7.

71 Garza D, Medhora MM, Hartl DL. Drosophila nonsense suppressors: functional analysis in Saccharomyces cerevisiae, Drosophila tissue culture cells and Drosophila melanogaster. Genetics 1990; 126:625–37.

72 Gautier A, Nguyen DP, Lusic H et al. Genetically encoded photocontrol of protein localization in mammalian cells. J Am Chem Soc 2010; 132:4086–8.

73 Gautier A, Deiters A, Chin JW. Light-activated kinases enable temporal dissection of signaling networks in living cells. J Am Chem Soc 2011; 133:2124–7.

74 Gehringer M, Laufer SA. Emerging and re-emerging warheads for targeted covalent inhibitors: applications in medicinal chemistry and chemical biology. J Med Chem 2019; 62:5673–724.

75 Greiss S, Chin JW. Expanding the genetic code of an animal. J Am Chem Soc 2011; 133:14196–9.

76 Grünewald J, Tsao M-L, Perera R et al. Immunochemical termination of self-tolerance. Proc Natl Acad Sci USA 2008; 105:11276–80.

77 Grünewald J, Hunt GS, Dong L et al. Mechanistic studies of the immunochemical termination of self-tolerance with unnatural amino acids. Proc Natl Acad Sci USA 2009; 106:4337–42.

78 Gupta K, Toombes GE, Swartz KJ. Exploring structural dynamics of a membrane protein by combining bioorthogonal chemistry and cysteine mutagenesis. ELife 2019; 8:e50776.

79 Hacker SM, Backus KM, Lazear MR et al. Global profiling of lysine reactivity and ligandability in the human proteome. Nat Chem 2017; 9:1181–90.

80 Han S, Yang A, Lee S et al. Expanding the genetic code of Mus musculus. Nat Commun 2017; 8:14568.

81 Hancock SM, Uprety R, Deiters A et al. Expanding the genetic code of yeast for incorporation of diverse unnatural amino acids via a pyrrolysyl-tRNA synthetase/tRNA pair. J Am Chem Soc 2010; 132:14819–24.

82 Hankore ED, Zhang L, Chen Y et al. Genetic incorporation of noncanonical amino acids using two mutually orthogonal quadruplet codons. ACS Synth Biol 2019; 8:1168–74.

83 Hao R, Ma K, Ru Y et al. Amber codon is genetically unstable in generation of premature termination codon (PTC)-harbouring Foot-and-mouth disease virus (FMDV) via genetic code expansion. RNA Biol 2021; 18:2330–41.

84 He X, Chen Y, Beltran DG et al. Functional genetic encoding of sulfotyrosine in mammalian cells. Nat Commun 2020; 11:4820.

85 He J, Fan Z, Tian Y et al. Spatiotemporal activation of protein O-GlcNAcylation in living cells. J Am Chem Soc 2022; 144:4289–93.

86 Heckler TG, Zama Y, Naka T et al. Dipeptide formation with misacylated tRNAPhes. J Biol Chem 1983; 258:4492–5.

87 Hemphill J, Chou C, Chin JW et al. Genetically encoded light-activated transcription for spatiotemporal control of gene expression and gene silencing in mammalian cells. J Am Chem Soc 2013; 135:13433–9.

88 Hemphill J, Borchardt EK, Brown K et al. Optical control of CRISPR/Cas9 gene editing. J Am Chem Soc 2015; 137:5642–5.

89 Herner A, Marjanovic J, Lewandowski TM et al. 2-Aryl-5-carboxytetrazole as a new photoaffinity label for drug target identification. J Am Chem Soc 2016; 138:14609–15.

90 Hino N, Oyama M, Sato A et al. Genetic incorporation of a photo-crosslinkable amino acid reveals novel protein complexes with GRB2 in mammalian cells. J Mol Biol 2011; 406:343–53.

91 Hoang DM, Pham PT, Bach TQ et al. Stem cell-based therapy for human diseases. Signal Transduct Target Ther 2022; 7:272.

92 Hodgkin J. Novel nematode amber suppressors. Genetics 1985; 111:287–310.

93 Hoppmann C, Wang L. Genetically encoding photoswitchable click amino acids for general optical control of conformation and function of proteins. Methods Enzymol 2019; 624:249–64.

94 Hoppmann C, Lacey VK, Louie GV et al. Genetically encoding photoswitchable click amino acids in Escherichia coli and mammalian cells. Angew Chem Int Ed Engl 2014; 53:3932–6.

95 Hoppmann C, Maslennikov I, Choe S et al. In Situ formation of an Azo Bridge on proteins controllable by visible light. J Am Chem Soc 2015; 137:11218–21.

96 Hutchins BM, Kazane SA, Staflin K et al. Site-specific coupling and sterically controlled formation of multimeric antibody fab fragments with unnatural amino acids. J Mol Biol 2011a; 406:595–603.

97 Hutchins BM, Kazane SA, Staflin K et al. Selective formation of covalent protein heterodimers with an unnatural amino acid. Chem Biol 2011b; 18:299–303.

98 Italia JS, Addy PS, Wrobel CJJ et al. An orthogonalized platform for genetic code expansion in both bacteria and eukaryotes. Nat Chem Biol 2017a; 13:446–50.

99 Italia JS, Zheng Y, Kelemen RE et al. Expanding the genetic code of mammalian cells. Biochem Soc Trans 2017b; 45:555–62.

100 Italia JS, Peeler JC, Hillenbrand CM et al. Genetically encoded protein sulfation in mammalian cells. Nat Chem Biol 2020; 16:379–82.

101 Jones LH. Recent advances in the molecular design of synthetic vaccines. Nat Chem 2015; 7:952–60.

102 Kang J-Y, Kawaguchi D, Coin I et al. In vivo expression of a light-activatable potassium channel using unnatural amino acids. Neuron 2013; 80:358–70.

103 Karijolich J, Yu Y-T. Therapeutic suppression of premature termination codons: mechanisms and clinical considerations (review). Int J Mol Med 2014; 34:355–62.

104 Kim SH, Jeong JH, Lee SH et al. LHRH receptor-mediated delivery of siRNA using polyelectrolyte complex micelles self-assembled from siRNA-PEG-LHRH conjugate and PEI. Bioconjug Chem 2008; 19:2156–62.

105 Kim CH, Axup JY, Dubrovska A et al. Synthesis of bispecific antibodies using genetically encoded unnatural amino acids. J Am Chem Soc 2012; 134:9918–21.

106 Kim H, Kim M, Im S-K et al. Mouse Cre-LoxP system: general principles to determine tissue-specific roles of target genes. Lab Anim Res 2018; 34:147–59.

107 Kimble J, Hirsh D. The postembryonic cell lineages of the hermaphrodite and male gonads in Caenorhabditis elegans. Dev Biol 1979; 70:396–417.

108 Kinser HE, Pincus Z. High-throughput screening in the C. elegans nervous system. Mol Cell Neurosci 2017; 80:192–7.

109 Kitada T, DiAndreth B, Teague B et al. Programming gene and engineered-cell therapies with synthetic biology. Science (New York, N.Y.) 2018; 359:eaad1067.

110 Kleiner P, Heydenreuter W, Stahl M et al. A whole proteome inventory of background photocrosslinker binding. Angew Chem Int Ed Engl 2017; 56:1396–401.

111 Klippenstein V, Hoppmann C, YePaoletti, SP et al. Optocontrol of glutamate receptor activity by single side-chain photoisomerization. ELife 2017; 6:e25808.

112 Ko W, Kumar R, Kim S et al. Construction of bacterial cells with an active transport system for unnatural amino acids. ACS Synth Biol 2019; 8:1195–203.

113 Kozlova EN, Berens C. Guiding differentiation of stem cells in vivo by tetracycline-controlled expression of key transcription factors. Cell Transplant 2012; 21:2537–54.

114 Krawczyk K, Xue S, Buchmann P et al. Electrogenetic cellular insulin release for real-time glycemic control in type 1 diabetic mice. Science (New York, N.Y.) 2020; 368:993–1001.

115 Kumar P, Ban H-S, Kim S-S et al. T cell-specific siRNA delivery suppresses HIV-1 infection in humanized mice. Cell 2008; 134:577–86.

116 Lacey VK, Louie GV, Noel JP et al. Expanding the library and substrate diversity of the pyrrolysyl-tRNA synthetase to incorporate unnatural amino acids containing conjugated rings. Chembiochem 2013; 14:2100–5.

117 Lang K, Davis L, Wallace S et al. Genetic encoding of bicyclononynes and trans-cyclooctenes for site-specific protein labeling in vitro and in live mammalian cells via rapid fluorogenic Diels–Alder reactions. J Am Chem Soc 2012a; 134:10317–20.

118 Lang K, Davis L, Torres-Kolbus J et al. Genetically encoded norbornene directs site-specific cellular protein labelling via a rapid bioorthogonal reaction. Nat Chem 2012b; 4:298–304.

119 Laski FA, Ganguly S, Sharp PA et al. Construction, stable transformation, and function of an amber suppressor tRNA gene in Drosophila melanogaster. Proc Natl Acad Sci USA 1989; 86:6696–8.

120 Ledsgaard L, Ljungars A, Rimbault C et al. Advances in antibody phage display technology. Drug Discov Today 2022; 27:2151–69.

121 Lee HS, Guo J, Lemke EA et al. Genetic incorporation of a small, environmentally sensitive, fluorescent probe into proteins in Saccharomyces cerevisiae. J Am Chem Soc 2009; 131:12921–3.

122 Lee J, Yun K-S, Choi CS et al. T cell-specific siRNA delivery using antibody-conjugated chitosan nanoparticles. Bioconjug Chem 2012; 23:1174–80.

123 Lee JM, Hammarén HM, Savitski MM et al. Control of protein stability by post-translational modifications. Nat Commun 2023; 14:201.

124 Lemke EA, Summerer D, Geierstanger BH et al. Control of protein phosphorylation with a genetically encoded photocaged amino acid. Nat Chem Biol 2007; 3:769–72.

125 Leong S, Lam HPJ, Kirkham Z et al. Antibody drug conjugates for the treatment of multiple myeloma. Am J Hematol 2023; 98:S22–34.

126 Lepthien S, Hoesl MG, Merkel L et al. Azatryptophans endow proteins with intrinsic blue fluorescence. Proc Natl Acad Sci USA 2008; 105:16095–100.

127 Lewis Phillips GD, Li G, Dugger DL et al. Targeting HER2-positive breast cancer with trastuzumab-DM1, an antibodycytotoxic drug conjugate. Cancer Res 2008; 68:9280–90.

128 Li F, Zhang H, Sun Y et al. Expanding the genetic code for photoclick chemistry in E. coli, mammalian cells, and A. thaliana. Angew Chem Int Ed Engl 2013; 52:9700–4.

129 Li J, Jia S, Chen PR. Diels–Alder reaction-triggered bioorthogonal protein decaging in living cells. Nat Chem Biol 2014a; 10:1003–5.

130 Li J, Yu J, Zhao J et al. Palladium-triggered deprotection chemistry for protein activation in living cells. Nat Chem 2014b; 6:352–61.

131 Li F, Li H, Zhai Q et al. A new vaccine targeting RANKL, prepared by incorporation of an unnatural amino acid into RANKL, prevents OVX-induced bone loss in mice. Biochem Biophys Res Commun 2018; 499:648–54.

132 Li Q, Chen Q, Klauser PC et al. Developing covalent protein drugs via proximity-enabled reactive therapeutics. Cell 2020; 182:85–97.e16.

133 Liaunardy-Jopeace A, Murton BL, Mahesh M et al. Encoding optical control in LCK kinase to quantitatively investigate its activity in live cells. Nat Struct Mol Biol 2017; 24:1155–63.

134 Lim RKV, Yu S, Cheng B et al. Targeted delivery of LXR agonist using a site-specific antibody-drug conjugate. Bioconjug Chem 2015; 26:2216–22.

135 Liu CC, Schultz PG. Adding new chemistries to the genetic code. Annu Rev Biochem 2010; 79:413–44.

136 Liu DR, Magliery TJ, Pastrnak M et al. Engineering a tRNA and aminoacyl-tRNA synthetase for the site-specific incorporation of unnatural amino acids into proteins in vivo. Proc Natl Acad Sci USA 1997; 94:10092–7.

137 Liu J, Hemphill J, Samanta S et al. Genetic code expansion in zebrafish embryos and its application to optical control of cell signaling. J Am Chem Soc 2017; 139:9100–3.

138 Liu J, Li S, Aslam NA et al. Genetically encoding photocaged quinone methide to multitarget protein residues covalently in vivo. J Am Chem Soc 2019; 141:9458–62.

139 Liu J, Cao L, Klauser PC et al. A genetically encoded fluorosulfonyloxybenzoyl-L-lysine for expansive covalent bonding of proteins via SuFEx chemistry. J Am Chem Soc 2021; 143:10341–51.

140 Liu J, Yang B, Wang L. Residue selective crosslinking of proteins through photoactivatable or proximity-enabled reactivity. Curr Opin Chem Biol 2023; 74:102285.

141 Long C, McAnally JR, Shelton JM et al. Prevention of muscular dystrophy in mice by CRISPR/Cas9-mediated editing of germline DNA. Science (New York, N.Y.) 2014; 345:1184–8.

142 Lu H, Wang D, Kazane S et al. Site-specific antibody-polymer conjugates for siRNA delivery. J Am Chem Soc 2013; 135:13885–91.

143 Luo J, Uprety R, Naro Y et al. Genetically encoded optochemical probes for simultaneous fluorescence reporting and light activation of protein function with two-photon excitation. J Am Chem Soc 2014; 136:15551–8.

144 Luo J, Arbely E, Zhang J et al. Genetically encoded optical activation of DNA recombination in human cells. Chem Commun (Cambridge, England) 2016a; 52:8529–32.

145 Luo J, Liu Q, Morihiro K et al. Small-molecule control of protein function through Staudinger reduction. Nat Chem 2016b; 8:1027–34.

146 Luo X, Fu G, Wang RE et al. Genetically encoding phosphotyrosine and its nonhydrolyzable analog in bacteria. Nat Chem Biol 2017a; 13:845–9.

147 Luo J, Torres-Kolbus J, Liu J et al. Genetic encoding of photocaged tyrosines with improved light-activation properties for the optical control of protease function. Chembiochem 2017b; 18:1442–7.

148 Ma JSY, Kim JY, Kazane SA et al. Versatile strategy for controlling the specificity and activity of engineered T cells. Proc Natl Acad Sci USA 2016; 113:E450–8.

149 Maalej KM, Merhi M, Inchakalody VP et al. CAR-cell therapy in the era of solid tumor treatment: current challenges and emerging therapeutic advances. Mol Cancer 2023; 22:20.

150 Mangubat-Medina AE, Ball ZT. Triggering biological processes: methods and applications of photocaged peptides and proteins. Chem Soc Rev 2021; 50:10403–21.

151 Mattheisen JM, Wollowitz JS, Huber T et al. Genetic code expansion to enable site-specific bioorthogonal labeling of functional G protein-coupled receptors in live cells. Protein Sci 2023; 32:e4550.

152 Mayer C. Selection, addiction and catalysis: emerging trends for the incorporation of noncanonical amino acids into peptides and proteins in vivo. Chembiochem 2019; 20:1357–64.

153 Mehl RA, Anderson JC, Santoro SW et al. Generation of a bacterium with a 21 amino acid genetic code. J Am Chem Soc 2003; 125:935–9.

154 Melançon CE, Schultz PG. One plasmid selection system for the rapid evolution of aminoacyl-tRNA synthetases. Bioorg Med Chem Lett 2009; 19:3845–7.

155 Mills EM, Barlow VL, Jones AT et al. Development of mammalian cell logic gates controlled by unnatural amino acids. Cell Rep Methods 2021; 1:100073.

156 Mitchell P, Tollervey D. An NMD pathway in yeast involving accelerated deadenylation and exosome-mediated 3′→5′ degradation. Mol Cell 2003; 11:1405–13.

157 Mukai T, Kobayashi T, Hino N et al. Adding L-lysine derivatives to the genetic code of mammalian cells with engineered pyrrolysyl-tRNA synthetases. Biochem Biophys Res Commun 2008; 371:818–22.

158 Mukai T, Lajoie MJ, Englert M et al. Rewriting the genetic code. Annu Rev Microbiol 2017; 71:557–77.

159 Muranaka N, Hohsaka T, Sisido M. Photoswitching of peroxidase activity by position-specific incorporation of a photoisomerizable non-natural amino acid into horseradish peroxidase. FEBS Lett 2002; 510:10–2.

160 Narancic T, Almahboub SA, O’Connor KE. Unnatural amino acids: production and biotechnological potential. World J Microbiol Biotechnol 2019; 35:67.

161 Neumann H, Wang K, Davis L et al. Encoding multiple unnatural amino acids via evolution of a quadruplet-decoding ribosome. Nature 2010; 464:441–4.

162 Nguyen DP, Mahesh M, Elsässer SJ et al. Genetic encoding of photocaged cysteine allows photoactivation of TEV protease in live mammalian cells. J Am Chem Soc 2014; 136:2240–3.

163 Nguyen T-A, Cigler M, Lang K. Expanding the genetic code to study protein–protein interactions. Angew Chem Int Ed Engl 2018; 57:14350–61.

164 Nikić I, Lemke EA. Genetic code expansion enabled site-specific dual-color protein labeling: superresolution microscopy and beyond. Curr Opin Chem Biol 2015; 28:164–73.

165 Nikić I, Estrada Girona G, Kang JH et al. Debugging eukaryotic genetic code expansion for site-specific click-PAINT super-resolution microscopy. Angew Chem Int Ed Engl 2016; 55:16172–6.

166 Niu W, Schultz PG, Guo J. An expanded genetic code in mammalian cells with a functional quadruplet codon. ACS Chem Biol 2013; 8:1640–5.

167 Nödling AR, Spear LA, Williams TL et al. Using genetically incorporated unnatural amino acids to control protein functions in mammalian cells. Essays Biochem 2019; 63:237–66.

168 Noren CJ, Anthony-Cahill SJ, Griffith MC et al. A general method for site-specific incorporation of unnatural amino acids into proteins. Science (New York, N.Y.) 1989; 244:182–8.

169 Osgood AO, Zheng Y, Roy SJS et al. An efficient opal-suppressor tryptophanyl pair creates new routes for simultaneously incorporating up to three distinct noncanonical amino acids into proteins in mammalian cells. Angew Chem Int Ed Engl 2023; 62:e202219269.

170 Palzer S, Bantel Y, Kazenwadel F et al. An expanded genetic code in Candida albicans to study protein–protein interactions in vivo. Eukaryot Cell 2013; 12:816–27.

171 Park S-H, Ko W, Lee HS et al. Analysis of protein–protein interaction in a single live cell by using a FRET system based on genetic code expansion technology. J Am Chem Soc 2019; 141:4273–81.

172 Park S-H, Ko W, Park S-H et al. Evaluation of the interaction between Bax and Hsp70 in cells by using a FRET system consisting of a fluorescent amino acid and YFP as a FRET pair. Chembiochem 2020; 21:59–63.

173 Parrish AR, She X, Xiang Z et al. Expanding the genetic code of Caenorhabditis elegans using bacterial aminoacyl-tRNA synthetase/tRNA pairs. ACS Chem Biol 2012; 7:1292–302.

174 Peeler JC, Falco JA, Kelemen RE et al. Generation of recombinant mammalian selenoproteins through genetic code expansion with photocaged Selenocysteine. ACS Chem Biol 2020; 15:1535–40.

175 Peng T, Hang HC. Site-specific bioorthogonal labeling for fluorescence imaging of intracellular proteins in living cells. J Am Chem Soc 2016; 138:14423–33.

176 Politano L. Read-through approach for stop mutations in Duchenne muscular dystrophy an update. Acta Myol 2021; 40:43–50.

177 Prasanth MI, Gayathri S, Bhaskar JP et al. Understanding the role of p38 and JNK mediated MAPK pathway in response to UV-A induced photoaging in Caenorhabditis elegans. J Photochem Photobiol B 2020; 205:111844.

178 Rader C. Bispecific antibodies in cancer immunotherapy. Curr Opin Biotechnol 2020; 65:9–16.

179 Ren W, Ji A, Ai H-w. Light activation of protein splicing with a photocaged fast intein. J Am Chem Soc 2015a; 137:2155–8.

180 Ren W, Ji A, Wang MX et al. Expanding the genetic code for a dinitrophenyl hapten. Chembiochem 2015b; 16:2007–10.

181 Rong L, Lim RM, Yin X et al. Site-specific dinitrophenylation of single-chain antibody fragments for redirecting a universal CAR-T cell against cancer antigens. J Mol Biol 2022; 434:167513.

182 Roy G, Reier J, Garcia A et al. Development of a high yielding expression platform for the introduction of non-natural amino acids in protein sequences. MAbs 2020; 12:1684749.

183 Ryan A, Janosko CP, Courtney TM et al. Engineering SHP2 phosphatase for optical control. Biochemistry 2022; 61:2687–97.

184 Sanders J, Hoffmann SA, Green AP et al. New opportunities for genetic code expansion in synthetic yeast. Curr Opin Biotechnol 2022; 75:102691.

185 Santoro SW, Schultz PG. Directed evolution of the substrate specificities of a site-specific recombinase and an aminoacyl-tRNA synthetase using fluorescence-activated cell sorting (FACS). Methods Mol Biol (Clifton, N.J.) 2003; 230:291–312.

186 Santoro SW, Wang L, Herberich B et al. An efficient system for the evolution of aminoacyl-tRNA synthetase specificity. Nat Biotechnol 2002; 20:1044–8.

187 Schumacher D, Hackenberger CPR, Leonhardt H et al. Current status: site-specific antibody drug conjugates. J Clin Immunol 2016; 36:100–7.

188 Segal I, Nachmias D, Konig A et al. A straightforward approach for bioorthogonal labeling of proteins and organelles in live mammalian cells, using a short peptide tag. BMC Biol 2020; 18:5.

189 Serfling R, Seidel L, Bock A et al. Quantitative single-residue bioorthogonal labeling of g protein-coupled receptors in live cells. ACS Chem Biol 2019; 14:1141–9.

190 Shandell MA, Quejada JR, Yazawa M et al. Detection of Nav15 conformational change in mammalian cells using the noncanonical amino acid ANAP. Biophys J 2019; 117:1352–63.

191 Shao N, Singh NS, Slade SE et al. Site specific genetic incorporation of Azidophenylalanine in Schizosaccharomyces pombe. Sci Rep 2015; 5:17196.

192 Shen B, Xiang Z, Miller B et al. Genetically encoding unnatural amino acids in neural stem cells and optically reporting voltage-sensitive domain changes in differentiated neurons. Stem Cells (Dayton, Ohio) 2011; 29:1231–40.

193 Shi N, Yang Q, Zhang H et al. Restoration of dystrophin expression in mice by suppressing a nonsense mutation through the incorporation of unnatural amino acids. Nat Biomed Eng 2022; 6:195–206.

194 Si L, Xu H, Zhou X et al. Generation of influenza A viruses as live but replication-incompetent virus vaccines. Science (New York, N.Y.) 2016; 354:1170–3.

195 Sibaud V, Beylot-Barry M, Protin C et al. Dermatological toxicities of Bruton’s tyrosine kinase inhibitors. Am J Clin Dermatol 2020; 21:799–812.

196 Siman P, Brik A. Chemical and semisynthesis of posttranslationally modified proteins. Org Biomol Chem 2012; 10:5684–97.

197 Singh J, Petter RC, Baillie TA et al. The resurgence of covalent drugs. Nat Rev Drug Discov 2011; 10:307–17.

198 Sletten EM, Bertozzi CR. From mechanism to mouse: a tale of two bioorthogonal reactions. Acc Chem Res 2011; 44:666–76.

199 Smolskaya S, Andreev YA. Site-specific incorporation of unnatural amino acids into Escherichia coli recombinant protein: methodology development and recent achievement. Biomolecules 2019; 9:255.

200 Song E, Zhu P, Lee S-K et al. Antibody mediated in vivo delivery of small interfering RNAs via cell-surface receptors. Nat Biotechnol 2005; 23:709–17.

201 Stokman G, Qin Y, Rácz Z et al. Application of siRNA in targeting protein expression in kidney disease. Adv Drug Deliv Rev 2010; 62:1378–89.

202 Sulston JE, Horvitz HR. Post-embryonic cell lineages of the nematode, Caenorhabditis elegans. Dev Biol 1977; 56:110–56.

203 Syed J, Palani S, Clarke ST et al. Expanding the zebrafish genetic code through site-specific introduction of azido-lysine, bicyclononyne-lysine, and diazirine-lysine. Int J Mol Sci 2019; 20:2577.

204 Takimoto JK, Adams KL, Xiang Z et al. Improving orthogonal tRNA-synthetase recognition for efficient unnatural amino acid incorporation and application in mammalian cells. Mol Biosyst 2009; 5:931–4.

205 Takimoto JK, Xiang Z, Kang J-Y et al. Esterification of an unnatural amino acid structurally deviating from canonical amino acids promotes its uptake and incorporation into proteins in mammalian cells. Chembiochem 2010; 11:2268–72.

206 Tanaka Y, Bond MR, Kohler JJ. Photocrosslinkers illuminate interactions in living cells. Mol Biosyst 2008; 4:473–80.

207 Tharp JM, Ehnbom A, Liu WR. tRNAPyl: structure, function, and applications. RNA Biol 2018; 15:441–52.

208 Teixeira AP, Fussenegger M. Engineering mammalian cells for disease diagnosis and treatment. Curr Opin Biotechnol 2019; 55:87–94.

209 Tian F, Lu Y, Manibusan A et al. A general approach to site-specific antibody drug conjugates. Proc Natl Acad Sci USA 2014; 111:1766–71.

210 Tian Y, Jacinto MP, Zeng Y et al. Genetically encoded 2-Aryl-5-carboxytetrazoles for site-selective protein photocross-linking. J Am Chem Soc 2017; 139:6078–81.

211 Tian X, Liu W-Q, Xu H et al. Cell-free expression of NO synthase and P450 enzyme for the biosynthesis of an unnatural amino acid L-4-nitrotryptophan. Synth Syst Biotechnol 2022; 7:775–83.

212 Tir N, Heistinger L, Grünwald-Gruber C et al. From strain engineering to process development: monoclonal antibody production with an unnatural amino acid in Pichia pastoris. Microb Cell Fact 2022; 21:157.

213 Toteva MM, Richard JP. The generation and reactions of quinone methides. Adv Phys Org Chem 2011; 45:39–91.

214 Tsai Y-H, Essig S, James JR et al. Selective, rapid and optically switchable regulation of protein function in live mammalian cells. Nat Chem 2015; 7:554–61.

215 Uprety R, Luo J, Liu J et al. Genetic encoding of caged cysteine and caged homocysteine in bacterial and mammalian cells. Chembiochem 2014; 15:1793–9.

216 Walker OS, Elsässer SJ, Mahesh M et al. Photoactivation of mutant isocitrate dehydrogenase 2 reveals rapid cancer-associated metabolic and epigenetic changes. J Am Chem Soc 2016; 138:718–21.

217 Walsh SJ, Bargh JD, Dannheim FM et al. Site-selective modification strategies in antibody-drug conjugates. Chem Soc Rev 2021; 50:1305–53.

218 Wan W, Tharp JM, Liu WR. Pyrrolysyl-tRNA synthetase: an ordinary enzyme but an outstanding genetic code expansion tool. Biochim Biophys Acta 2014; 1844:1059–70.

219 Wang L. Engineering the genetic code in cells and animals: biological considerations and impacts. Acc Chem Res 2017; 50:2767–75.

220 Wang L, Audhya A. In vivo imaging of C. elegans endocytosis. Methods (San Diego, Calif.) 2014; 68:518–28.

221 Wang Q, Wang L. New methods enabling efficient incorporation of unnatural amino acids in yeast. J Am Chem Soc 2008; 130:6066–7.

222 Wang Q, Wang L. Genetic incorporation of unnatural amino acids into proteins in yeast. Methods Mol Biol (Clifton, N.J.) 2012; 794:199–213.

223 Wang N, Wang L. Genetically encoding latent bioreactive amino acids and the development of covalent protein drugs. Curr Opin Chem Biol 2022; 66:102106.

224 Wang L, Brock A, Herberich B et al. Expanding the genetic code of Escherichia coli. Science (New York, N.Y.) 2001; 292:498–500.

225 Wang W, Takimoto JK, Louie GV et al. Genetically encoding unnatural amino acids for cellular and neuronal studies. Nat Neurosci 2007; 10:1063–72.

226 Wang K, Schmied WH, Chin JW. Reprogramming the genetic code: from triplet to quadruplet codes. Angew Chem Int Ed Engl 2012; 51:2288–97.

227 Wang RE, Liu T, Wang Y et al. An immunosuppressive antibodydrug conjugate. J Am Chem Soc 2015; 137:3229–32.

228 Wang J, Zheng S, Liu Y et al. Palladium-triggered chemical rescue of intracellular proteins via genetically encoded Allene-Caged tyrosine. J Am Chem Soc 2016a; 138:15118–21.

229 Wang WW, Zeng Y, Wu B et al. A chemical biology approach to reveal Sirt6-targeted Histone H3 sites in nucleosomes. ACS Chem Biol 2016b; 11:1973–81.

230 Wang N, Yang B, Fu C et al. Genetically encoding Fluorosulfate-l-tyrosine to react with lysine, histidine, and tyrosine via SuFEx in proteins in vivo. J Am Chem Soc 2018; 140:4995–9.

231 Wang WW, Angulo-Ibanez M, Lyu J et al. A click chemistry approach reveals the chromatin-dependent histone H3K36 Deacylase nature of SIRT7. J Am Chem Soc 2019; 141:2462–73.

232 Wang T-Y, Sang G-J, Wang Q et al. Generation of premature termination codon (PTC)-harboring pseudorabies virus (PRV) via genetic code expansion technology. Viruses 2022; 14:572.

233 Washburn T, O’Tousa JE. Nonsense suppression of the major rhodopsin gene of Drosophila. Genetics 1992; 130:585–95.

234 Weigle WO. The induction of autoimmunity in rabbits following injection of heterologous or altered homologous thyroglobulin. J Exp Med 1965; 121:289–308.

235 Wesalo JS, Luo J, Morihiro K et al. Phosphine-activated lysine analogues for fast chemical control of protein subcellular localization and protein SUMOylation. Chembiochem 2020; 21:141–8.

236 Wu AM. Antibodies and antimatter: the resurgence of immuno-PET. J Nucl Med 2009; 50:2–5.

237 Wu N, Deiters A, Cropp TA et al. A genetically encoded photocaged amino acid. J Am Chem Soc 2004; 126:14306–7.

238 Wu Y, Zhu H, Zhang B et al. Synthesis of site-specific radiolabeled antibodies for radioimmunotherapy via genetic code expansion. Bioconjug Chem 2016; 27:2460–8.

239 Wu Y, Zhu R-Y, Mitchell LA et al. In vitro DNA SCRaMbLE. Nat Commun 2018a; 9:1935.

240 Wu T, Li F, Sha X et al. A novel recombinant RANKL vaccine prepared by incorporation of an unnatural amino acid into RANKL and its preventive effect in a murine model of collagen-induced arthritis. Int Immunopharmacol 2018b; 64:326–32.

241 Wu K-L, Moore JA, Miller MD et al. Expanding the eukaryotic genetic code with a biosynthesized 21st amino acid. Protein Sci 2022a; 31:e4443.

242 Wu D, Zhang Y, Tang Z et al. Creation of a yeast strain with co-translationally acylated nucleosomes. Angew Chem Int Ed Engl 2022b; 61:e202205570.

243 Wu X-Y, Li M-Y, Yang S-J et al. Controlled genetic encoding of unnatural amino acids in a protein nanopore. Angew Chem Int Ed Engl 2023; 62:e202300582.

244 Wulf M, Pless SA. High-sensitivity fluorometry to resolve ion channel conformational dynamics. Cell Rep 2018; 22:1615–26.

245 Xi Z, Davis L, Baxter K et al. Using a quadruplet codon to expand the genetic code of an animal. Nucleic Acids Res 2022; 50:4801–12.

246 Xiang Z, Ren H, Hu YS et al. Adding an unnatural covalent bond to proteins through proximity-enhanced bioreactivity. Nat Methods 2013; 10:885–8.

247 Xiang Z, Lacey VK, Ren H et al. Proximity-enabled protein crosslinking through genetically encoding haloalkane unnatural amino acids. Angew Chem Int Ed Engl 2014; 53:2190–3.

248 Xiao H, Chatterjee A, Choi S-hyun et al. Genetic incorporation of multiple unnatural amino acids into proteins in mammalian cells. Angew Chem Int Ed Engl 2013; 52:14080–3.

249 Xue Y-P, Cao C-H, Zheng Y-G. Enzymatic asymmetric synthesis of chiral amino acids. Chem Soc Rev 2018; 47:1516–61.

250 Yanagisawa T, Hino N, Iraha F et al. Wide-range protein photo-crosslinking achieved by a genetically encoded N(ϵ)- (benzyloxycarbonyl)lysine derivative with a diazirinyl moiety. Mol Biosyst 2012; 8:1131–5.

251 Yang Y, Luo S, Huang J et al. Photoactivation of innate immunity receptor TLR8 in live mammalian cells by genetic encoding of photocaged tyrosine. Chembiochem 2022; 23:e202100344.

252 Yang J, Wang K, Zhang S et al. Site-specific introduction of bioorthogonal handles to nanopores by genetic code expansion. Angew Chem Int Ed Engl 2023; 62:e202216115.

253 Yao Y-d, Sun T-meng, Huang S-yin et al. Targeted delivery of PLK1-siRNA by ScFv suppresses Her2+ breast cancer growth and metastasis. Sci Transl Med 2012; 4:130ra48.

254 Young TS, Ahmad I, Brock A et al. Expanding the genetic repertoire of the methylotrophic yeast Pichia pastoris. Biochemistry 2009; 48:2643–53.

255 Young TS, Young DD, Ahmad I et al. Evolution of cyclic peptide protease inhibitors. Proc Natl Acad Sci USA 2011; 108:11052–6.

256 Yu B, Li S, Tabata T et al. Accelerating PERx reaction enables covalent nanobodies for potent neutralization of SARS-CoV-2 and variants. Chem 2022; 8:2766–83.

257 Yuan Z, Wang N, Kang G et al. Controlling multicycle replication of live-attenuated HIV-1 using an unnatural genetic switch. ACS Synth Biol 2017; 6:721–31.

258 Zang J, Chen Y, Liu C et al. Genetic code expansion reveals aminoacylated lysine ubiquitination mediated by UBE2W. Nat Struct Mol Biol 2023; 30:62–71.

259 Zaykov AN, Mayer JP, DiMarchi RD. Pursuit of a perfect insulin. Nat Rev Drug Discov 2016; 15:425–39.

260 Zhang Z, Alfonta L, Tian F et al. Selective incorporation of 5-hydroxytryptophan into proteins in mammalian cells. Proc Natl Acad Sci USA 2004; 101:8882–7.

261 Zhang G, Li J, Xie R et al. Bioorthogonal chemical activation of kinases in living systems. ACS Cent Sci 2016; 2:325–31.

262 Zhao S, Shi J, Yu G et al. Photosensitive tyrosine analogues unravel site-dependent phosphorylation in TrkA initiated MAPK/ERK signaling. Commun Biol 2020; 3:706.

263 Zheng Y, Lewis TL, Igo P et al. Virus-enabled optimization and delivery of the genetic machinery for efficient unnatural amino acid mutagenesis in mammalian cells and tissues. ACS Synth Biol 2017; 6:13–8.

264 Zhu C, Xu L, Chen L et al. Epitope-directed antibody elicitation by genetically encoded chemical cross-linking reactivity in the antigen. ACS Cent Sci 2023; 9:1229–40.

265 Zuany-Amorim C, Manlius C, Dalum I et al. Induction of TNF-alpha autoantibody production by AutoVac TNF106: a novel therapeutic approach for the treatment of allergic diseases. Int Arch Allergy Immunol 2004; 133:154–63.