Abstract

Genetic code expansion allows unnatural (non-canonical) amino acid incorporation into proteins of interest by repurposing the cellular translation machinery. The development of this technique has enabled site-specific incorporation of many structurally and chemically diverse amino acids, facilitating a plethora of applications, including protein imaging, engineering, mechanistic and structural investigations, and functional regulation. Particularly, genetic code expansion provides great tools to study mammalian proteins, of which dysregulations often have important implications in health. In recent years, a series of methods has been developed to modulate protein function through genetically incorporated unnatural amino acids. In this review, we will first discuss the basic concept of genetic code expansion and give an up-to-date list of amino acids that can be incorporated into proteins in mammalian cells. We then focus on the use of unnatural amino acids to activate, inhibit, or reversibly modulate protein function by translational, optical or chemical control. The features of each approach will also be highlighted.

Introduction

Knowledge of protein function is of pivotal importance to life science research. It can guide conventional drug development programmes and lead to novel strategies to address currently non-targetable systems [1–3]. In order to understand the precise role and interacting network of a protein, it is essential to analyse it within its native environment. For a mammalian protein, its function often also depends on its host cell (e.g. cell type and cell cycle stage), specific subcellular location and post-translational modifications. In addition, a protein of interest often exists in the presence of other closely related homologues (e.g. proteins within the same family), making it difficult to decipher the precise function of a specific protein in cells. Targeting the protein by small-molecule inhibition is often not possible in these cases, as protein homologues will also be affected. To tackle this problem, over the last two decades there has been a drive to develop and refine the technique of genetic code expansion which allows researchers to exploit the cellular translation machinery for site-specific incorporation of unnatural (non-canonical) amino acids into target proteins [4–14]. Consequently, this enables the use of building blocks beyond the 20 canonical amino acids and incorporation of unnatural amino acids with unprecedented functionality into target proteins in live cells. The repurposing of the translational machinery by this approach has paved the way for revealing the functions of proteins under physiological conditions [15–19]. For example, the technique can be used to site-specifically introduce an unnatural amino acid into the homologue of interest, whereby unique functionality (on the unnatural amino acid) can be used for selective activation, inhibition, or reversible regulation of the target homologue [7].

At the molecular level, the mechanism of protein translation is highly conserved in all organisms, where the cellular machinery ‘translates’ every nucleotide triplet as a codon consecutively on the mRNA into the corresponding amino acid. In nature, the endogenous aminoacyl-tRNA synthetase (aaRS)/tRNA pairs within the cell decode 61 of the total 64 codons to 20 canonical amino acids. The remaining three codons (UAG, UGA and UAA) are used for translation termination, and hence they are also known as ‘stop’ codons. In order to achieve site-specific incorporation of an unnatural amino acid, an orthogonal aaRS/tRNA pair is needed, which must decode a codon that does not correspond to any canonical amino acid, a so-called blank codon (Figure 1). Stop codons are most commonly used as a blank codon in genetic code expansion, and decoding of a stop codon is known as ‘suppression’ because it suppresses the translation termination. The amber stop codon (UAG) is often used as the blank codon due to its minimal occurrence in most organisms.

Mechanism of genetic code expansion for site-specific incorporation of an unnatural amino acid by amber suppression

Figure 1
Mechanism of genetic code expansion for site-specific incorporation of an unnatural amino acid by amber suppression
Figure 1
Mechanism of genetic code expansion for site-specific incorporation of an unnatural amino acid by amber suppression

Within the concept of genetic code expansion, ‘orthogonality’ refers to the non-reactivity of the orthogonal aaRS/tRNA pair with the endogenous pair and canonical amino acids in the host cell. The orthogonal synthetase must only acylate the orthogonal tRNA with the designated unnatural amino acid; neither canonical amino acids nor endogenous tRNAs are substrates of the orthogonal synthetase; similarly, neither the unnatural amino acid nor orthogonal tRNA is a substrate of the endogenous synthetases (Figure 2).

Allowed and not allowed reactivities between the orthogonal and endogenous aaRS/tRNA pairs

Figure 2
Allowed and not allowed reactivities between the orthogonal and endogenous aaRS/tRNA pairs

(A) Matching amino acid and aaRS/tRNA pairs; (B) mismatched amino acids; (C) mismatched aaRS/tRNA pairs.

Figure 2
Allowed and not allowed reactivities between the orthogonal and endogenous aaRS/tRNA pairs

(A) Matching amino acid and aaRS/tRNA pairs; (B) mismatched amino acids; (C) mismatched aaRS/tRNA pairs.

Besides the amber codon, other stop codons [20–26] and different four-nucleotide codons [27,28] have been used as a blank codon. The use of four-nucleotide codons expands the theoretical codon numbers from 43 (64) to 44 (256) so that multiple different unnatural amino acids can be incorporated at the same time. However, decoding a four-nucleotide codon by the ribosome is less efficient than decoding the normal three-nucleotide codons. Although this issue has been addressed in Escherichia coli through ribosome engineering [29–31], the lower efficiency in decoding four-nucleotide codons remains an issue in mammalian systems [27,28].

To date, many unnatural amino acids (1110, Table 1) can be site-specifically incorporated into proteins produced by mammalian cells using genetic code expansion [5,32]. While the amino acids are structurally diverse, the majority of them can be incorporated through only a few orthogonal synthetases and their mutants. The Pyrrolysyl-tRNA synthetase (PylRS)/tRNAPyl pairs from archaea species Methanosarcina barkeri (Mb) and Methanosarcina mazei (Mm) have proven to be extraordinarily useful pairs [4]. The tRNAPyl naturally recognises the UAG codon and thus engineering of this tRNA is not needed. In addition, this pair is orthogonal in both E. coli and mammalian cells; hence, it facilitates the engineering of PylRS in E. coli and subsequently using the engineered PylRS mutant for incorporation of the designated unnatural amino acid in mammalian systems. As shown in Table 1, a wide range of amino acids has been incorporated into proteins in mammalian cells through only a few point mutations on the PylRS gene.

Table 1
Overview of unnatural amino acids that have been successfully incorporated into proteins in mammalian cells and used for a variety of applications to date
Amino acidaaRSMutationstRNAApplication
Cysteine and selenocysteine derivatives 
 EcLeuRS [24M40I T252A Y499I Y527A H529G EctRNACUALeu Method development 
 EcLeuRS [24M40I T252A Y499I Y527A H529G EctRNACUALeu Method development 
 EcLeuRS [24M40I T252A Y499I Y527A H529G EctRNACUALeu Method development 
 EcLeuRS [24M40I T252A Y499I Y527A H529G EctRNACUALeu Method development 
 EcLeuRS [24M40I T252A Y499I Y527A H529G EctRNACUALeu Method development 
 EcLeuRS [24M40I T252A Y499I Y527A H529G EctRNACUALeu Method development 
 EcLeuRS [71M40G L41Q T252A Y499L Y527G H537F EctRNACUALeu Photoactivation 
 EcLeuRS [72M40G L41Q Y499L Y527G H537F EctRNACUALeu Photoactivation 
 MbPylRS [73,74N311Q C313A V366M MbtRNACUAPyl Photoactivation 
 MbPylRS [75M241F A267S Y271C L274M MbtRNACUAPyl Photoactivation 
 MbPylRS [75M241F A267S Y271C L274M MbtRNACUAPyl Photoactivation 
 MbPylRS [76C313W W382T MbPyltRNA Method development 
 MbPylRS [40L274A C313S Y349F MbtRNACUAPyl Photocrosslinking 
 MbPylRS [40L274A C313S Y349F MbtRNACUAPyl Photocrosslinking 
Phenylalanine derivatives 
 EcTyrRS [77Y37V D182S F183M D265R EctRNACUATyr Method development 
BstRNACUATyr 
 EcTyrRS [35,37,43,77Y37V D182S F183M D265R [77]
Y37I D182S F183M [37,43]
Y37V D165G D182S F183M L186A D265R [35
EctRNACUATyr [35,77Method development [35,37,77]
Protein engineering [43
BstRNACUATyr [37,43,77
 EcTyrRS [77Y37V D182S F183M D265R EctRNACUATyr Method development 
BstRNACUATyr 
 EcTyrRS [77Y37V D182S F183M D265R EctRNACUATyr Method development 
BstRNACUATyr 
 EcTyrRSCUA [16,17,35,37,38,43,49,55,68,77–96Y37L D182S F183A L186A D265R [78,81,84,85]
Y37V D182S F183M D265R [77,90]
Y37L D182S F183M L186A [16,17,37,38,43,49,55,68,79,80,82,83,86–89,91–96]
Y37V D165G D182S F183M L186A D265R [35
EctRNACUATyr [35,77,78,81,86,90Bioorthogonal labelling [38,79,83,87–89,96]
Method development [17,25,35,37,77,78,90]
Photocrosslinking [38,68,81,84,85,91–95]
Protein engineering [43,49,55,83]
Spectroscopic probe [16,80,82,86
BstRNACUATyr [16,17,37,38,43,49,55,68,77,79,80,82–85,87–96
EcTyrRSUCA [25Y37V D182S F183M EctRNAUCATyr 
BstRNAUCATyr 
 EcTyrRS [35,97Y37I D182S F183M D265R [97]
Y37S D182S F183A L186E D265R [35]
Y37G D182S F183I L186E D265R [35]
Y37S D182S F183I L186E D265R [35
EctRNACUATyr [35,97Method development [35]
Spectroscopic probe [97
BstRNACUATyr [97
 MmPylRS [50L301M Y306L L309A C348F MmtRNACUAPyl Method development 
 MmPylRS [98N346A C348A MmtRNACUAPyl Method development 
 MmPylRS [98N346A C348A MmtRNACUAPyl Method development 
 MmPylRS [98N346A C348A MmtRNACUAPyl Method development 
 MmPylRS [98N346A C348A MmtRNACUAPyl Method development 
 MmPylRS [98N346A C348A MmtRNACUAPyl Method development 
 MmPylRS [98N346A C348A MmtRNACUAPyl Method development 
 MmPylRS [98N346A C348A MmtRNACUAPyl Method development 
 EcTyrRSCUA [21,35,37,43,77,83,87,90,99,100Y37I N165G D182G F183M L186A [83,99]
Y37I D182G F183M L186A [37,43,87,100]
Y37V D182S F183M D265R [21,77,90]
Y37V D165G D182S F183M L186A D265R [35
EctRNACUATyr [21,35,77,90Bioorthogonal labelling [83,87,100]
Method development [25,35,37,77,83,90,99]
Protein engineering [21,43
BstRNACUATyr [37,43,77,83,87,99,100
EcTyrRSUCA [25Y37V D182S F183M EctRNAUCATyr 
BstRNAUCATyr 
 EcTyrRS [84,101Y37I D182G F183M L186A D265R BstRNACUATyr Chemical crosslinking [84,101]
Method development [101
 EcTyrRS [15,37,55,78,85,92,94,95,99,102–105Y37G D182G L186A
D265R [78,85,103]
Y37G D182G L186A [15,55,92,94,95,99,102,104,105]
Y37G D182G F183Y L186M [37
EctRNACUATyr [15,78,103Mechanistic studies [15]
Method development [37,78,95,99,103,106]
Photocrosslinking [85,92,94,102,104,105]
Photoinhibition [55
BstRNACUATyr [37,55,85,92,94,95,99,102,104,105
MmPylRS [106A302T N346T C348T W417C [106MmtRNACUAPyl [106
 MmPylRS [106A302T N346G C348T V401I W417Y MmtRNACUAPyl Method development 
 EcLeuRS [107–109L38F M40G L41P Y499V Y500L Y527A H537E L538S F541C A560V EctRNACUALeu Method development [107,108]
Spectroscopic probe [109
 MmPylRS [110N346Q C348S V401G W417T MmtRNACUAPyl Spectroscopic Probe 
 MbPylRS [111L270F L274M N311G C313G Y349F MbtRNACUAPyl Photoswitching 
 MbPylRS [111L270F L274M N311G C313G Y349F MbtRNACUAPyl Photoswitching 
 MbPylRS [111L270F L274M N311G C313G Y349F MbtRNACUAPyl Photoswitching 
 MmPylRS [56,112A302T L309S N346V C348G MmtRNACUAPyl Method development [112]
Photoswitching [56
Histidine derivatives 
 MaPylRS [26L121M L125I Y126F M129A V168F MatRNACUAPyl Method development 
MbPylRS [113L270I Y271F L274G C313F Y349F MbtRNACUAPyl 
 MbPylRS [113L270I Y271F L274G C313F Y349F MbtRNACUAPyl Method development 
Lysine derivatives 
 MbPylRS [114L266M L270I Y271F L274A C313F MbtRNACUAPyl Method development 
 MbPylRS [115–117D76G L266V L270I Y271F L274A C313F [115]
D76G L266M L270I Y271F L274A C313F [116,117
MbtRNACUAPyl Method development [44,50,115,117,118]
Spectroscopic probe [116
MmPylRS [44,50,118L305I Y306F L309A C348F [118]
L301M Y306L L309A C348F [44,50
MmtRNACUAPyl [50,118]
MmtRNAUUAPyl [44
 MmPylRS [50L301M Y306L L309A C348F MmtRNACUAPyl Method development 
 MbPylRS [119D76G L266M L270I Y271F L274A C313F MbtRNACUAPyl Method development 
 MbPylRS [77,120L274A C313F Y349F [120]
wt [77
MbtRNACUAPyl Method development 
 MbPylRS [121Y271M L274A C313A MbtRNACUAPyl Photocrosslinking 
 MmPylRS [122Y306V L309A C348F Y384F MmtRNACUAPyl Photocrosslinking 
 MaPylRS [26wt MatRNACUAPyl Method development [21,22,25,26,44,47,69,73,77,106,115,117,118,123–130
MbPylRS [21,25,44,125,129,130wt MbtRNACUAPyl [25,44,125,129,130]
MbtRNAUUAPyl [44]
MbtRNAUCAPyl [25,44
MmPylRS [22,26,44,47,69,73,77,106,115,117,118,123,124,126–128wt MmtRNACUAPyl [21,22,26,47,69,73,77,106,115,118,123,124,126,127]
MmtRNACUAPylBU25CB[22,117,128]
MmtRNAUUAPyl [21,44]
MmtRNAUCAPyl [21
 MbPylRS [21,24,25,48,77,131–137Wt [21,24,25,48,77,132–136]
L274A C313S Y349F [131]

Y349F [137
MbtRNACUAPyl [25,77,131]
MbtRNAUCAPyl [24,25,48,132–136
Bioorthogonal labelling [127,131,137]
Imaging [136]
Method development [24,25,77,134,137]
Protein engineering [21,48,132,133,135–137
MmPylRS [127wt MmtRNACUAPyl [127,137]
MmtRNAUUAPyl [21
 MbPylRS [57,69,77,138,139wt MbtRNACUAPyl Bioorthogonal labelling [131,139]
Chemical decaging [57]
Imaging [69,129,138]
Method development [69,77
MmPylRS [69,129,131wt MmtRNACUAPyl 
 MbPylRS [25,130wt MbtRNACUAPyl [25Bioorthogonal labelling [130]
Method development [25
MmPylRS [130wt MmtRNACUAPyl 
 MbPylRS [131,140L274A C313S Y349F MbtRNACUAPyl Bioorthogonal labelling 
 MmPylRS [141wt MmtRNACUAPyl Method development 
 MbPylRS [140wt MbtRNACUAPyl Method development 
 MmPylRS [118,142,143R61K G131E L309A C348V Y384F [118]
Y306A Y384F [142]
R61K G131E Y306A Y384F [143
MmtRNACUAPyl [118,142]
MmtRNACUAPylBU25C [143
Method development [118,142,143
 MbPylRS [140Y271I L274A C313A Y349F MbtRNACUAPyl Method development [140,141]
Photoactivation [61,144
MmPylRS [61,141,144Y306M L309A C348A Y384F MmtRNACUAPyl 
 MbPylRS [145Y271M L274T C313A Y349F MbtRNACUAPyl Method development 
 MbPylRS [146Y271I 274M C313A MbtRNACUAPyl Method development 
 MbPylRS [63Y271A Y349F MbtRNACUAPyl Chemical decaging 
 MbPylRS [62L274A C313S Y349F MbtRNACUAPyl Bioorthogonal labelling
Chemical decaging 
 MbPylRS [66,67,69,75,125,147–152M241F A267S Y271C L274M [66,67,69,75,125,147–152MbtRNACUAPyl [66,67,69,75,125,147–152]
MbtRNACUAPylU25C [66
Method development [69]
Photoactivation [66,67,75,125,147–152
 MbPylRS [153Y271A L274M MbtRNACUAPyl Photoactivation 
 MbPylRS [153Y271A L274M MbtRNACUAPyl Photoactivation 
 MbPylRS [153Y271A L274M MbtRNACUAPyl Method development 
 MbPylRS [154L266M L270I Y271L L274A C313 MbtRNACUAPyl Method development 
 MbPylRS [24,25wt MbtRNACUAPyl [25]
MbtRNAUCAPyl [24
Imaging [123]
Method
development [22,24,25,69,115,155,156
MmPylRS [22,69,115,123,155,156Wt [22,69,115,123,155,156]
Y306A Y384F [155
MmtRNACUAPylBU25CB[22,155]
MmtRNACUAPyl [69,115,123,156
Mx1201PylRS [155wt Mx1201tRNACUAPylMx1201tRNACUAPylC41CA 
 MmPylRS [124wt MmtRNACUAPyl Bioorthogonal labelling 
 MbPylRS [77,157,158Wt [77,158]
L274M 313A Y349F [157
MbtRNACUAPyl Method development [77,155,159]
Photocrosslinking [157,158
MmPylRS [155,159Y306A Y384F MmtRNACUAPyl [159]
MmtRNACUAPylU25C [155
 MmPylRS [159Y306A Y384F MmtRNACUAPyl Method development 
 MbPylRS [132,140,160L274A C313S Y349F MbtRNACUAPyl Method development [140]
Photocrosslinking [132,160]
Protein engineering [132
 MmPylRS [159Y306A Y384F MmtRNACUAPyl Method development 
 MmPylRS [142Y306A Y384F MmtRNACUAPyl Photocrosslinking 
 MmPylRS [143R61K G131E Y306A Y384F MmtRNACUAPylBU25C Photocrosslinking 
 MmPylRS [18,39,59–61,123,155,161–166Y306A Y384F [18,39,59–61,123,155,161–166MmtRNACUAPyl [18,39,59–61,123,161–166]
MmtRNACUAPylBU25CB[155
Imaging [123,161,162,164,166]
Chemical decaging [18,59–61]
Chemical crosslinking [163]
Method development [155,165]
Protein labelling [39
Mx1201PylRS [155Y126A Mx1201tRNACUAPyl 
 MbPylRS [123,167,168Y271A L274M C313A MbtRNACUAPyl Bioorthogonal labelling [124,167]
Imaging [123,168]
Method development [169
MmPylRS [124,169Y306A Y384F [169]
Y306A L309M C348A [124
MmtRNACUAPyl 
 MmPylRS [165Y306A Y384F MmtRNACUAPyl Method development 
 MbPylRS [167,168Y271A L274M C313A MbtRNACUAPyl Bioorthogonal labelling [167]
Method development [167,169
MmPylRS [169Y306A Y384F MmtRNACUAPyl 
 MbPylRS [24wt MbtRNAUCAPyl Bioorthogonal labelling [39,127,131]
Method development [24,169
MmPylRS [39,127,131,169Wt [127,131]
Y306A Y384F [39,169
MmtRNACUAPyl 
 MmPylRS [169Y306A Y384F MmtRNACUAPyl Method development 
 MmPylRS [39,161,166,169,170Y306A Y384F MmtRNACUAPyl Bioorthogonal labelling [39]
Imaging [161,166,170]
Method development [169
 MbPylRS [19,64,140,168,171,172Y271M L274G C313A [19,64,168,171,172]
M241F A267S Y271C L274M [140
MbtRNACUAPyl [64,140,168,171,172]
MbtRNACUAPylU25C [19
Chemical inhibition [64]
Bioorthogonal labelling [39,131,167]
Imaging [123,128,161,166,168,171,172]
Method development [155,159,165]
Protein engineering [140]
Spectroscopic probe [19
 MmPylRS [39,123,128,131,155,159,161,165–167Y306A 384F [39,123,128,131,155,159,161, 165–167MmtRNACUAPyl [39,123,128,131,159,161, 165–167]
MmtRNACUAPylBU25C [155
 
Tryptophan derivatives 
 EcTrpRS [34S8A V144S V146A
S8A V144G V146C 
EctRNACUATrp Method development 
 EcTrpRS [34S8A V144S V146A EctRNACUATrp Method development 
 EcTrpRS [34S8A V144G V146C EctRNACUATrp Method development 
 EcTrpRS [34S8A V144G V146C EctRNACUATrp Method development 
 EcTrpRS [34S8A V144G V146C EctRNACUATrp Method development 
Tyrosine derivatives 
 EcTyrRS [46Y37V Q195C BstRNACUATyr Method development 
 EcTyrRSCUA [15,21,35,37,77,78,90Y37T D182T F183M D265R [78]
Y37V D182S F183M [37]
Y37V D182S F183M D265R [21,77,90]
Y37T D182T F183M [15]
Y37V D165G D182S F183M L186A D265R [35
EctRNACUATyr [15,21,35,77,78,90Mechanistic studies [15]
Method development [21,25,35,37,77,78,90
BstRNACUATyr [21,37,77,90
EcTyrRSUCA [25Y37V D182S F183M EctRNAUCATyr 
BstRNAUCATyr 
 EcTyrRS [77Y37V D182S F183M D265R EctRNACUATyr Method development 
BstRNACUATyr 
 EcTyrRS [25,35,37,77Y37V D182S F183M D265R [77]
Y37S D182T F183M L186V [37]
Y37V D182S F183M [25]
Y37V D165G D182S F183M L186A D265R [35
EctRNACUATyr [25,35,77Method development 
BstRNACUATyr [37,77
 MbPylRS [126,148,173L270F L274M N311G C313G Y349F [173]
L270F L274M N311G C313G [126,148
MbtRNACUAPyl Photoactivation 
 MbPylRS [173L270F L274M N311G C313G Y349F MbtRNACUAPyl Photoactivation 
 MbPylRS [173L270F L274M N311G C313G Y349F MbtRNACUAPyl Photoactivation 
 MbPylRS [173L270F L274M N311G C313G Y349F MbtRNACUAPyl Photoactivation 
 MmPylRS [174N346T C348I Y384L W417K MmtRNACUAPyl Bioorthogonal labelling 
 EcTyrRS [35Y37V D182S F183M D265R
Y37V D165G D182S F183M L186A D265R 
EctRNACUATyr Method development 
Miscellaneous unnatural amino acids 
 EcLeuRS [24,25M40I T252A Y499I Y527A H529G [24]
E20K M40V L41S T252R Y499S Y527L H529G H537G [25
EctRNACUALeu Method development 
 EcLeuRS [15,103,175M40A L41N T252A Y499I Y527G H537T EctRNACUALeu Mechanistic studies [15,103]
Method development [175
 MmSepRS [176wt MjtRNACUACys Method development 
Amino acidaaRSMutationstRNAApplication
Cysteine and selenocysteine derivatives 
 EcLeuRS [24M40I T252A Y499I Y527A H529G EctRNACUALeu Method development 
 EcLeuRS [24M40I T252A Y499I Y527A H529G EctRNACUALeu Method development 
 EcLeuRS [24M40I T252A Y499I Y527A H529G EctRNACUALeu Method development 
 EcLeuRS [24M40I T252A Y499I Y527A H529G EctRNACUALeu Method development 
 EcLeuRS [24M40I T252A Y499I Y527A H529G EctRNACUALeu Method development 
 EcLeuRS [24M40I T252A Y499I Y527A H529G EctRNACUALeu Method development 
 EcLeuRS [71M40G L41Q T252A Y499L Y527G H537F EctRNACUALeu Photoactivation 
 EcLeuRS [72M40G L41Q Y499L Y527G H537F EctRNACUALeu Photoactivation 
 MbPylRS [73,74N311Q C313A V366M MbtRNACUAPyl Photoactivation 
 MbPylRS [75M241F A267S Y271C L274M MbtRNACUAPyl Photoactivation 
 MbPylRS [75M241F A267S Y271C L274M MbtRNACUAPyl Photoactivation 
 MbPylRS [76C313W W382T MbPyltRNA Method development 
 MbPylRS [40L274A C313S Y349F MbtRNACUAPyl Photocrosslinking 
 MbPylRS [40L274A C313S Y349F MbtRNACUAPyl Photocrosslinking 
Phenylalanine derivatives 
 EcTyrRS [77Y37V D182S F183M D265R EctRNACUATyr Method development 
BstRNACUATyr 
 EcTyrRS [35,37,43,77Y37V D182S F183M D265R [77]
Y37I D182S F183M [37,43]
Y37V D165G D182S F183M L186A D265R [35
EctRNACUATyr [35,77Method development [35,37,77]
Protein engineering [43
BstRNACUATyr [37,43,77
 EcTyrRS [77Y37V D182S F183M D265R EctRNACUATyr Method development 
BstRNACUATyr 
 EcTyrRS [77Y37V D182S F183M D265R EctRNACUATyr Method development 
BstRNACUATyr 
 EcTyrRSCUA [16,17,35,37,38,43,49,55,68,77–96Y37L D182S F183A L186A D265R [78,81,84,85]
Y37V D182S F183M D265R [77,90]
Y37L D182S F183M L186A [16,17,37,38,43,49,55,68,79,80,82,83,86–89,91–96]
Y37V D165G D182S F183M L186A D265R [35
EctRNACUATyr [35,77,78,81,86,90Bioorthogonal labelling [38,79,83,87–89,96]
Method development [17,25,35,37,77,78,90]
Photocrosslinking [38,68,81,84,85,91–95]
Protein engineering [43,49,55,83]
Spectroscopic probe [16,80,82,86
BstRNACUATyr [16,17,37,38,43,49,55,68,77,79,80,82–85,87–96
EcTyrRSUCA [25Y37V D182S F183M EctRNAUCATyr 
BstRNAUCATyr 
 EcTyrRS [35,97Y37I D182S F183M D265R [97]
Y37S D182S F183A L186E D265R [35]
Y37G D182S F183I L186E D265R [35]
Y37S D182S F183I L186E D265R [35
EctRNACUATyr [35,97Method development [35]
Spectroscopic probe [97
BstRNACUATyr [97
 MmPylRS [50L301M Y306L L309A C348F MmtRNACUAPyl Method development 
 MmPylRS [98N346A C348A MmtRNACUAPyl Method development 
 MmPylRS [98N346A C348A MmtRNACUAPyl Method development 
 MmPylRS [98N346A C348A MmtRNACUAPyl Method development 
 MmPylRS [98N346A C348A MmtRNACUAPyl Method development 
 MmPylRS [98N346A C348A MmtRNACUAPyl Method development 
 MmPylRS [98N346A C348A MmtRNACUAPyl Method development 
 MmPylRS [98N346A C348A MmtRNACUAPyl Method development 
 EcTyrRSCUA [21,35,37,43,77,83,87,90,99,100Y37I N165G D182G F183M L186A [83,99]
Y37I D182G F183M L186A [37,43,87,100]
Y37V D182S F183M D265R [21,77,90]
Y37V D165G D182S F183M L186A D265R [35
EctRNACUATyr [21,35,77,90Bioorthogonal labelling [83,87,100]
Method development [25,35,37,77,83,90,99]
Protein engineering [21,43
BstRNACUATyr [37,43,77,83,87,99,100
EcTyrRSUCA [25Y37V D182S F183M EctRNAUCATyr 
BstRNAUCATyr 
 EcTyrRS [84,101Y37I D182G F183M L186A D265R BstRNACUATyr Chemical crosslinking [84,101]
Method development [101
 EcTyrRS [15,37,55,78,85,92,94,95,99,102–105Y37G D182G L186A
D265R [78,85,103]
Y37G D182G L186A [15,55,92,94,95,99,102,104,105]
Y37G D182G F183Y L186M [37
EctRNACUATyr [15,78,103Mechanistic studies [15]
Method development [37,78,95,99,103,106]
Photocrosslinking [85,92,94,102,104,105]
Photoinhibition [55
BstRNACUATyr [37,55,85,92,94,95,99,102,104,105
MmPylRS [106A302T N346T C348T W417C [106MmtRNACUAPyl [106
 MmPylRS [106A302T N346G C348T V401I W417Y MmtRNACUAPyl Method development 
 EcLeuRS [107–109L38F M40G L41P Y499V Y500L Y527A H537E L538S F541C A560V EctRNACUALeu Method development [107,108]
Spectroscopic probe [109
 MmPylRS [110N346Q C348S V401G W417T MmtRNACUAPyl Spectroscopic Probe 
 MbPylRS [111L270F L274M N311G C313G Y349F MbtRNACUAPyl Photoswitching 
 MbPylRS [111L270F L274M N311G C313G Y349F MbtRNACUAPyl Photoswitching 
 MbPylRS [111L270F L274M N311G C313G Y349F MbtRNACUAPyl Photoswitching 
 MmPylRS [56,112A302T L309S N346V C348G MmtRNACUAPyl Method development [112]
Photoswitching [56
Histidine derivatives 
 MaPylRS [26L121M L125I Y126F M129A V168F MatRNACUAPyl Method development 
MbPylRS [113L270I Y271F L274G C313F Y349F MbtRNACUAPyl 
 MbPylRS [113L270I Y271F L274G C313F Y349F MbtRNACUAPyl Method development 
Lysine derivatives 
 MbPylRS [114L266M L270I Y271F L274A C313F MbtRNACUAPyl Method development 
 MbPylRS [115–117D76G L266V L270I Y271F L274A C313F [115]
D76G L266M L270I Y271F L274A C313F [116,117
MbtRNACUAPyl Method development [44,50,115,117,118]
Spectroscopic probe [116
MmPylRS [44,50,118L305I Y306F L309A C348F [118]
L301M Y306L L309A C348F [44,50
MmtRNACUAPyl [50,118]
MmtRNAUUAPyl [44
 MmPylRS [50L301M Y306L L309A C348F MmtRNACUAPyl Method development 
 MbPylRS [119D76G L266M L270I Y271F L274A C313F MbtRNACUAPyl Method development 
 MbPylRS [77,120L274A C313F Y349F [120]
wt [77
MbtRNACUAPyl Method development 
 MbPylRS [121Y271M L274A C313A MbtRNACUAPyl Photocrosslinking 
 MmPylRS [122Y306V L309A C348F Y384F MmtRNACUAPyl Photocrosslinking 
 MaPylRS [26wt MatRNACUAPyl Method development [21,22,25,26,44,47,69,73,77,106,115,117,118,123–130
MbPylRS [21,25,44,125,129,130wt MbtRNACUAPyl [25,44,125,129,130]
MbtRNAUUAPyl [44]
MbtRNAUCAPyl [25,44
MmPylRS [22,26,44,47,69,73,77,106,115,117,118,123,124,126–128wt MmtRNACUAPyl [21,22,26,47,69,73,77,106,115,118,123,124,126,127]
MmtRNACUAPylBU25CB[22,117,128]
MmtRNAUUAPyl [21,44]
MmtRNAUCAPyl [21
 MbPylRS [21,24,25,48,77,131–137Wt [21,24,25,48,77,132–136]
L274A C313S Y349F [131]

Y349F [137
MbtRNACUAPyl [25,77,131]
MbtRNAUCAPyl [24,25,48,132–136
Bioorthogonal labelling [127,131,137]
Imaging [136]
Method development [24,25,77,134,137]
Protein engineering [21,48,132,133,135–137
MmPylRS [127wt MmtRNACUAPyl [127,137]
MmtRNAUUAPyl [21
 MbPylRS [57,69,77,138,139wt MbtRNACUAPyl Bioorthogonal labelling [131,139]
Chemical decaging [57]
Imaging [69,129,138]
Method development [69,77
MmPylRS [69,129,131wt MmtRNACUAPyl 
 MbPylRS [25,130wt MbtRNACUAPyl [25Bioorthogonal labelling [130]
Method development [25
MmPylRS [130wt MmtRNACUAPyl 
 MbPylRS [131,140L274A C313S Y349F MbtRNACUAPyl Bioorthogonal labelling 
 MmPylRS [141wt MmtRNACUAPyl Method development 
 MbPylRS [140wt MbtRNACUAPyl Method development 
 MmPylRS [118,142,143R61K G131E L309A C348V Y384F [118]
Y306A Y384F [142]
R61K G131E Y306A Y384F [143
MmtRNACUAPyl [118,142]
MmtRNACUAPylBU25C [143
Method development [118,142,143
 MbPylRS [140Y271I L274A C313A Y349F MbtRNACUAPyl Method development [140,141]
Photoactivation [61,144
MmPylRS [61,141,144Y306M L309A C348A Y384F MmtRNACUAPyl 
 MbPylRS [145Y271M L274T C313A Y349F MbtRNACUAPyl Method development 
 MbPylRS [146Y271I 274M C313A MbtRNACUAPyl Method development 
 MbPylRS [63Y271A Y349F MbtRNACUAPyl Chemical decaging 
 MbPylRS [62L274A C313S Y349F MbtRNACUAPyl Bioorthogonal labelling
Chemical decaging 
 MbPylRS [66,67,69,75,125,147–152M241F A267S Y271C L274M [66,67,69,75,125,147–152MbtRNACUAPyl [66,67,69,75,125,147–152]
MbtRNACUAPylU25C [66
Method development [69]
Photoactivation [66,67,75,125,147–152
 MbPylRS [153Y271A L274M MbtRNACUAPyl Photoactivation 
 MbPylRS [153Y271A L274M MbtRNACUAPyl Photoactivation 
 MbPylRS [153Y271A L274M MbtRNACUAPyl Method development 
 MbPylRS [154L266M L270I Y271L L274A C313 MbtRNACUAPyl Method development 
 MbPylRS [24,25wt MbtRNACUAPyl [25]
MbtRNAUCAPyl [24
Imaging [123]
Method
development [22,24,25,69,115,155,156
MmPylRS [22,69,115,123,155,156Wt [22,69,115,123,155,156]
Y306A Y384F [155
MmtRNACUAPylBU25CB[22,155]
MmtRNACUAPyl [69,115,123,156
Mx1201PylRS [155wt Mx1201tRNACUAPylMx1201tRNACUAPylC41CA 
 MmPylRS [124wt MmtRNACUAPyl Bioorthogonal labelling 
 MbPylRS [77,157,158Wt [77,158]
L274M 313A Y349F [157
MbtRNACUAPyl Method development [77,155,159]
Photocrosslinking [157,158
MmPylRS [155,159Y306A Y384F MmtRNACUAPyl [159]
MmtRNACUAPylU25C [155
 MmPylRS [159Y306A Y384F MmtRNACUAPyl Method development 
 MbPylRS [132,140,160L274A C313S Y349F MbtRNACUAPyl Method development [140]
Photocrosslinking [132,160]
Protein engineering [132
 MmPylRS [159Y306A Y384F MmtRNACUAPyl Method development 
 MmPylRS [142Y306A Y384F MmtRNACUAPyl Photocrosslinking 
 MmPylRS [143R61K G131E Y306A Y384F MmtRNACUAPylBU25C Photocrosslinking 
 MmPylRS [18,39,59–61,123,155,161–166Y306A Y384F [18,39,59–61,123,155,161–166MmtRNACUAPyl [18,39,59–61,123,161–166]
MmtRNACUAPylBU25CB[155
Imaging [123,161,162,164,166]
Chemical decaging [18,59–61]
Chemical crosslinking [163]
Method development [155,165]
Protein labelling [39
Mx1201PylRS [155Y126A Mx1201tRNACUAPyl 
 MbPylRS [123,167,168Y271A L274M C313A MbtRNACUAPyl Bioorthogonal labelling [124,167]
Imaging [123,168]
Method development [169
MmPylRS [124,169Y306A Y384F [169]
Y306A L309M C348A [124
MmtRNACUAPyl 
 MmPylRS [165Y306A Y384F MmtRNACUAPyl Method development 
 MbPylRS [167,168Y271A L274M C313A MbtRNACUAPyl Bioorthogonal labelling [167]
Method development [167,169
MmPylRS [169Y306A Y384F MmtRNACUAPyl 
 MbPylRS [24wt MbtRNAUCAPyl Bioorthogonal labelling [39,127,131]
Method development [24,169
MmPylRS [39,127,131,169Wt [127,131]
Y306A Y384F [39,169
MmtRNACUAPyl 
 MmPylRS [169Y306A Y384F MmtRNACUAPyl Method development 
 MmPylRS [39,161,166,169,170Y306A Y384F MmtRNACUAPyl Bioorthogonal labelling [39]
Imaging [161,166,170]
Method development [169
 MbPylRS [19,64,140,168,171,172Y271M L274G C313A [19,64,168,171,172]
M241F A267S Y271C L274M [140
MbtRNACUAPyl [64,140,168,171,172]
MbtRNACUAPylU25C [19
Chemical inhibition [64]
Bioorthogonal labelling [39,131,167]
Imaging [123,128,161,166,168,171,172]
Method development [155,159,165]
Protein engineering [140]
Spectroscopic probe [19
 MmPylRS [39,123,128,131,155,159,161,165–167Y306A 384F [39,123,128,131,155,159,161, 165–167MmtRNACUAPyl [39,123,128,131,159,161, 165–167]
MmtRNACUAPylBU25C [155
 
Tryptophan derivatives 
 EcTrpRS [34S8A V144S V146A
S8A V144G V146C 
EctRNACUATrp Method development 
 EcTrpRS [34S8A V144S V146A EctRNACUATrp Method development 
 EcTrpRS [34S8A V144G V146C EctRNACUATrp Method development 
 EcTrpRS [34S8A V144G V146C EctRNACUATrp Method development 
 EcTrpRS [34S8A V144G V146C EctRNACUATrp Method development 
Tyrosine derivatives 
 EcTyrRS [46Y37V Q195C BstRNACUATyr Method development 
 EcTyrRSCUA [15,21,35,37,77,78,90Y37T D182T F183M D265R [78]
Y37V D182S F183M [37]
Y37V D182S F183M D265R [21,77,90]
Y37T D182T F183M [15]
Y37V D165G D182S F183M L186A D265R [35
EctRNACUATyr [15,21,35,77,78,90Mechanistic studies [15]
Method development [21,25,35,37,77,78,90
BstRNACUATyr [21,37,77,90
EcTyrRSUCA [25Y37V D182S F183M EctRNAUCATyr 
BstRNAUCATyr 
 EcTyrRS [77Y37V D182S F183M D265R EctRNACUATyr Method development 
BstRNACUATyr 
 EcTyrRS [25,35,37,77Y37V D182S F183M D265R [77]
Y37S D182T F183M L186V [37]
Y37V D182S F183M [25]
Y37V D165G D182S F183M L186A D265R [35
EctRNACUATyr [25,35,77Method development 
BstRNACUATyr [37,77