Using genetically incorporated unnatural amino acids to control protein functions in mammalian cells

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

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

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 (1–110, 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)/tRNA^Pyl pairs from archaea species Methanosarcina barkeri (Mb) and Methanosarcina mazei (Mm) have proven to be extraordinarily useful pairs [4]. The tRNA^Pyl 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 acid	aaRS	Mutations	tRNA	Application
Cysteine and selenocysteine derivatives
	EcLeuRS [24]	M40I T252A Y499I Y527A H529G	${E c t R N A}_{C U A}^{L e u}$	Method development
	EcLeuRS [24]	M40I T252A Y499I Y527A H529G	${E c t R N A}_{C U A}^{L e u}$	Method development
	EcLeuRS [24]	M40I T252A Y499I Y527A H529G	${E c t R N A}_{C U A}^{L e u}$	Method development
	EcLeuRS [24]	M40I T252A Y499I Y527A H529G	${E c t R N A}_{C U A}^{L e u}$	Method development
	EcLeuRS [24]	M40I T252A Y499I Y527A H529G	${E c t R N A}_{C U A}^{L e u}$	Method development
	EcLeuRS [24]	M40I T252A Y499I Y527A H529G	${E c t R N A}_{C U A}^{L e u}$	Method development
	EcLeuRS [71]	M40G L41Q T252A Y499L Y527G H537F	${E c t R N A}_{C U A}^{L e u}$	Photoactivation
	EcLeuRS [72]	M40G L41Q Y499L Y527G H537F	${E c t R N A}_{C U A}^{L e u}$	Photoactivation
	MbPylRS [73,74]	N311Q C313A V366M	${M b t R N A}_{C U A}^{P y l}$	Photoactivation
	MbPylRS [75]	M241F A267S Y271C L274M	${M b t R N A}_{C U A}^{P y l}$	Photoactivation
	MbPylRS [75]	M241F A267S Y271C L274M	${M b t R N A}_{C U A}^{P y l}$	Photoactivation
	MbPylRS [76]	C313W W382T	MbPyltRNA	Method development
	MbPylRS [40]	L274A C313S Y349F	${M b t R N A}_{C U A}^{P y l}$	Photocrosslinking
	MbPylRS [40]	L274A C313S Y349F	${M b t R N A}_{C U A}^{P y l}$	Photocrosslinking
Phenylalanine derivatives
	EcTyrRS [77]	Y37V D182S F183M D265R	${E c t R N A}_{C U A}^{T y r}$	Method development
	EcTyrRS [77]	Y37V D182S F183M D265R	${B s t R N A}_{C U A}^{T y r}$	Method development
	EcTyrRS [35,37,43,77]	Y37V D182S F183M D265R [77] Y37I D182S F183M [37,43] Y37V D165G D182S F183M L186A D265R [35]	${E c t R N A}_{C U A}^{T y r}$ [35,77]	Method development [35,37,77] Protein engineering [43]
	EcTyrRS [35,37,43,77]		${B s t R N A}_{C U A}^{T y r}$ [37,43,77]	Method development [35,37,77] Protein engineering [43]
	EcTyrRS [77]	Y37V D182S F183M D265R	${E c t R N A}_{C U A}^{T y r}$	Method development
	EcTyrRS [77]	Y37V D182S F183M D265R	${B s t R N A}_{C U A}^{T y r}$	Method development
	EcTyrRS [77]	Y37V D182S F183M D265R	${E c t R N A}_{C U A}^{T y r}$	Method development
	EcTyrRS [77]	Y37V D182S F183M D265R	${B s t R N A}_{C U A}^{T y r}$	Method development
	EcTyrRS_CUA [16,17,35,37,38,43,49,55,68,77–96]	Y37L 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]	${E c t R N A}_{C U A}^{T y r}$ [35,77,78,81,86,90]	Bioorthogonal 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]
	EcTyrRS_CUA [16,17,35,37,38,43,49,55,68,77–96]		${B s t R N A}_{C U A}^{T y r}$ [16,17,37,38,43,49,55,68,77,79,80,82–85,87–96]
	EcTyrRS_UCA [25]	Y37V D182S F183M	${E c t R N A}_{U C A}^{T y r}$
	EcTyrRS_UCA [25]	Y37V D182S F183M	${B s t R N A}_{U C A}^{T y r}$
	EcTyrRS [35,97]	Y37I D182S F183M D265R [97] Y37S D182S F183A L186E D265R [35] Y37G D182S F183I L186E D265R [35] Y37S D182S F183I L186E D265R [35]	${E c t R N A}_{C U A}^{T y r}$ [35,97]	Method development [35] Spectroscopic probe [97]
	EcTyrRS [35,97]		${B s t R N A}_{C U A}^{T y r}$ [97]	Method development [35] Spectroscopic probe [97]
	MmPylRS [50]	L301M Y306L L309A C348F	${M m t R N A}_{C U A}^{P y l}$	Method development
	MmPylRS [98]	N346A C348A	${M m t R N A}_{C U A}^{P y l}$	Method development
	MmPylRS [98]	N346A C348A	${M m t R N A}_{C U A}^{P y l}$	Method development
	MmPylRS [98]	N346A C348A	${M m t R N A}_{C U A}^{P y l}$	Method development
	MmPylRS [98]	N346A C348A	${M m t R N A}_{C U A}^{P y l}$	Method development
	MmPylRS [98]	N346A C348A	${M m t R N A}_{C U A}^{P y l}$	Method development
	MmPylRS [98]	N346A C348A	${M m t R N A}_{C U A}^{P y l}$	Method development
	MmPylRS [98]	N346A C348A	${M m t R N A}_{C U A}^{P y l}$	Method development
	EcTyrRS_CUA [21,35,37,43,77,83,87,90,99,100]	Y37I 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]	${E c t R N A}_{C U A}^{T y r}$ [21,35,77,90]	Bioorthogonal labelling [83,87,100] Method development [25,35,37,77,83,90,99] Protein engineering [21,43]
	EcTyrRS_CUA [21,35,37,43,77,83,87,90,99,100]		${B s t R N A}_{C U A}^{T y r}$ [37,43,77,83,87,99,100]
	EcTyrRS_UCA [25]	Y37V D182S F183M	${E c t R N A}_{U C A}^{T y r}$
	EcTyrRS_UCA [25]	Y37V D182S F183M	${B s t R N A}_{U C A}^{T y r}$
	EcTyrRS [84,101]	Y37I D182G F183M L186A D265R	${B s t R N A}_{C U A}^{T y r}$	Chemical crosslinking [84,101] Method development [101]
	EcTyrRS [15,37,55,78,85,92,94,95,99,102–105]	Y37G D182G L186A D265R [78,85,103] Y37G D182G L186A [15,55,92,94,95,99,102,104,105] Y37G D182G F183Y L186M [37]	${E c t R N A}_{C U A}^{T y r}$ [15,78,103]	Mechanistic studies [15] Method development [37,78,95,99,103,106] Photocrosslinking [85,92,94,102,104,105] Photoinhibition [55]
	EcTyrRS [15,37,55,78,85,92,94,95,99,102–105]		${B s t R N A}_{C U A}^{T y r}$ [37,55,85,92,94,95,99,102,104,105]
	MmPylRS [106]	A302T N346T C348T W417C [106]	${M m t R N A}_{C U A}^{P y l}$ [106]
	MmPylRS [106]	A302T N346G C348T V401I W417Y	${M m t R N A}_{C U A}^{P y l}$	Method development
	EcLeuRS [107–109]	L38F M40G L41P Y499V Y500L Y527A H537E L538S F541C A560V	${E c t R N A}_{C U A}^{L e u}$	Method development [107,108] Spectroscopic probe [109]
	MmPylRS [110]	N346Q C348S V401G W417T	${M m t R N A}_{C U A}^{P y l}$	Spectroscopic Probe
	MbPylRS [111]	L270F L274M N311G C313G Y349F	${M b t R N A}_{C U A}^{P y l}$	Photoswitching
	MbPylRS [111]	L270F L274M N311G C313G Y349F	${M b t R N A}_{C U A}^{P y l}$	Photoswitching
	MbPylRS [111]	L270F L274M N311G C313G Y349F	${M b t R N A}_{C U A}^{P y l}$	Photoswitching
	MmPylRS [56,112]	A302T L309S N346V C348G	${M m t R N A}_{C U A}^{P y l}$	Method development [112] Photoswitching [56]
Histidine derivatives
	MaPylRS [26]	L121M L125I Y126F M129A V168F	${M a t R N A}_{C U A}^{P y l}$	Method development
	MbPylRS [113]	L270I Y271F L274G C313F Y349F	${M b t R N A}_{C U A}^{P y l}$	Method development
	MbPylRS [113]	L270I Y271F L274G C313F Y349F	${M b t R N A}_{C U A}^{P y l}$	Method development
Lysine derivatives
	MbPylRS [114]	L266M L270I Y271F L274A C313F	${M b t R N A}_{C U A}^{P y l}$	Method development
	MbPylRS [115–117]	D76G L266V L270I Y271F L274A C313F [115] D76G L266M L270I Y271F L274A C313F [116,117]	${M b t R N A}_{C U A}^{P y l}$	Method development [44,50,115,117,118] Spectroscopic probe [116]
	MmPylRS [44,50,118]	L305I Y306F L309A C348F [118] L301M Y306L L309A C348F [44,50]	${M m t R N A}_{C U A}^{P y l}$ [50,118] ${M m t R N A}_{U U A}^{P y l}$ [44]
	MmPylRS [50]	L301M Y306L L309A C348F	${M m t R N A}_{C U A}^{P y l}$	Method development
	MbPylRS [119]	D76G L266M L270I Y271F L274A C313F	${M b t R N A}_{C U A}^{P y l}$	Method development
	MbPylRS [77,120]	L274A C313F Y349F [120] wt [77]	${M b t R N A}_{C U A}^{P y l}$	Method development
	MbPylRS [121]	Y271M L274A C313A	${M b t R N A}_{C U A}^{P y l}$	Photocrosslinking
	MmPylRS [122]	Y306V L309A C348F Y384F	${M m t R N A}_{C U A}^{P y l}$	Photocrosslinking
	MaPylRS [26]	wt	${M a t R N A}_{C U A}^{P y l}$	Method development [21,22,25,26,44,47,69,73,77,106,115,117,118,123–130]
	MbPylRS [21,25,44,125,129,130]	wt	${M b t R N A}_{C U A}^{P y l}$ [25,44,125,129,130] ${M b t R N A}_{U U A}^{P y l}$ [44] ${M b t R N A}_{U C A}^{P y l}$ [25,44]
	MmPylRS [22,26,44,47,69,73,77,106,115,117,118,123,124,126–128]	wt	${M m t R N A}_{C U A}^{P y l}$ [21,22,26,47,69,73,77,106,115,118,123,124,126,127] ${M m t R N A}_{C U A}^{P y l} B U 25 C B$ [22,117,128] ${M m t R N A}_{U U A}^{P y l}$ [21,44] ${M m t R N A}_{U C A}^{P y l}$ [21]
	MbPylRS [21,24,25,48,77,131–137]	Wt [21,24,25,48,77,132–136] L274A C313S Y349F [131] Y349F [137]	${M b t R N A}_{C U A}^{P y l}$ [25,77,131] ${M b t R N A}_{U C A}^{P y l}$ [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 [127]	wt	${M m t R N A}_{C U A}^{P y l}$ [127,137] ${M m t R N A}_{U U A}^{P y l}$ [21]
	MbPylRS [57,69,77,138,139]	wt	${M b t R N A}_{C U A}^{P y l}$	Bioorthogonal labelling [131,139] Chemical decaging [57] Imaging [69,129,138] Method development [69,77]
	MmPylRS [69,129,131]	wt	${M m t R N A}_{C U A}^{P y l}$
	MbPylRS [25,130]	wt	${M b t R N A}_{C U A}^{P y l}$ [25]	Bioorthogonal labelling [130] Method development [25]
	MmPylRS [130]	wt	${M m t R N A}_{C U A}^{P y l}$	Bioorthogonal labelling [130] Method development [25]
	MbPylRS [131,140]	L274A C313S Y349F	${M b t R N A}_{C U A}^{P y l}$	Bioorthogonal labelling
	MmPylRS [141]	wt	${M m t R N A}_{C U A}^{P y l}$	Method development
	MbPylRS [140]	wt	${M b t R N A}_{C U A}^{P y l}$	Method development
	MmPylRS [118,142,143]	R61K G131E L309A C348V Y384F [118] Y306A Y384F [142] R61K G131E Y306A Y384F [143]	${M m t R N A}_{C U A}^{P y l}$ [118,142] ${M m t R N A}_{C U A}^{P y l} B U 25 C$ [143]	Method development [118,142,143]
	MbPylRS [140]	Y271I L274A C313A Y349F	${M b t R N A}_{C U A}^{P y l}$	Method development [140,141] Photoactivation [61,144]
	MmPylRS [61,141,144]	Y306M L309A C348A Y384F	${M m t R N A}_{C U A}^{P y l}$	Method development [140,141] Photoactivation [61,144]
	MbPylRS [145]	Y271M L274T C313A Y349F	${M b t R N A}_{C U A}^{P y l}$	Method development
	MbPylRS [146]	Y271I 274M C313A	${M b t R N A}_{C U A}^{P y l}$	Method development
	MbPylRS [63]	Y271A Y349F	${M b t R N A}_{C U A}^{P y l}$	Chemical decaging
	MbPylRS [62]	L274A C313S Y349F	${M b t R N A}_{C U A}^{P y l}$	Bioorthogonal labelling Chemical decaging
	MbPylRS [66,67,69,75,125,147–152]	M241F A267S Y271C L274M [66,67,69,75,125,147–152]	${M b t R N A}_{C U A}^{P y l}$ [66,67,69,75,125,147–152] ${M b t R N A}_{C U A}^{P y l} U 25 C$ [66]	Method development [69] Photoactivation [66,67,75,125,147–152]
	MbPylRS [153]	Y271A L274M	${M b t R N A}_{C U A}^{P y l}$	Photoactivation
	MbPylRS [153]	Y271A L274M	${M b t R N A}_{C U A}^{P y l}$	Photoactivation
	MbPylRS [153]	Y271A L274M	${M b t R N A}_{C U A}^{P y l}$	Method development
	MbPylRS [154]	L266M L270I Y271L L274A C313	${M b t R N A}_{C U A}^{P y l}$	Method development
	MbPylRS [24,25]	wt	${M b t R N A}_{C U A}^{P y l}$ [25] ${M b t R N A}_{U C A}^{P y l}$ [24]	Imaging [123] Method development [22,24,25,69,115,155,156]
	MmPylRS [22,69,115,123,155,156]	Wt [22,69,115,123,155,156] Y306A Y384F [155]	${M m t R N A}_{C U A}^{P y l} B U 25 C B$ [22,155] ${M m t R N A}_{C U A}^{P y l}$ [69,115,123,156]
	Mx1201PylRS [155]	wt	${M x 1201 t R N A}_{C U A}^{P y l}$ ${M x 1201 t R N A}_{C U A}^{P y l}$ C41CA
	MmPylRS [124]	wt	${M m t R N A}_{C U A}^{P y l}$	Bioorthogonal labelling
	MbPylRS [77,157,158]	Wt [77,158] L274M 313A Y349F [157]	${M b t R N A}_{C U A}^{P y l}$	Method development [77,155,159] Photocrosslinking [157,158]
	MmPylRS [155,159]	Y306A Y384F	${M m t R N A}_{C U A}^{P y l}$ [159] ${M m t R N A}_{C U A}^{P y l}$ U25C [155]	Method development [77,155,159] Photocrosslinking [157,158]
	MmPylRS [159]	Y306A Y384F	${M m t R N A}_{C U A}^{P y l}$	Method development
	MbPylRS [132,140,160]	L274A C313S Y349F	${M b t R N A}_{C U A}^{P y l}$	Method development [140] Photocrosslinking [132,160] Protein engineering [132]
	MmPylRS [159]	Y306A Y384F	${M m t R N A}_{C U A}^{P y l}$	Method development
	MmPylRS [142]	Y306A Y384F	${M m t R N A}_{C U A}^{P y l}$	Photocrosslinking
	MmPylRS [143]	R61K G131E Y306A Y384F	${M m t R N A}_{C U A}^{P y l} B U 25 C$	Photocrosslinking
	MmPylRS [18,39,59–61,123,155,161–166]	Y306A Y384F [18,39,59–61,123,155,161–166]	${M m t R N A}_{C U A}^{P y l}$ [18,39,59–61,123,161–166] ${M m t R N A}_{C U A}^{P y l} B U 25 C B$ [155]	Imaging [123,161,162,164,166] Chemical decaging [18,59–61] Chemical crosslinking [163] Method development [155,165] Protein labelling [39]
	Mx1201PylRS [155]	Y126A	${M x 1201 t R N A}_{C U A}^{P y l}$
	MbPylRS [123,167,168]	Y271A L274M C313A	${M b t R N A}_{C U A}^{P y l}$	Bioorthogonal labelling [124,167] Imaging [123,168] Method development [169]
	MmPylRS [124,169]	Y306A Y384F [169] Y306A L309M C348A [124]	${M m t R N A}_{C U A}^{P y l}$
	MmPylRS [165]	Y306A Y384F	${M m t R N A}_{C U A}^{P y l}$	Method development
	MbPylRS [167,168]	Y271A L274M C313A	${M b t R N A}_{C U A}^{P y l}$	Bioorthogonal labelling [167] Method development [167,169]
	MmPylRS [169]	Y306A Y384F	${M m t R N A}_{C U A}^{P y l}$	Bioorthogonal labelling [167] Method development [167,169]
	MbPylRS [24]	wt	${M b t R N A}_{U C A}^{P y l}$	Bioorthogonal labelling [39,127,131] Method development [24,169]
	MmPylRS [39,127,131,169]	Wt [127,131] Y306A Y384F [39,169]	${M m t R N A}_{C U A}^{P y l}$
	MmPylRS [169]	Y306A Y384F	${M m t R N A}_{C U A}^{P y l}$	Method development
	MmPylRS [39,161,166,169,170]	Y306A Y384F	${M m t R N A}_{C U A}^{P y l}$	Bioorthogonal labelling [39] Imaging [161,166,170] Method development [169]
	MbPylRS [19,64,140,168,171,172]	Y271M L274G C313A [19,64,168,171,172] M241F A267S Y271C L274M [140]	${M b t R N A}_{C U A}^{P y l}$ [64,140,168,171,172] ${M b t R N A}_{C U A}^{P y l} U 25 C$ [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 [39,123,128,131,155,159,161,165–167]	Y306A 384F [39,123,128,131,155,159,161, 165–167]	${M m t R N A}_{C U A}^{P y l}$ [39,123,128,131,159,161, 165–167] ${M m t R N A}_{C U A}^{P y l} B U 25 C$ [155]
Tryptophan derivatives
	EcTrpRS [34]	S8A V144S V146A S8A V144G V146C	${E c t R N A}_{C U A}^{T r p}$	Method development
	EcTrpRS [34]	S8A V144S V146A	${E c t R N A}_{C U A}^{T r p}$	Method development
	EcTrpRS [34]	S8A V144G V146C	${E c t R N A}_{C U A}^{T r p}$	Method development
	EcTrpRS [34]	S8A V144G V146C	${E c t R N A}_{C U A}^{T r p}$	Method development
	EcTrpRS [34]	S8A V144G V146C	${E c t R N A}_{C U A}^{T r p}$	Method development
Tyrosine derivatives
	EcTyrRS [46]	Y37V Q195C	${B s t R N A}_{C U A}^{T y r}$	Method development
	EcTyrRS_CUA [15,21,35,37,77,78,90]	Y37T 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]	${E c t R N A}_{C U A}^{T y r}$ [15,21,35,77,78,90]	Mechanistic studies [15] Method development [21,25,35,37,77,78,90]
	EcTyrRS_CUA [15,21,35,37,77,78,90]		${B s t R N A}_{C U A}^{T y r}$ [21,37,77,90]
	EcTyrRS_UCA [25]	Y37V D182S F183M	${E c t R N A}_{U C A}^{T y r}$
	EcTyrRS_UCA [25]	Y37V D182S F183M	${B s t R N A}_{U C A}^{T y r}$
	EcTyrRS [77]	Y37V D182S F183M D265R	${E c t R N A}_{C U A}^{T y r}$	Method development
	EcTyrRS [77]	Y37V D182S F183M D265R	${B s t R N A}_{C U A}^{T y r}$	Method development
	EcTyrRS [25,35,37,77]	Y37V D182S F183M D265R [77] Y37S D182T F183M L186V [37] Y37V D182S F183M [25] Y37V D165G D182S F183M L186A D265R [35]	${E c t R N A}_{C U A}^{T y r}$ [25,35,77]	Method development
	EcTyrRS [25,35,37,77]		${B s t R N A}_{C U A}^{T y r}$ [37,77]	Method development
	MbPylRS [126,148,173]	L270F L274M N311G C313G Y349F [173] L270F L274M N311G C313G [126,148]	${M b t R N A}_{C U A}^{P y l}$	Photoactivation
	MbPylRS [173]	L270F L274M N311G C313G Y349F	${M b t R N A}_{C U A}^{P y l}$	Photoactivation
	MbPylRS [173]	L270F L274M N311G C313G Y349F	${M b t R N A}_{C U A}^{P y l}$	Photoactivation
	MbPylRS [173]	L270F L274M N311G C313G Y349F	${M b t R N A}_{C U A}^{P y l}$	Photoactivation
	MmPylRS [174]	N346T C348I Y384L W417K	${M m t R N A}_{C U A}^{P y l}$	Bioorthogonal labelling
	EcTyrRS [35]	Y37V D182S F183M D265R Y37V D165G D182S F183M L186A D265R	${E c t R N A}_{C U A}^{T y r}$	Method development
Miscellaneous unnatural amino acids
	EcLeuRS [24,25]	M40I T252A Y499I Y527A H529G [24] E20K M40V L41S T252R Y499S Y527L H529G H537G [25]	${E c t R N A}_{C U A}^{L e u}$	Method development
	EcLeuRS [15,103,175]	M40A L41N T252A Y499I Y527G H537T	${E c t R N A}_{C U A}^{L e u}$	Mechanistic studies [15,103] Method development [175]
	MmSepRS [176]	wt	${M j t R N A}_{C U A}^{C y s}$	Method development

Amino acid	aaRS	Mutations	tRNA	Application
Cysteine and selenocysteine derivatives
	EcLeuRS [24]	M40I T252A Y499I Y527A H529G	${E c t R N A}_{C U A}^{L e u}$	Method development
	EcLeuRS [24]	M40I T252A Y499I Y527A H529G	${E c t R N A}_{C U A}^{L e u}$	Method development
	EcLeuRS [24]	M40I T252A Y499I Y527A H529G	${E c t R N A}_{C U A}^{L e u}$	Method development
	EcLeuRS [24]	M40I T252A Y499I Y527A H529G	${E c t R N A}_{C U A}^{L e u}$	Method development
	EcLeuRS [24]	M40I T252A Y499I Y527A H529G	${E c t R N A}_{C U A}^{L e u}$	Method development
	EcLeuRS [24]	M40I T252A Y499I Y527A H529G	${E c t R N A}_{C U A}^{L e u}$	Method development
	EcLeuRS [71]	M40G L41Q T252A Y499L Y527G H537F	${E c t R N A}_{C U A}^{L e u}$	Photoactivation
	EcLeuRS [72]	M40G L41Q Y499L Y527G H537F	${E c t R N A}_{C U A}^{L e u}$	Photoactivation
	MbPylRS [73,74]	N311Q C313A V366M	${M b t R N A}_{C U A}^{P y l}$	Photoactivation
	MbPylRS [75]	M241F A267S Y271C L274M	${M b t R N A}_{C U A}^{P y l}$	Photoactivation
	MbPylRS [75]	M241F A267S Y271C L274M	${M b t R N A}_{C U A}^{P y l}$	Photoactivation
	MbPylRS [76]	C313W W382T	MbPyltRNA	Method development
	MbPylRS [40]	L274A C313S Y349F	${M b t R N A}_{C U A}^{P y l}$	Photocrosslinking
	MbPylRS [40]	L274A C313S Y349F	${M b t R N A}_{C U A}^{P y l}$	Photocrosslinking
Phenylalanine derivatives
	EcTyrRS [77]	Y37V D182S F183M D265R	${E c t R N A}_{C U A}^{T y r}$	Method development
	EcTyrRS [77]	Y37V D182S F183M D265R	${B s t R N A}_{C U A}^{T y r}$	Method development
	EcTyrRS [35,37,43,77]	Y37V D182S F183M D265R [77] Y37I D182S F183M [37,43] Y37V D165G D182S F183M L186A D265R [35]	${E c t R N A}_{C U A}^{T y r}$ [35,77]	Method development [35,37,77] Protein engineering [43]
	EcTyrRS [35,37,43,77]		${B s t R N A}_{C U A}^{T y r}$ [37,43,77]	Method development [35,37,77] Protein engineering [43]
	EcTyrRS [77]	Y37V D182S F183M D265R	${E c t R N A}_{C U A}^{T y r}$	Method development
	EcTyrRS [77]	Y37V D182S F183M D265R	${B s t R N A}_{C U A}^{T y r}$	Method development
	EcTyrRS [77]	Y37V D182S F183M D265R	${E c t R N A}_{C U A}^{T y r}$	Method development
	EcTyrRS [77]	Y37V D182S F183M D265R	${B s t R N A}_{C U A}^{T y r}$	Method development
	EcTyrRS_CUA [16,17,35,37,38,43,49,55,68,77–96]	Y37L 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]	${E c t R N A}_{C U A}^{T y r}$ [35,77,78,81,86,90]	Bioorthogonal 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]
	EcTyrRS_CUA [16,17,35,37,38,43,49,55,68,77–96]		${B s t R N A}_{C U A}^{T y r}$ [16,17,37,38,43,49,55,68,77,79,80,82–85,87–96]
	EcTyrRS_UCA [25]	Y37V D182S F183M	${E c t R N A}_{U C A}^{T y r}$
	EcTyrRS_UCA [25]	Y37V D182S F183M	${B s t R N A}_{U C A}^{T y r}$
	EcTyrRS [35,97]	Y37I D182S F183M D265R [97] Y37S D182S F183A L186E D265R [35] Y37G D182S F183I L186E D265R [35] Y37S D182S F183I L186E D265R [35]	${E c t R N A}_{C U A}^{T y r}$ [35,97]	Method development [35] Spectroscopic probe [97]
	EcTyrRS [35,97]		${B s t R N A}_{C U A}^{T y r}$ [97]	Method development [35] Spectroscopic probe [97]
	MmPylRS [50]	L301M Y306L L309A C348F	${M m t R N A}_{C U A}^{P y l}$	Method development
	MmPylRS [98]	N346A C348A	${M m t R N A}_{C U A}^{P y l}$	Method development
	MmPylRS [98]	N346A C348A	${M m t R N A}_{C U A}^{P y l}$	Method development
	MmPylRS [98]	N346A C348A	${M m t R N A}_{C U A}^{P y l}$	Method development
	MmPylRS [98]	N346A C348A	${M m t R N A}_{C U A}^{P y l}$	Method development
	MmPylRS [98]	N346A C348A	${M m t R N A}_{C U A}^{P y l}$	Method development
	MmPylRS [98]	N346A C348A	${M m t R N A}_{C U A}^{P y l}$	Method development
	MmPylRS [98]	N346A C348A	${M m t R N A}_{C U A}^{P y l}$	Method development
	EcTyrRS_CUA [21,35,37,43,77,83,87,90,99,100]	Y37I 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]	${E c t R N A}_{C U A}^{T y r}$ [21,35,77,90]	Bioorthogonal labelling [83,87,100] Method development [25,35,37,77,83,90,99] Protein engineering [21,43]
	EcTyrRS_CUA [21,35,37,43,77,83,87,90,99,100]		${B s t R N A}_{C U A}^{T y r}$ [37,43,77,83,87,99,100]
	EcTyrRS_UCA [25]	Y37V D182S F183M	${E c t R N A}_{U C A}^{T y r}$
	EcTyrRS_UCA [25]	Y37V D182S F183M	${B s t R N A}_{U C A}^{T y r}$
	EcTyrRS [84,101]	Y37I D182G F183M L186A D265R	${B s t R N A}_{C U A}^{T y r}$	Chemical crosslinking [84,101] Method development [101]
	EcTyrRS [15,37,55,78,85,92,94,95,99,102–105]	Y37G D182G L186A D265R [78,85,103] Y37G D182G L186A [15,55,92,94,95,99,102,104,105] Y37G D182G F183Y L186M [37]	${E c t R N A}_{C U A}^{T y r}$ [15,78,103]	Mechanistic studies [15] Method development [37,78,95,99,103,106] Photocrosslinking [85,92,94,102,104,105] Photoinhibition [55]
	EcTyrRS [15,37,55,78,85,92,94,95,99,102–105]		${B s t R N A}_{C U A}^{T y r}$ [37,55,85,92,94,95,99,102,104,105]
	MmPylRS [106]	A302T N346T C348T W417C [106]	${M m t R N A}_{C U A}^{P y l}$ [106]
	MmPylRS [106]	A302T N346G C348T V401I W417Y	${M m t R N A}_{C U A}^{P y l}$	Method development
	EcLeuRS [107–109]	L38F M40G L41P Y499V Y500L Y527A H537E L538S F541C A560V	${E c t R N A}_{C U A}^{L e u}$	Method development [107,108] Spectroscopic probe [109]
	MmPylRS [110]	N346Q C348S V401G W417T	${M m t R N A}_{C U A}^{P y l}$	Spectroscopic Probe
	MbPylRS [111]	L270F L274M N311G C313G Y349F	${M b t R N A}_{C U A}^{P y l}$	Photoswitching
	MbPylRS [111]	L270F L274M N311G C313G Y349F	${M b t R N A}_{C U A}^{P y l}$	Photoswitching
	MbPylRS [111]	L270F L274M N311G C313G Y349F	${M b t R N A}_{C U A}^{P y l}$	Photoswitching
	MmPylRS [56,112]	A302T L309S N346V C348G	${M m t R N A}_{C U A}^{P y l}$	Method development [112] Photoswitching [56]
Histidine derivatives
	MaPylRS [26]	L121M L125I Y126F M129A V168F	${M a t R N A}_{C U A}^{P y l}$	Method development
	MbPylRS [113]	L270I Y271F L274G C313F Y349F	${M b t R N A}_{C U A}^{P y l}$	Method development
	MbPylRS [113]	L270I Y271F L274G C313F Y349F	${M b t R N A}_{C U A}^{P y l}$	Method development
Lysine derivatives
	MbPylRS [114]	L266M L270I Y271F L274A C313F	${M b t R N A}_{C U A}^{P y l}$	Method development
	MbPylRS [115–117]	D76G L266V L270I Y271F L274A C313F [115] D76G L266M L270I Y271F L274A C313F [116,117]	${M b t R N A}_{C U A}^{P y l}$	Method development [44,50,115,117,118] Spectroscopic probe [116]
	MmPylRS [44,50,118]	L305I Y306F L309A C348F [118] L301M Y306L L309A C348F [44,50]	${M m t R N A}_{C U A}^{P y l}$ [50,118] ${M m t R N A}_{U U A}^{P y l}$ [44]
	MmPylRS [50]	L301M Y306L L309A C348F	${M m t R N A}_{C U A}^{P y l}$	Method development
	MbPylRS [119]	D76G L266M L270I Y271F L274A C313F	${M b t R N A}_{C U A}^{P y l}$	Method development
	MbPylRS [77,120]	L274A C313F Y349F [120] wt [77]	${M b t R N A}_{C U A}^{P y l}$	Method development
	MbPylRS [121]	Y271M L274A C313A	${M b t R N A}_{C U A}^{P y l}$	Photocrosslinking
	MmPylRS [122]	Y306V L309A C348F Y384F	${M m t R N A}_{C U A}^{P y l}$	Photocrosslinking
	MaPylRS [26]	wt	${M a t R N A}_{C U A}^{P y l}$	Method development [21,22,25,26,44,47,69,73,77,106,115,117,118,123–130]
	MbPylRS [21,25,44,125,129,130]	wt	${M b t R N A}_{C U A}^{P y l}$ [25,44,125,129,130] ${M b t R N A}_{U U A}^{P y l}$ [44] ${M b t R N A}_{U C A}^{P y l}$ [25,44]
	MmPylRS [22,26,44,47,69,73,77,106,115,117,118,123,124,126–128]	wt	${M m t R N A}_{C U A}^{P y l}$ [21,22,26,47,69,73,77,106,115,118,123,124,126,127] ${M m t R N A}_{C U A}^{P y l} B U 25 C B$ [22,117,128] ${M m t R N A}_{U U A}^{P y l}$ [21,44] ${M m t R N A}_{U C A}^{P y l}$ [21]
	MbPylRS [21,24,25,48,77,131–137]	Wt [21,24,25,48,77,132–136] L274A C313S Y349F [131] Y349F [137]	${M b t R N A}_{C U A}^{P y l}$ [25,77,131] ${M b t R N A}_{U C A}^{P y l}$ [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 [127]	wt	${M m t R N A}_{C U A}^{P y l}$ [127,137] ${M m t R N A}_{U U A}^{P y l}$ [21]
	MbPylRS [57,69,77,138,139]	wt	${M b t R N A}_{C U A}^{P y l}$	Bioorthogonal labelling [131,139] Chemical decaging [57] Imaging [69,129,138] Method development [69,77]
	MmPylRS [69,129,131]	wt	${M m t R N A}_{C U A}^{P y l}$
	MbPylRS [25,130]	wt	${M b t R N A}_{C U A}^{P y l}$ [25]	Bioorthogonal labelling [130] Method development [25]
	MmPylRS [130]	wt	${M m t R N A}_{C U A}^{P y l}$	Bioorthogonal labelling [130] Method development [25]
	MbPylRS [131,140]	L274A C313S Y349F	${M b t R N A}_{C U A}^{P y l}$	Bioorthogonal labelling
	MmPylRS [141]	wt	${M m t R N A}_{C U A}^{P y l}$	Method development
	MbPylRS [140]	wt	${M b t R N A}_{C U A}^{P y l}$	Method development
	MmPylRS [118,142,143]	R61K G131E L309A C348V Y384F [118] Y306A Y384F [142] R61K G131E Y306A Y384F [143]	${M m t R N A}_{C U A}^{P y l}$ [118,142] ${M m t R N A}_{C U A}^{P y l} B U 25 C$ [143]	Method development [118,142,143]
	MbPylRS [140]	Y271I L274A C313A Y349F	${M b t R N A}_{C U A}^{P y l}$	Method development [140,141] Photoactivation [61,144]
	MmPylRS [61,141,144]	Y306M L309A C348A Y384F	${M m t R N A}_{C U A}^{P y l}$	Method development [140,141] Photoactivation [61,144]
	MbPylRS [145]	Y271M L274T C313A Y349F	${M b t R N A}_{C U A}^{P y l}$	Method development
	MbPylRS [146]	Y271I 274M C313A	${M b t R N A}_{C U A}^{P y l}$	Method development
	MbPylRS [63]	Y271A Y349F	${M b t R N A}_{C U A}^{P y l}$	Chemical decaging
	MbPylRS [62]	L274A C313S Y349F	${M b t R N A}_{C U A}^{P y l}$	Bioorthogonal labelling Chemical decaging
	MbPylRS [66,67,69,75,125,147–152]	M241F A267S Y271C L274M [66,67,69,75,125,147–152]	${M b t R N A}_{C U A}^{P y l}$ [66,67,69,75,125,147–152] ${M b t R N A}_{C U A}^{P y l} U 25 C$ [66]	Method development [69] Photoactivation [66,67,75,125,147–152]
	MbPylRS [153]	Y271A L274M	${M b t R N A}_{C U A}^{P y l}$	Photoactivation
	MbPylRS [153]	Y271A L274M	${M b t R N A}_{C U A}^{P y l}$	Photoactivation
	MbPylRS [153]	Y271A L274M	${M b t R N A}_{C U A}^{P y l}$	Method development
	MbPylRS [154]	L266M L270I Y271L L274A C313	${M b t R N A}_{C U A}^{P y l}$	Method development
	MbPylRS [24,25]	wt	${M b t R N A}_{C U A}^{P y l}$ [25] ${M b t R N A}_{U C A}^{P y l}$ [24]	Imaging [123] Method development [22,24,25,69,115,155,156]
	MmPylRS [22,69,115,123,155,156]	Wt [22,69,115,123,155,156] Y306A Y384F [155]	${M m t R N A}_{C U A}^{P y l} B U 25 C B$ [22,155] ${M m t R N A}_{C U A}^{P y l}$ [69,115,123,156]
	Mx1201PylRS [155]	wt	${M x 1201 t R N A}_{C U A}^{P y l}$ ${M x 1201 t R N A}_{C U A}^{P y l}$ C41CA
	MmPylRS [124]	wt	${M m t R N A}_{C U A}^{P y l}$	Bioorthogonal labelling
	MbPylRS [77,157,158]	Wt [77,158] L274M 313A Y349F [157]	${M b t R N A}_{C U A}^{P y l}$	Method development [77,155,159] Photocrosslinking [157,158]
	MmPylRS [155,159]	Y306A Y384F	${M m t R N A}_{C U A}^{P y l}$ [159] ${M m t R N A}_{C U A}^{P y l}$ U25C [155]	Method development [77,155,159] Photocrosslinking [157,158]
	MmPylRS [159]	Y306A Y384F	${M m t R N A}_{C U A}^{P y l}$	Method development
	MbPylRS [132,140,160]	L274A C313S Y349F	${M b t R N A}_{C U A}^{P y l}$	Method development [140] Photocrosslinking [132,160] Protein engineering [132]
	MmPylRS [159]	Y306A Y384F	${M m t R N A}_{C U A}^{P y l}$	Method development
	MmPylRS [142]	Y306A Y384F	${M m t R N A}_{C U A}^{P y l}$	Photocrosslinking
	MmPylRS [143]	R61K G131E Y306A Y384F	${M m t R N A}_{C U A}^{P y l} B U 25 C$	Photocrosslinking
	MmPylRS [18,39,59–61,123,155,161–166]	Y306A Y384F [18,39,59–61,123,155,161–166]	${M m t R N A}_{C U A}^{P y l}$ [18,39,59–61,123,161–166] ${M m t R N A}_{C U A}^{P y l} B U 25 C B$ [155]	Imaging [123,161,162,164,166] Chemical decaging [18,59–61] Chemical crosslinking [163] Method development [155,165] Protein labelling [39]
	Mx1201PylRS [155]	Y126A	${M x 1201 t R N A}_{C U A}^{P y l}$
	MbPylRS [123,167,168]	Y271A L274M C313A	${M b t R N A}_{C U A}^{P y l}$	Bioorthogonal labelling [124,167] Imaging [123,168] Method development [169]
	MmPylRS [124,169]	Y306A Y384F [169] Y306A L309M C348A [124]	${M m t R N A}_{C U A}^{P y l}$
	MmPylRS [165]	Y306A Y384F	${M m t R N A}_{C U A}^{P y l}$	Method development
	MbPylRS [167,168]	Y271A L274M C313A	${M b t R N A}_{C U A}^{P y l}$	Bioorthogonal labelling [167] Method development [167,169]
	MmPylRS [169]	Y306A Y384F	${M m t R N A}_{C U A}^{P y l}$	Bioorthogonal labelling [167] Method development [167,169]
	MbPylRS [24]	wt	${M b t R N A}_{U C A}^{P y l}$	Bioorthogonal labelling [39,127,131] Method development [24,169]
	MmPylRS [39,127,131,169]	Wt [127,131] Y306A Y384F [39,169]	${M m t R N A}_{C U A}^{P y l}$
	MmPylRS [169]	Y306A Y384F	${M m t R N A}_{C U A}^{P y l}$	Method development
	MmPylRS [39,161,166,169,170]	Y306A Y384F	${M m t R N A}_{C U A}^{P y l}$	Bioorthogonal labelling [39] Imaging [161,166,170] Method development [169]
	MbPylRS [19,64,140,168,171,172]	Y271M L274G C313A [19,64,168,171,172] M241F A267S Y271C L274M [140]	${M b t R N A}_{C U A}^{P y l}$ [64,140,168,171,172] ${M b t R N A}_{C U A}^{P y l} U 25 C$ [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 [39,123,128,131,155,159,161,165–167]	Y306A 384F [39,123,128,131,155,159,161, 165–167]	${M m t R N A}_{C U A}^{P y l}$ [39,123,128,131,159,161, 165–167] ${M m t R N A}_{C U A}^{P y l} B U 25 C$ [155]
Tryptophan derivatives
	EcTrpRS [34]	S8A V144S V146A S8A V144G V146C	${E c t R N A}_{C U A}^{T r p}$	Method development
	EcTrpRS [34]	S8A V144S V146A	${E c t R N A}_{C U A}^{T r p}$	Method development
	EcTrpRS [34]	S8A V144G V146C	${E c t R N A}_{C U A}^{T r p}$	Method development
	EcTrpRS [34]	S8A V144G V146C	${E c t R N A}_{C U A}^{T r p}$	Method development
	EcTrpRS [34]	S8A V144G V146C	${E c t R N A}_{C U A}^{T r p}$	Method development
Tyrosine derivatives
	EcTyrRS [46]	Y37V Q195C	${B s t R N A}_{C U A}^{T y r}$	Method development
	EcTyrRS_CUA [15,21,35,37,77,78,90]	Y37T 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]	${E c t R N A}_{C U A}^{T y r}$ [15,21,35,77,78,90]	Mechanistic studies [15] Method development [21,25,35,37,77,78,90]
	EcTyrRS_CUA [15,21,35,37,77,78,90]		${B s t R N A}_{C U A}^{T y r}$ [21,37,77,90]
	EcTyrRS_UCA [25]	Y37V D182S F183M	${E c t R N A}_{U C A}^{T y r}$
	EcTyrRS_UCA [25]	Y37V D182S F183M	${B s t R N A}_{U C A}^{T y r}$
	EcTyrRS [77]	Y37V D182S F183M D265R	${E c t R N A}_{C U A}^{T y r}$	Method development
	EcTyrRS [77]	Y37V D182S F183M D265R	${B s t R N A}_{C U A}^{T y r}$	Method development
	EcTyrRS [25,35,37,77]	Y37V D182S F183M D265R [77] Y37S D182T F183M L186V [37] Y37V D182S F183M [25] Y37V D165G D182S F183M L186A D265R [35]	${E c t R N A}_{C U A}^{T y r}$ [25,35,77]	Method development
	EcTyrRS [25,35,37,77]		${B s t R N A}_{C U A}^{T y r}$ [37,77]	Method development
	MbPylRS [126,148,173]	L270F L274M N311G C313G Y349F [173] L270F L274M N311G C313G [126,148]	${M b t R N A}_{C U A}^{P y l}$	Photoactivation
	MbPylRS [173]	L270F L274M N311G C313G Y349F	${M b t R N A}_{C U A}^{P y l}$	Photoactivation
	MbPylRS [173]	L270F L274M N311G C313G Y349F	${M b t R N A}_{C U A}^{P y l}$	Photoactivation
	MbPylRS [173]	L270F L274M N311G C313G Y349F	${M b t R N A}_{C U A}^{P y l}$	Photoactivation
	MmPylRS [174]	N346T C348I Y384L W417K	${M m t R N A}_{C U A}^{P y l}$	Bioorthogonal labelling
	EcTyrRS [35]	Y37V D182S F183M D265R Y37V D165G D182S F183M L186A D265R	${E c t R N A}_{C U A}^{T y r}$	Method development
Miscellaneous unnatural amino acids
	EcLeuRS [24,25]	M40I T252A Y499I Y527A H529G [24] E20K M40V L41S T252R Y499S Y527L H529G H537G [25]	${E c t R N A}_{C U A}^{L e u}$	Method development
	EcLeuRS [15,103,175]	M40A L41N T252A Y499I Y527G H537T	${E c t R N A}_{C U A}^{L e u}$	Mechanistic studies [15,103] Method development [175]
	MmSepRS [176]	wt	${M j t R N A}_{C U A}^{C y s}$	Method development

Method development includes demonstration of incorporation, optimisation of incorporation efficiency, application as a control substrate, proof-of-principle of a technique for subsequent studies etc. Abbreviations: Ma, Methanomethylophilus alvus; wt, wild type.

Many engineered E. coli aaRS/tRNA pairs have also been used as orthogonal pairs in mammalian cells. The most successful ones are the E. coli tyrosine, leucine and tryptophan pairs [33]. However, as all these synthetases naturally recognise a canonical amino acid, it is necessary to abolish their natural activity towards the canonical amino acid and to recognise only the designated unnatural amino acid. As it is technically difficult to perform directed evolution in mammalian cells due to low efficiency in transfection and screening, synthetase engineering is normally carried out in E. coli [34,35] or yeast [15,36,37] so that large mutant libraries can be easily screened. It is also necessary to modify the E. coli tRNA so that it decodes a blank codon instead of a codon corresponding to a canonical amino acid.

Based on the simplicity of the established methodology [38–40] and the promiscuity of many orthogonal synthetases towards different unnatural amino acids (vide infra) [41], the number of genetically incorporable unnatural amino acids has steadily increased. In addition, some orthogonal aaRS/tRNA pairs are mutually orthogonal [21,24–26,42] and can be used at the same time to incorporate multiple different unnatural amino acids into a protein of interest.

As recent reviews cover fundamental aspects of genetic code expansion [4,6,9,13], the engineering of new orthogonal synthetases [8], and general [5,10,11,14] or specific [7,12] applications of genetic code expansion in eukaryotic systems, we will focus on recent advances and applications of genetic code expansion for controlling protein function in mammalian cells through translational, optical or chemical means.

Translational control by amber suppression

Genetic code expansion by unnatural amino acid incorporation in response to an amber stop codon provides the simplest way to ‘switch on’ protein production. In this case, an amber stop codon is placed into the gene of interest (Figure 3) for incorporation of the unnatural amino acid into a permissive site of the target protein [43]. In the absence of the designated unnatural amino acid, protein translation stops prematurely at the amber stop codon, generating truncated and non-functional protein product and thus giving an effect as a nonsense mutation (Figure 3A). In the presence of the unnatural amino acid, the orthogonal tRNA is acylated and decodes the amber codon, leading to amber suppression and generation of full-length, functional protein product (Figure 3B). Thus, simple addition of the unnatural amino acid into the growth medium ‘switches on’ the protein production and function [44]. In contrast with commonly used systems for inducible mammalian protein expression (e.g. the tetracycline transcriptional transactivation) [45], the lag time is shorter in the translational control by amber suppression (i.e. time for translation and folding) than the gene activation approaches (i.e. time for transcription, mRNA processing, translation and folding). Amber suppression also allows a more stringent control, as the background activity (if any) can be further minimised by including multiple amber codons into the gene of interest. The translational control by amber suppression approach is fully complementary to conventional genetic approaches (e.g. knockout, knockdown) that deplete a protein in cells to ‘switch off’ its function. In addition, the unnatural amino acid approach is reversible, as removing the unnatural amino acid in the growth medium will ‘switch off’ the translation of the protein of interest.

Use of amber suppression to switch on protein production

Figure 3

(A) Absence of the unnatural amino acid leads to recognition of the UAG codon for translation termination. (B) Addition of the unnatural amino acid leads to amber suppression and successful production of the full-length and functional protein.

Use of amber suppression to switch on protein production

(A) Absence of the unnatural amino acid leads to recognition of the UAG codon for translation termination. (B) Addition of the unnatural amino acid leads to amber suppression and successful production of the full-length and functional protein.

The translational switch-on process has been widely employed as a reporter system to test incorporation of new unnatural amino acids by using luminescent proteins like green fluorescent protein [46] or luciferase [20]. Upon successful incorporation, cells can emit light, whose intensity directly correlates to the unnatural amino acid incorporation efficiency. Apart from the reporter strategy, this approach has also been used to regulate function of other proteins, such as Cas9 for controllable gene editing in mouse embryos [47].

Besides the general use of the ‘translational activation’ approach to study protein function, this principle has been proven to be powerful in controlling virus replication [43,48,49]. By introducing TAG codons within the virus genes, viruses can only be generated using cell lines containing an orthogonal synthetase/tRNA_CUA pair, and the resulting viruses are replication-incompetent in normal cells due to the lack of amber suppressor tRNA (Figure 4A) [43,48]. Such replication-incompetent viruses offer an additional tier of control for live-attenuated vaccines and significantly increase their safety. This concept has been further developed by including the genes encoding the orthogonal aaRS/tRNA pair into the viral genome (Figure 4B) [49]. In this case, viruses can be replicated in wild-type cells and the native hosts, as long as the unnatural amino acid is supplemented. Here, spatial control can also be achieved by local administration of the unnatural amino acid as demonstrated in examples of mice with an expanded genetic code [50,51]. Thus, the approach can be used for controlling viral vectors in gene therapy, where spatiotemporal virus replication and gene editing are highly desirable.

Translational activation approaches to control virus replication

Figure 4

(A) Use of genetic code expansion to control replication of an amber codon tagged virus within transgenic host cells containing the orthogonal tRNA/synthetase pairs [43,48]. (B) Use of genetic code expansion to control replication of an amber codon tagged virus within normal host cells with the orthogonal tRNA/synthetase pair gene encoded by the viral genome [49].

Translational activation approaches to control virus replication

(A) Use of genetic code expansion to control replication of an amber codon tagged virus within transgenic host cells containing the orthogonal tRNA/synthetase pairs [43,48]. (B) Use of genetic code expansion to control replication of an amber codon tagged virus within normal host cells with the orthogonal tRNA/synthetase pair gene encoded by the viral genome [49].

While the translational control approach is quite simple, the response is not instantaneous. There is always a lag time from when the unnatural amino acid is administered into the culture medium until the full-length protein is produced and folded. Similarly, depleting the unnatural amino acid in the growth medium will stop production of new proteins, but the protein function will only be completely switched off when all previously produced proteins are degraded in the cells. Thus, the kinetics of the switching off process largely depend on the half-life of the protein, so the response rate is the same as with genetic knockdown.

Light-induced activation or inhibition

The slower kinetics of the translational control approach limit its applicability to study biological processes where fast response is needed. This can be addressed by using light to unmask or modify unnatural amino acids and subsequently regulate protein function. Depending on the nature of the light-responsive group, it is possible to either activate, inhibit, or reversibly switch on/off protein function (Table 2). Unnatural amino acids containing a photocage (i.e. a photolabile protecting group) [52] have been widely used for protein activation. When replacing a functionally critical amino acid residue with the corresponding photocaged amino acid, the target protein becomes inactive; upon light irradiation, the photocage is removed, thereby restoring the protein’s function. To date, photocaged cysteines (17–21), lysines (67, 72–74) and tyrosines (102–105) have been used to control enzyme function, intein splicing, protein subcellular localisation, virus–host interactions, and cell signalling cascades [13]. The light-activation approach is particularly useful for kinetic studies as it provides extreme spatiotemporal resolution. Spatial control can be achieved to even subcellular locations using focused light beams, which is virtually impossible when using the translational control approach. Theoretically, it is also possible to incorporate photocaged serine in mammalian cells as it has been demonstrated in yeast [53]. Therefore, the light-activation approach is applicable to regulate any protein that has a functionally critical cysteine, lysine, tyrosine, or serine residue in mammalian cells, including but not limited to kinases, DNA- and RNA-binding proteins, proteases, phosphatases, oxidoreductases, isomerases, and ubiquitin-modifying enzymes [54].

Table 2

Overview of photocaged unnatural amino acids that have been incorporated in mammalian cell systems for activating protein function upon irradiation with light of specific wavelength λ

System	Proteins	λ_decag (nm)
HEK293T	eGFP, potassium channel Kir2.1 [71]	385
	eGFP [72]	Long wavelength UV
	eGFP, mCherry, Npu DnaE intein, Src kinase [72]	Long wavelength UV
	TEV protease [73,74], Npu DnaE intein [74]	365
	sfGFP, luciferase [75]	365
HEK 293T	sfGFP, luciferase [75]	365
HEK293 [67,125] HEK293T [66,75,147–150,152] HEK293ET [151] HeLa [150,152]	Nuclear localisation peptide for subcellular localisation of SATB1 and FOXO3 transcription factors, and TEV protease [149]	350
	sfGFP, luciferase [75]	365
	p53 transcription factor [125]
	Isocitrate dehydrogenase [67]
	Cas9 endonuclease [150]
	Cre recombinase [148]
	Capsid of adeno-associated virus 2 [147]
	T7RNA polymerase [152]
	MEK1 kinase [151]
	LCK kinase [66]	405
HEK293T	Luciferase [144]	365
HEK293T or CHO K1	eGFP and luciferase [153]	365 405
HEK293T or CHO K1	eGFP and luciferase [153]	365 405 760¹
HEK293 [126] HEK293T [148,173]	Cre recombinase [148]STAT1transcription factor [126]Luciferase [173]	365
HEK293T	luciferase, TEV protease [173]

System	Proteins	λ_decag (nm)
HEK293T	eGFP, potassium channel Kir2.1 [71]	385
	eGFP [72]	Long wavelength UV
	eGFP, mCherry, Npu DnaE intein, Src kinase [72]	Long wavelength UV
	TEV protease [73,74], Npu DnaE intein [74]	365
	sfGFP, luciferase [75]	365
HEK 293T	sfGFP, luciferase [75]	365
HEK293 [67,125] HEK293T [66,75,147–150,152] HEK293ET [151] HeLa [150,152]	Nuclear localisation peptide for subcellular localisation of SATB1 and FOXO3 transcription factors, and TEV protease [149]	350
	sfGFP, luciferase [75]	365
	p53 transcription factor [125]
	Isocitrate dehydrogenase [67]
	Cas9 endonuclease [150]
	Cre recombinase [148]
	Capsid of adeno-associated virus 2 [147]
	T7RNA polymerase [152]
	MEK1 kinase [151]
	LCK kinase [66]	405
HEK293T	Luciferase [144]	365
HEK293T or CHO K1	eGFP and luciferase [153]	365 405
HEK293T or CHO K1	eGFP and luciferase [153]	365 405 760¹
HEK293 [126] HEK293T [148,173]	Cre recombinase [148]STAT1transcription factor [126]Luciferase [173]	365
HEK293T	luciferase, TEV protease [173]

1

Decaging is only achieved at this wavelength via a two-photon activation using a specialised multiphoton laser setup. Abbreviation: Npu, Nostoc punctiforme.

In contrast, the incorporation of a photocrosslinking amino acid can be used to inhibit protein function upon light irradiation [55]. In this case, a photocrosslinking amino acid is placed in the interior of the target protein. Upon light irradiation, a highly reactive functionality (e.g. radical, nitrene, carbene) is generated and reacted non-specifically with a nearby amino acid residue, causing cross-linking of the protein and subsequent abolishment of the protein’s activity. The feasibility of this approach has been demonstrated with the use of p-benzoylphenylalanine (41) in the study of glutamate receptors, GluA1 and GluA2 [55]. When compared with the use of a photocaged amino acid, inhibiting a protein by photocrosslinking does not rely on the existence of a functionally critical residue, and thus theoretically, it can be used to investigate any protein in mammalian cells. Nevertheless, for each protein target it is necessary to screen a suitable site for placing the photocrosslinking amino acid. Protein variants containing the photocrosslinking amino acid must (i) behave in the same way as the wild-type protein (i.e. phenotypically silent) before light irradiation; and (ii) be fully inhibited after light irradiation causing the photocrosslinking. Due to these criteria, the screening process can be laborious and time-consuming.

In addition to light-induced activation and inhibition, reversible regulation of a protein function can be achieved through incorporation of a photoswitchable amino acid (Table 3). For example, 48, containing an azobenzene functionality which undergoes reversible cis-trans isomerisation upon irradiation with blue and UV light, has been used to control the activity of a glutamate receptor [56]. However, the general applicability of this approach suffers from similar constraints as inhibition by photocrosslinking. Extensive screening is often needed to identify a suitable site for incorporation, such that the resulting protein variant is fully active or inactive upon irradiation with light of a specific wavelength. At the current state of the art, there is no guarantee that such a site can be found in the target protein.

Table 3

Overview of photoswitchable unnatural amino acids that have been incorporated in mammalian cell systems used to modulate protein function upon irradiation with the given wavelengths λ

System	Proteins	λ_trans-cis (nm)	λ_cis-trans (nm)
HEK 293T	Luciferase [111]	355	450
		530	405
		530	405
	NMDAR glutamate receptor [56]	365	460

System	Proteins	λ_trans-cis (nm)	λ_cis-trans (nm)
HEK 293T	Luciferase [111]	355	450
		530	405
		530	405
	NMDAR glutamate receptor [56]	365	460

Overall, the use of light-responsive amino acids offers superior temporal control of protein function as the response is significantly faster (seconds) than the translational control by amber suppression (minutes to hours). Additionally, spatial control can be achieved at subcellular level, which is not possible with the translational approach. Generally, UV light at approximately 360 nm (i.e. UVA) is required (Tables 2 and 3) to induce the change (i.e. decaging, cross-linking, or isomerisation). However, UVA light has been shown to alter cellular signalling processes [57] or influence proper cellular function, if high intensity irradiation is applied (i.e. 50 J.cm⁻²) [58]. Though not necessarily problematic, this has to be considered when planning to apply light-responsive unnatural amino acids. Thus, there is a trend to develop new functionalities that can be modulated by light of higher wavelengths [52]. In particular, red and near-infrared light (650–750 nm) are appealing because they cause no harm to cells even under excessive exposure, and they can penetrate tissues for in vivo applications. To date, coumarin-caged lysines (73 and 74) are the only genetically incorporable unnatural amino acids that can be decaged within these wavelengths, although by two-photon approach that requires a specialised multiphoton laser setup [52]. Nevertheless, with the continuous advances in light-responsive chemical functionalities and orthogonal aaRS engineering, it is expected that more unnatural amino acids with the desired photophysical properties can be incorporated through genetic code expansion.

Small-molecule induced activation or inhibition

In addition to light, small molecules can also be used to unmask or modify unnatural amino acids and subsequently regulate protein function with prompt response. For example, several protecting groups can be removed bioorthogonally inside live mammalian cells, and these chemistries have been used to switch on protein function by genetic code expansion. Intracellular bioorthogonal reactions that have been used in this purpose include inverse electron demand Diels–Alder reactions [18,59–61], 1,3-dipolar cycloadditions [62], Staudinger reactions [63], and palladium-catalysed propargyl removal (Table 4) [57]. Currently, all of these have only been demonstrated in caged lysine molecules (61, 70, 71, 85) through a number of examples, including activation of luciferases, kinases, nucleases etc. Theoretically, all these protecting groups can be applied to other nucleophilic amino acids (e.g. cysteine, serine, threonine, tyrosine) subjected to successful engineering of the corresponding orthogonal synthetases.

Table 4

Overview of bioorthogonally protected unnatural amino acids tested in mammalian cell systems and their deprotection conditions

System	Proteins	Reaction	Reagent
HeLa, CHO, HEK293T, IH3T3, Caco-2, A549, HeLa	GFP, OspF phosphothreonine lyase	Pd-catalysed Tsuji–Trost-like reaction	Pd(II) complexes [57]
HEK293T	eGFP, SATB1 transcription factor, Cre recombinase, Cas9 endonuclease	Staudinger	Various phosphines [63]
HEK293T	eGFR, luciferase, OspF phosphothreonine lyase, Src kinase	1,3-dipolar cycloaddition	trans-cyclooctenes [62]
HEK293T	GFP [61], Luciferase [59,61], MEK1 [18,60] and MEK2 [18] and FAK [60] and Src [60] kinases	Inverse electron demand Diels–Alder reactions	Tetrazines [18,59–61]

System	Proteins	Reaction	Reagent
HeLa, CHO, HEK293T, IH3T3, Caco-2, A549, HeLa	GFP, OspF phosphothreonine lyase	Pd-catalysed Tsuji–Trost-like reaction	Pd(II) complexes [57]
HEK293T	eGFP, SATB1 transcription factor, Cre recombinase, Cas9 endonuclease	Staudinger	Various phosphines [63]
HEK293T	eGFR, luciferase, OspF phosphothreonine lyase, Src kinase	1,3-dipolar cycloaddition	trans-cyclooctenes [62]
HEK293T	GFP [61], Luciferase [59,61], MEK1 [18,60] and MEK2 [18] and FAK [60] and Src [60] kinases	Inverse electron demand Diels–Alder reactions	Tetrazines [18,59–61]

On the other hand, bioorthogonal amino acids (e.g. 77 and 92, Table 5) have been used for rapid and selective inhibition of a specific enzyme in live mammalian cells [64]. In this case, a bioorthogonal amino acid is placed into the target enzyme without affecting the enzyme function. Upon contact with an inhibitor conjugate bearing the complementary bioorthogonal group, the enzyme variant is tethered to the conjugate and thus the enzyme activity is inhibited (Figure 5). The inhibition is exquisitely selective and can even discriminate between isoforms that differ by a single amino acid residue. Using this approach, selective inhibition of an intracellular kinase for which no selective small-molecule inhibitor exists was achieved. In addition, placing a photoswitchable moiety (i.e. azobenzene) into the inhibitor conjugate enables reversible modulation of enzyme activity by light.

Table 5

Overview of bioorthogonally reactive unnatural amino acids that have been incorporated in mammalian cell systems used to deactivate protein function upon reaction with the specified reagents

Amino acid		System	Proteins	Reaction	Reagent
		HEK293T	MEK1 and LCK kinases	inverse electron demand Diels–Alder reactions	Inhibitor–tetrazine conjugates [64]
		HEK293T	MEK1 and MEK2 kinases	inverse electron demand Diels–Alder reactions	Inhibitor–tetrazine conjugates [64]

Amino acid		System	Proteins	Reaction	Reagent
		HEK293T	MEK1 and LCK kinases	inverse electron demand Diels–Alder reactions	Inhibitor–tetrazine conjugates [64]
		HEK293T	MEK1 and MEK2 kinases	inverse electron demand Diels–Alder reactions	Inhibitor–tetrazine conjugates [64]

Example of the small-molecule approach to protein inhibition

Figure 5

Use of unnatural amino acid incorporation for selective inhibition of protein function by bioorthogonal tethering [64].

https://doi.org/10.1021/acs.jmedchem.5b00760

Example of the small-molecule approach to protein inhibition

Use of unnatural amino acid incorporation for selective inhibition of protein function by bioorthogonal tethering [64].

In comparison with light-induced activation or inhibition, small molecules can be used to activate or inhibit the target protein in deep animal tissue or intact animals which are not easily accessible by light. However, as mentioned above, only a few reactions have so far been shown to be bioorthogonal with high reaction rates to allow fast response [65]. The extension of this methodology is therefore tied to the development of novel bioorthogonal reactions. In contrast to the use of light for activation or inhibition, the small-molecule approach, similar to the translational approach, only allows spatial control by using cell-compartment selective compounds or reactions, or local injections.

Conclusion

Genetic code expansion has matured into a technique that can be routinely used in mammalian systems. Controlling protein function is currently mostly achieved by translation, light, and small molecules. These methods have been summarised and discussed, and their features have been compared (Table 6). Translational control is arguably the easiest to perform but suffers from the longer response time (up to several hours). On the other hand, both light and small-molecule induced methods have a faster response (seconds to minutes). The light approach is particularly appealing where subcellular spatial resolution is needed. While all three approaches have shown promise, most of the reviewed applications are so far proof-of-principle studies. The dissemination of this technique could be enhanced by the community simplifying access to plasmids (e.g. through plasmid repository), standardising the reporting format of aaRS mutants with full sequencing information, and providing protocols with extensive details. Only general implementation of protein control by genetic code expansion in the wider scientific community to unravel new biological insights will truly demonstrate the power of these techniques [55,56,66–68]. Since genetic code expansion has also been recently demonstrated in mice [47,50,51,60,69,70], we foresee that optimisation will be tailored to target cells, tissues, and mammalian models and many of the aforementioned approaches will be applied in vivo, providing the complete native environment to study function of mammalian proteins. With further promotion and adaptation of genetic code expansion, this is to be expected.

Table 6

Comparison of protein function control approaches currently enabled by genetic code expansion

Approach	Temporal Control	Spatial control	Reversibility
Translational control	Yes, slow	Only by local administration	Yes, but high lag time
Optical control	Yes, fast	Yes, very high (to subcellular levels)	Yes, for photoswitchable amino acids
Chemical control	Yes, medium	Only by local administration	Yet to be established

Approach	Temporal Control	Spatial control	Reversibility
Translational control	Yes, slow	Only by local administration	Yes, but high lag time
Optical control	Yes, fast	Yes, very high (to subcellular levels)	Yes, for photoswitchable amino acids
Chemical control	Yes, medium	Only by local administration	Yet to be established

Summary

Genetic code expansion can now be routinely used for incorporation of more than 100 different unnatural amino acids in mammalian cells using only four orthogonal aaRS/tRNA pairs and their mutants as shown in Table 1.
Protein function can be temporally regulated (activation or inhibition) by simple translational control, i.e. supplementation of the desired unnatural amino acid to allow full-length, functional protein production.
More rapid control can be achieved by incorporating stimuli responsive amino acids which allow activation, inhibition, or reversible regulation of protein function by light or small molecules.
Most of the approaches have been demonstrated in proof-of-principle studies, but are ready for adaptation by the broader scientific community.

Funding

This work was supported by the BBSRC [grant number BB/P009506/1 (to Y.-H.T.)]; and the Wellcome Trust [grant numbers 202056/Z/16/Z (to L.Y.P.L.), 200730/Z/16/Z (to Y.-H.T.)]. The funders play no role in design, decision to publish, or preparation of this manuscript.

Competing interests

The authors declare that there are no competing interests associated with the manuscript.

Abbreviations

aaRS: aminoacyl-tRNA synthetase
Ec: Escherichia coli
Mx: Methanomethylophilus alvus Mx1201
PylRS: Pyrrolysyl-tRNA synthetase

References

1.

McDonnell

D.P.

,

Wardell

S.E.

and

Norris

J.D.

(

2015

)

Oral selective estrogen receptor downregulators (SERDs), a breakthrough endocrine therapy for breast cancer

.

J. Med. Chem.

58

,

4883

–

4887

[PubMed]

https://doi.org/10.1038/mt.2015.214

2.

Petta

I.

,

Lievens

S.

,

Libert

C.

,

Tavernier

J.

and

De Bosscher

K.

(

2016

)

Modulation of protein-protein interactions for the development of novel therapeutics

.

Mol. Ther.

24

,

707

–

718

[PubMed]

https://doi.org/10.1042/EBC20170052

3.

van Montfort

R.L.M.

and

Workman

P.

(

2017

)

Structure-based drug design: aiming for a perfect fit

.

Essays Biochem

61

,

431

–

437

[PubMed]

https://doi.org/10.1016/j.bbapap.2014.03.002

4.

Wan

W.

,

Tharp

J.M.

and

Liu

W.R.

(

2014

)

Pyrrolysyl-tRNA synthetase: an ordinary enzyme but an outstanding genetic code expansion tool

.

Biochim. Biophys. Acta

1844

,

1059

–

1070

[PubMed]

https://doi.org/10.1039/C4SC01534G

5.

Dumas

A.

,

Lercher

L.

,

Spicer

C.D.

and

Davis

B.G.

(

2015

)

Designing logical codon reassignment - Expanding the chemistry in biology

.

Chem. Sci.

6

,

50

–

69

[PubMed]

https://doi.org/10.1007/978-1-4939-2845-3_7

6.

Leisle

L.

,

Valiyaveetil

F.

,

Mehl

R.A.

and

Ahern

C.A.

(

2015

)

Incorporation of non-canonical amino acids

.

Adv. Exp. Med. Biol.

869

,

119

–

151

[PubMed]

7.

Neumann-Staubitz

P.

and

Neumann

H.

(

2016

)

The use of unnatural amino acids to study and engineer protein function

.

Curr. Opin. Chem. Biol.

38

,

119

–

128

https://doi.org/10.1021/acs.accounts.7b00376

8.

Wang

L.

(

2017

)

Engineering the genetic code in cells and animals: biological considerations and impacts

.

Acc. Chem. Res.

50

,

2767

–

2775

[PubMed]

https://doi.org/10.1042/BST20160336

9.

Italia

J.S.

,

Zheng

Y.

,

Kelemen

R.E.

,

Erickson

S.B.

,

Addy

P.S.

and

Chatterjee

A.

(

2017

)

Expanding the genetic code of mammalian cells

.

Biochem. Soc. Trans.

45

,

555

–

562

[PubMed]

https://doi.org/10.1017/S0033583517000051

10.

Debelouchina

G.T.

and

Muir

T.W.

(

2017

)

A molecular engineering toolbox for the structural biologist

.

Q. Rev. Biophys.

50

,

e7

[PubMed]

https://doi.org/10.1038/nature24031

11.

Chin

J.W.

(

2017

)

Expanding and reprogramming the genetic code

.

Nature

550

,

53

–

60

[PubMed]

https://doi.org/10.1021/acs.accounts.6b00647

12.

Zhang

S.

,

He

D.

,

Lin

Z.

,

Yang

Y.

,

Song

H.

and

Chen

P.R.

(

2017

)

Conditional chaperone-client interactions revealed by genetically encoded photo-cross-linkers

.

Acc. Chem. Res.

50

,

1184

–

1192

[PubMed]

https://doi.org/10.1021/acschembio.7b00974

13.

Young

D.D.

and

Schultz

P.G.

(

2018

)

Playing with the molecules of life

.

ACS Chem. Biol.

13

,

854

–

870

[PubMed]

https://doi.org/10.1021/acschembio.8b00520

14.

Brown

W.

,

Liu

J.

and

Deiters

A.

(

2018

)

Genetic code expansion in animals

.

ACS Chem. Biol.

13

,

2375

–

2386

[PubMed]

https://doi.org/10.1038/nn1932

15.

Wang

W.

,

Takimoto

J.K.

,

Louie

G.V.

,

Baiga

T.J.

,

Noel

J.P.

,

Lee

K.F.

et al.

(

2007

)

Genetically encoding unnatural amino acids for cellular and neuronal studies

.

Nat. Neurosci.

10

,

1063

–

1072

[PubMed]

https://doi.org/10.1038/nature08948

16.

Ye

S.

,

Zaitseva

E.

,

Caltabiano

G.

,

Schertler

G.F.X.

,

Sakmar

T.P.

,

Deupi

X.

et al.

(

2010

)

Tracking G-protein-coupled receptor activation using genetically encoded infrared probes

.

Nature

464

,

1386

–

1390

[PubMed]

https://doi.org/10.1073/pnas.1318808111

17.

Zhu

S.

,

Riou

M.

,

Yao

C.A.

,

Carvalho

S.

,

Rodriguez

P.C.

,

Bensaude

O.

et al.

(

2014

)

Genetically encoding a light switch in an ionotropic glutamate receptor reveals subunit-specific interfaces

.

Proc. Natl. Acad. Sci. U.S.A.

111

,

6081

–

6086

https://doi.org/10.1002/cbic.201700255

18.

Zheng

S.

,

Fan

X.

,

Wang

J.

,

Zhao

J.

and

Chen

P.R.

(

2017

)

Dissection of kinase isoforms through orthogonal and chemical inducible signaling cascades

.

ChemBioChem

18

,

1593

–

1598

[PubMed]

https://doi.org/10.1038/s41467-018-06299-7

19.

Baumdick

M.

,

Gelleri

M.

,

Uttamapinant

C.

,

Beránek

V.

,

Chin

J.W.

and

Bastiaens

P.I.H.

(

2018

)

A conformational sensor based on genetic code expansion reveals an autocatalytic component in EGFR activation

.

Nat. Commun.

9

,

3847

[PubMed]

https://doi.org/10.1093/nar/gkh959

20.

Köhrer

C.

,

Sullivan

E.L.

and

RajBhandary

U.L.

(

2004

)

Complete set of orthogonal 21st aminoacyl-tRNA synthetase-amber, ochre and opal suppressor tRNA pairs: concomitant suppression of three different termination codons in an mRNA in mammalian cells

.

Nucleic Acids Res.

32

,

6200

–

6211

[PubMed]

https://doi.org/10.1002/anie.201308137

21.

Xiao

H.

,

Chatterjee

A.

,

Choi

S.H.

,

Bajjuri

K.M.

,

Sinha

S.C.

and

Schultz

P.G.

(

2013

)

Genetic incorporation of multiple unnatural amino acids into proteins in mammalian cells

.

Angew. Chem. Int. Ed.

52

,

14080

–

14083

https://doi.org/10.1021/ja5069728

22.

Schmied

W.H.

,

Elsässer

S.J.

,

Uttamapinant

C.

and

Chin

J.W.

(

2014

)

Efficient multisite unnatural amino acid incorporation in mammalian cells via optimized pyrrolysyl tRNA synthetase/tRNA expression and engineered eRF1

.

J. Am. Chem. Soc.

136

,

15577

–

15583

[PubMed]

https://doi.org/10.1093/nar/gkx1156

23.

Serfling

R.

,

Lorenz

C.

,

Etzel

M.

,

Schicht

G.

,

Böttke

T.

,

Mörl

M.

et al.

(

2018

)

Designer tRNAs for efficient incorporation of non-canonical amino acids by the pyrrolysine system in mammalian cells

.

Nucleic Acids Res.

46

,

1

–

10

[PubMed]

https://doi.org/10.1021/acs.biochem.7b00952

24.

Zheng

Y.

,

Mukherjee

R.

,

Chin

M.A.

,

Igo

P.

,

Gilgenast

M.J.

and

Chatterjee

A.

(

2018

)

Expanding the scope of single- and double-noncanonical amino acid mutagenesis in mammalian cells using orthogonal polyspecific leucyl-tRNA synthetases

.

Biochemistry

57

,

441

–

445

[PubMed]

https://doi.org/10.1039/C7SC02560B

25.

Zheng

Y.

,

Addy

P.S.

,

Mukherjee

R.

and

Chatterjee

A.

(

2017

)

Defining the current scope and limitations of dual noncanonical amino acid mutagenesis in mammalian cells

.

Chem. Sci.

8

,

7211

–

7217

[PubMed]

https://doi.org/10.1021/acs.biochem.8b00808

26.

Beranek

V.

,

Willis

J. C.W.

and

Chin

J.W.

(

2019

)

An evolved Methanomethylophilus alvus pyrrolysyl-tRNA Synthetase/tRNA pair is highly active and orthogonal in mammalian cells

.

Biochemistry

58

,

387

–

390

[PubMed]

https://doi.org/10.1021/cb4001662

27.

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

[PubMed]

https://doi.org/10.1002/cbic.200500360

28.

Taki

M.

,

Matsushita

J.

and

Sisido

M.

(

2006

)

Expanding the genetic code in a mammalian cell line by the introduction of four-base codon/anticodon pairs

.

ChemBioChem

7

,

425

–

428

[PubMed]

https://doi.org/10.1038/nature08817

29.

Neumann

H.

,

Wang

K.

,

Davis

L.

,

Garcia-Alai

M.

and

Chin

J.W.

(

2010

)

Encoding multiple unnatural amino acids via evolution of a quadruplet-decoding ribosome

.

Nature

464

,

441

–

444

[PubMed]

https://doi.org/10.1038/nchem.1919

30.

Wang

K.

,

Sachdeva

A.

,

Cox

D.J.

,

Wilf

N.M.

,

Lang

K.

,

Wallace

S.

et al.

(

2014

)

Optimized orthogonal translation of unnatural amino acids enables spontaneous protein double-labelling and FRET

.

Nat. Chem.

6

,

393

–

403

[PubMed]

https://doi.org/10.1038/s41586-018-0773-z

31.

Schmied

W.H.

,

Tnimov

Z.

,

Uttamapinant

C.

,

Rae

C.D.

,

Fried

S.D.

and

Chin

J.W.

(

2018

)

Controlling orthogonal ribosome subunit interactions enables evolution of new function

.

Nature

564

,

444

–

448

[PubMed]

https://doi.org/10.1101/cshperspect.a023945

32.

Xiao

H.

and

Schultz

P.G.

(

2016

)

At the interface of chemical and biological synthesis: an expanded genetic code

.

Cold Spring Harbor Perspect. Biol.

8

,

a023945

https://doi.org/10.1016/j.cbpa.2018.07.014

33.

Vargas-Rodriguez

O.

,

Sevostyanova

A.

,

Söll

D.

and

Crnković

A.

(

2018

)

Upgrading aminoacyl-tRNA synthetases for genetic code expansion

.

Curr. Opin. Chem. Biol.

46

,

115

–

122

[PubMed]

https://doi.org/10.1038/nchembio.2312

34.

Italia

J.S.

,

Addy

P.S.

,

Wrobel

C.J.

,

Crawford

L.A.

,

Lajoie

M.J.

,

Zheng

Y.

et al.

(

2017

)

An orthogonalized platform for genetic code expansion in both bacteria and eukaryotes

.

Nat. Chem. Biol.

13

,

446

–

450

[PubMed]

https://doi.org/10.1016/j.chembiol.2018.07.002

35.

Italia

J.S.

,

Latour

C.

,

Wrobel

C.J.J.

and

Chatterjee

A.

(

2018

)

Resurrecting the bacterial tyrosyl-tRNA Synthetase/tRNA pair for expanding the genetic code of both E. coli and eukaryotes

.

Cell Chem. Biol.

25

,

1304

–

1312

[PubMed]

https://doi.org/10.1126/science.1084772

36.

Chin

J.W.

,

Cropp

T.A.

,

Anderson

J.C.

,

Mukherji

M.

,

Zhang

Z.

and

Schultz

P.G.

(

2003

)

An expanded eukaryotic genetic code

.

Science

301

,

964

–

967

[PubMed]

https://doi.org/10.1038/nmeth1016

37.

Liu

W.

,

Brock

A.

,

Chen

S.

,

Chen

S.

and

Schultz

P.G.

(

2007

)

Genetic incorporation of unnatural amino acids into proteins in mammalian cells

.

Nat. Methods

4

,

239

–

244

[PubMed]

https://doi.org/10.3791/50588

38.

Naganathan

S.

,

Grunbeck

A.

,

Tian

H.

,

Huber

T.

and

Sakmar

T.P.

(

2013

)

Genetically-encoded molecular probes to study G protein-coupled receptors

.

J. Vis. Exp.

https://doi.org/10.1038/nprot.2015.045

39.

Nikić

I.

,

Kang

J.H.

,

Girona

G.E.

,

Aramburu

I.V.

and

Lemke

E.A.

(

2015

)

Labeling proteins on live mammalian cells using click chemistry

.

Nat. Protoc.

10

,

780

–

791

[PubMed]

https://doi.org/10.1038/nprot.2017.090

40.

Yang

Y.

,

Song

H.

,

He

D.

,

Zhang

S.

,

Dai

S.

,

Xie

X.

et al.

(

2017

)

Genetically encoded releasable photo-cross-linking strategies for studying protein-protein interactions in living cells

.

Nat. Protoc.

12

,

2147

–

2168

[PubMed]

https://doi.org/10.1021/bi101929e

41.

Young

D.D.

,

Young

T.S.

,

Jahnz

M.

,

Ahmad

I.

,

Spraggon

G.

and

Schultz

P.G.

(

2011

)

An evolved aminoacyl-tRNA synthetase with atypical polysubstrate specificity

.

Biochemistry

50

,

1894

–

1900

[PubMed]

https://doi.org/10.1038/s41557-018-0052-5

42.

Willis

J.C.W.

and

Chin

J.W.

(

2018

)

Mutually orthogonal pyrrolysyl-tRNA synthetase/tRNA pairs

.

Nat. Chem.

10

,

831

–

837

[PubMed]

https://doi.org/10.1002/anie.201402092

43.

Wang

N.

,

Li

Y.

,

Niu

W.

,

Sun

M.

,

Cerny

R.

,

Li

Q.

et al.

(

2014

)

Construction of a live-attenuated HIV-1 vaccine through genetic code expansion

.

Angew. Chem. Int. Ed.

53

,

4867

–

4871

44.

Mali

P.

and

Katrekar

D.

(

2017

)

In vivo RNA targeting of point mutations via suppressor tRNAs and adenosine deaminases

.

bioRxiv

https://doi.org/10.1126/science.7792603

45.

Gossen

M.

,

Freundlieb

S.

,

Bender

G.

,

Muller

G.

,

Hillen

W.

and

Bujard

H.

(

1995

)

Transcriptional activation by tetracyclines in mammalian cells

.

Science

268

,

1766

–

1769

[PubMed]

https://doi.org/10.1093/nar/gkf589

46.

Sakamoto

K.

,

Hayashi

A.

,

Sakamoto

A.

,

Kiga

D.

,

Nakayama

H.

,

Soma

A.

et al.

(

2002

)

Site-specific incorporation of an unnatural amino acid into proteins in mammalian cells

.

Nucleic Acids Res.

30

,

4692

–

4699

[PubMed]

https://doi.org/10.1038/s41598-018-28178-3

47.

Suzuki

T.

,

Asami

M.

,

Patel

S.G.

,

Luk

L.Y.P.

,

Tsai

Y.-H.

and

Perry

A.C.F.

(

2018

)

Switchable genome editing via genetic code expansion

.

Sci. Rep.

8

,

10051

[PubMed]

https://doi.org/10.1126/science.aah5869

48.

Si

L.

,

Xu

H.

,

Zhou

X.

,

Zhang

Z.

,

Tian

Z.

,

Wang

Y.

et al.

(

2016

)

Generation of influenza A viruses as live but replication-incompetent virus vaccines

.

Science

354

,

1170

–

1173

[PubMed]

https://doi.org/10.1021/acssynbio.6b00373

49.

Yuan

Z.

,

Wang

N.

,

Kang

G.

,

Niu

W.

,

Li

Q.

and

Guo

J.

(

2017

)

Controlling multicycle replication of live-attenuated HIV-1 using an unnatural genetic switch

.

ACS Synth. Biol.

6

,

721

–

731

[PubMed]

https://doi.org/10.1038/ncomms14568

50.

Han

S.

,

Yang

A.

,

Lee

S.

,

Lee

H.W.

,

Park

C.B.

and

Park

H.S.

(

2017

)

Expanding the genetic code of Mus musculus

.

Nat. Commun.

8

,

14568

[PubMed]

https://doi.org/10.1038/nbt.4056

51.

Krogager

T.P.

,

Ernst

R.J.

,

Elliott

T.S.

,

Calo

L.

,

Beránek

V.

,

Ciabatti

E.

et al.

(

2018

)

Labeling and identifying cell-specific proteomes in the mouse brain

.

Nat. Biotechnol.

36

,

156

–

159

[PubMed]

https://doi.org/10.1021/cr300177k

52.

Klán

P.

,

Šolomek

T.

,

Bochet

C.G.

,

Blanc

A.

,

Givens

R.

,

Rubina

M.

et al.

(

2013

)

Photoremovable protecting groups in chemistry and biology: reaction mechanisms and efficacy

.

Chem. Rev.

113

,

119

–

191

[PubMed]

https://doi.org/10.1038/nchembio.2007.44

53.

Lemke

E.A.

,

Summerer

D.

,

Geierstanger

B.H.

,

Brittain

S.M.

and

Schultz

P.G.

(

2007

)

Control of protein phosphorylation with a genetically encoded photocaged amino acid

.

Nat. Chem. Biol.

3

,

769

–

772

[PubMed]

https://doi.org/10.1093/nar/gky1049

54.

The UniProt Consortium

,

2019

,

UniProt: a worldwide hub of protein knowledge

,

Nucleic Acids Res.

,

47

,

D506

–

D515

,

,

[PubMed]

55.

Klippenstein

V.

,

Ghisi

V.

,

Wietstruk

M.

and

Plested

A.J.

(

2014

)

Photoinactivation of glutamate receptors by genetically encoded unnatural amino acids

.

J. Neurosci.

34

,

980

–

991

https://doi.org/10.1523/JNEUROSCI.3725-13.2014

[PubMed]

https://doi.org/10.7554/eLife.25808

56.

Klippenstein

V.

,

Hoppmann

C.

,

Ye

S.

,

Wang

L.

and

Paoletti

P.

(

2017

)

Optocontrol of glutamate receptor activity by single side-chain photoisomerization

.

Elife

6

,

e25808

[PubMed]

https://doi.org/10.1038/nchem.1887

57.

Li

J.

,

Yu

J.

,

Zhao

J.

,

Wang

J.

,

Zheng

S.

,

Lin

S.

et al.

(

2014

)

Palladium-triggered deprotection chemistry for protein activation in living cells

.

Nat. Chem.

6

,

352

–

361

[PubMed]

https://doi.org/10.1038/srep15348

58.

Wäldchen

S.

,

Lehmann

J.

,

Klein

T.

,

van de Linde

S.

and

Sauer

M.

(

2015

)

Light-induced cell damage in live-cell super-resolution microscopy

.

Sci. Rep.

5

,

15348

[PubMed]

https://doi.org/10.1038/nchembio.1656

59.

Li

J.

,

Jia

S.

and

Chen

P.R.

(

2014

)

Diels-Alder reaction-triggered bioorthogonal protein decaging in living cells

.

Nat. Chem. Biol.

10

,

1003

–

1005

[PubMed]

https://doi.org/10.1021/acscentsci.6b00024

60.

Zhang

G.

,

Li

J.

,

Xie

R.

,

Fan

X.

,

Liu

Y.

,

Zheng

S.

et al.

(

2016

)

Bioorthogonal chemical activation of kinases in living systems

.

ACS Cent. Sci.

2

,

325

–

331

[PubMed]

https://doi.org/10.1002/anie.201608009

61.

Fan

X.

,

Ge

Y.

,

Lin

F.

,

Yang

Y.

,

Zhang

G.

,

Ngai

W.S.

et al.

(

2016

)

Optimized tetrazine derivatives for rapid bioorthogonal decaging in living cells

.

Angew. Chem. Int. Ed.

55

,

14046

–

14050

https://doi.org/10.1039/C6SC02615J

62.

Ge

Y.

,

Fan

X.

and

Chen

P.R.

(

2016

)

A genetically encoded multifunctional unnatural amino acid for versatile protein manipulations in living cells

.

Chem. Sci.

7

,

7055

–

7060

[PubMed]

https://doi.org/10.1038/nchem.2573

63.

Luo

J.

,

Liu

Q.

,

Morihiro

K.

and

Deiters

A.

(

2016

)

Small-molecule control of protein function through Staudinger reduction

.

Nat. Chem.

8

,

1027

–

1034

[PubMed]

https://doi.org/10.1038/nchem.2253

64.

Tsai

Y.-H.

,

Essig

S.

,

James

J.R.

,

Lang

K.

and

Chin

J.W.

(

2015

)

Selective, rapid and optically switchable regulation of protein function in live mammalian cells

.

Nat. Chem.

7

,

554

–

561

[PubMed]

https://doi.org/10.1021/cb4009292

65.

Lang

K.

and

Chin

J.W.

(

2014

)

Bioorthogonal reactions for labeling proteins

.

ACS Chem. Biol.

9

,

16

–

20

[PubMed]

https://doi.org/10.1038/nsmb.3492

66.

Liaunardy-Jopeace

A.

,

Murton

B.L.

,

Mahesh

M.

,

Chin

J.W.

and

James

J.R.

(

2017

)

Encoding optical control in LCK kinase to quantitatively investigate its activity in live cells

.

Nat. Struct. Mol. Biol.

24

,

1155

–

1163

[PubMed]

https://doi.org/10.1021/jacs.5b07627

67.

Walker

O.S.

,

Elsässer

S.J.

,

Mahesh

M.

,

Bachman

M.

,

Balasubramanian

S.

and

Chin

J.W.

(

2016

)

Photoactivation of mutant isocitrate dehydrogenase 2 reveals rapid cancer-associated metabolic and epigenetic changes

.

J. Am. Chem. Soc.

138

,

718

–

721

[PubMed]

https://doi.org/10.1021/acs.biochem.8b00502

68.

Simms

J.

,

Uddin

R.

,

Sakmar

T.P.

,

Gingell

J.J.

,

Garelja

M.L.

,

Hay

D.L.

et al.

(

2018

)

Photoaffinity cross-linking and unnatural amino acid mutagenesis reveal insights into calcitonin gene-related peptide binding to the calcitonin receptor-like receptor/receptor activity-modifying protein 1 (CLR/RAMP1) complex

.

Biochemistry

57

,

4915

–

4922

[PubMed]

https://doi.org/10.1038/nchembio.2160

69.

Ernst

R.J.

,

Krogager

T.P.

,

Maywood

E.S.

,

Zanchi

R.

,

Beránek

V.

,

Elliott

T.S.

et al.

(

2016

)

Genetic code expansion in the mouse brain

.

Nat. Chem. Biol.

12

,

776

–

778

[PubMed]

https://doi.org/10.1038/cr.2016.145

70.

Chen

Y.

,

Ma

J.

,

Lu

W.

,

Tian

M.

,

Thauvin

M.

,

Yuan

C.

et al.

(

2017

)

Heritable expansion of the genetic code in mouse and zebrafish

.

Cell Res

27

,

294

–

297

[PubMed]

https://doi.org/10.1016/j.neuron.2013.08.016

71.

Kang

J.Y.

,

Kawaguchi

D.

,

Coin

I.

,

Xiang

Z.

,

O’Leary

D.D.

,

Slesinger

P.A.

et al.

(

2013

)

In vivo expression of a light-activatable potassium channel using unnatural amino acids

.

Neuron

80

,

358

–

370

[PubMed]

https://doi.org/10.1021/ja508597d

72.

Ren

W.

,

Ji

A.

and

Ai

H.-W.

(

2015

)

Light activation of protein splicing with a photocaged fast intein

.

J. Am. Chem. Soc.

137

,

2155

–

2158

[PubMed]

https://doi.org/10.1021/ja412191m

73.

Nguyen

D.P.

,

Mahesh

M.

,

Elsässer

S.J.

,

Hancock

S.M.

,

Uttamapinant

C.

and

Chin

J.W.

(

2014

)

Genetic encoding of photocaged cysteine allows photoactivation of TEV protease in live mammalian cells

.

J. Am. Chem. Soc.

136

,

2240

–

2243

[PubMed]

https://doi.org/10.1021/jacs.7b02618

74.

Gramespacher

J.A.

,

Stevens

A.J.

,

Nguyen

D.P.

,

Chin

J.W.

and

Muir

T.W.

(

2017

)

Intein zymogens: conditional assembly and splicing of split inteins via targeted proteolysis

.

J. Am. Chem. Soc.

139

,

8074

–

8077

[PubMed]

https://doi.org/10.1002/cbic.201400073

75.

Uprety

R.

,

Luo

J.

,

Liu

J.

,

Naro

Y.

,

Samanta

S.

and

Deiters

A.

(

2014

)

Genetic encoding of caged cysteine and caged homocysteine in bacterial and mammalian cells

.

ChemBioChem

15

,

1793

–

1799

[PubMed]

https://doi.org/10.1021/jacs.8b04603

76.

Liu

J.

,

Zheng

F.

,

Cheng

R.

,

Li

S.

,

Rozovsky

S.

,

Wang

Q.

et al.

(

2018

)

Site-specific incorporation of selenocysteine using an expanded genetic code and palladium-mediated chemical deprotection

.

J. Am. Chem. Soc.

140

,

8807

–

8816

[PubMed]

https://doi.org/10.1073/pnas.1309584110

77.

Chatterjee

A.

,

Xiao

H.

,

Bollong

M.

,

Ai

H.-W.

and

Schultz

P.G.

(

2013

)

Efficient viral delivery system for unnatural amino acid mutagenesis in mammalian cells

.

Proc. Natl. Acad. Sci. U.S.A.

110

,

11803

–

11808

https://doi.org/10.1039/b904228h

78.

Takimoto

J.K.

,

Adams

K.L.

,

Xiang

Z.

and

Wang

L.

(

2009

)

Improving orthogonal tRNA-synthetase recognition for efficient unnatural amino acid incorporation and application in mammalian cells

.

Mol. BioSyst.

5

,

931

–

934

[PubMed]

https://doi.org/10.1021/acschembio.6b00520

79.

Smits

A.H.

,

Borrmann

A.

,

Roosjen

M.

,

van Hest

J.C.

and

Vermeulen

M.

(

2016

)

Click-MS: tagless protein enrichment using bioorthogonal chemistry for quantitative proteomics

.

ACS Chem. Biol.

11

,

3245

–

3250

[PubMed]

https://doi.org/10.1038/nchembio.167

80.

Ye

S.

,

Huber

T.

,

Vogel

R.

and

Sakmar

T.P.

(

2009

)

FTIR analysis of GPCR activation using azido probes

.

Nat. Chem. Biol.

5

,

397

–

399

[PubMed]

https://doi.org/10.1002/anie.201102646

81.

Coin

I.

,

Perrin

M.H.

,

Vale

W.W.

and

Wang

L.

(

2011

)

Photo-cross-linkers incorporated into G-protein-coupled receptors in mammalian cells: a ligand comparison

.

Angew. Chem. Int. Ed.

50

,

8077

–

8081

https://doi.org/10.1021/ja303261d

82.

Chen

S.

,

Chen

Z.-j.

,

Ren

W.

and

Ai

H.-W.

(

2012

)

Reaction-based genetically encoded fluorescent hydrogen sulfide sensors

.

J. Am. Chem. Soc.

134

,

9589

–

9592

[PubMed]

https://doi.org/10.1021/bi301292h

83.

Naganathan

S.

,

Ye

S.

,

Sakmar

T.P.

and

Huber

T.

(

2013

)

Site-specific epitope tagging of G protein-coupled receptors by bioorthogonal modification of a genetically encoded unnatural amino acid

.

Biochemistry

52

,

1028

–

1036

[PubMed]

https://doi.org/10.1016/j.cell.2013.11.008

84.

Coin

I.

,

Katritch

V.

,

Sun

T.

,

Xiang

Z.

,

Siu

F.Y.

,

Beyermann

M.

et al.

(

2013

)

Genetically encoded chemical probes in cells reveal the binding path of urocortin-I to CRF class B GPCR

.

Cell

155

,

1258

–

1269

[PubMed]

https://doi.org/10.1021/cb500351s

85.

Wang

W.

,

Li

T.

,

Felsovalyi

K.

,

Chen

C.

,

Cardozo

T.

and

Krogsgaard

M.

(

2014

)

Quantitative analysis of T cell receptor complex interaction sites using genetically encoded photo-cross-linkers

.

ACS Chem. Biol.

9

,

2165

–

2172

[PubMed]

https://doi.org/10.1021/bi500830d

86.

Chen

Z.J.

and

Ai

H.-W.

(

2014

)

A highly responsive and selective fluorescent probe for imaging physiological hydrogen sulfide

.

Biochemistry

53

,

5966

–

5974

[PubMed]

https://doi.org/10.1002/cbic.201402193

87.

Tian

H.

,

Naganathan

S.

,

Kazmi

M.A.

,

Schwartz

T.W.

,

Sakmar

T.P.

and

Huber

T.

(

2014

)

Bioorthogonal fluorescent labeling of functional G-protein-coupled receptors

.

ChemBioChem

15

,

1820

–

1829

[PubMed]

https://doi.org/10.1021/bi501267x

88.

Naganathan

S.

,

Ray-Saha

S.

,

Park

M.

,

Tian

H.

,

Sakmar

T.P.

and

Huber

T.

(

2015

)

Multiplex detection of functional G protein-coupled receptors harboring site-specifically modified unnatural amino acids

.

Biochemistry

54

,

776

–

786

[PubMed]

https://doi.org/10.1002/cbic.201500030

89.

Tian

H.

,

Sakmar

T.P.

and

Huber

T.

(

2015

)

Micelle-enhanced bioorthogonal labeling of genetically encoded azido groups on the lipid-embedded surface of a GPCR

.

ChemBioChem

16

,

1314

–

1322

[PubMed]

https://doi.org/10.1021/acssynbio.6b00092

90.

Zheng

Y.

,

Lewis

T.L.

Jr,

Igo

P.

,

Polleux

F.

and

Chatterjee

A.

(

2017

)

Virus-enabled optimization and delivery of the genetic machinery for efficient unnatural amino acid mutagenesis in mammalian cells and tissues

.

ACS Synth. Biol.

6

,

13

–

18

[PubMed]

https://doi.org/10.1074/jbc.M117.779496

91.

Koole

C.

,

Reynolds

C.A.

,

Mobarec

J.C.

,

Hick

C.

,

Sexton

P.M.

and

Sakmar

T.P.

(

2017

)

Genetically encoded photocross-linkers determine the biological binding site of exendin-4 peptide in the N-terminal domain of the intact human glucagon-like peptide-1 receptor (GLP-1R)

.

J. Biol. Chem.

292

,

7131

–

7144

[PubMed]

https://doi.org/10.1021/bi200214r

92.

Grunbeck

A.

,

Huber

T.

,

Sachdev

P.

and

Sakmar

T.P.

(

2011

)

Mapping the ligand-binding site on a G protein-coupled receptor (GPCR) using genetically encoded photocrosslinkers

.

Biochemistry

50

,

3411

–

3413

[PubMed]

https://doi.org/10.1021/bi401289p

93.

Ray-Saha

S.

,

Huber

T.

and

Sakmar

T.P.

(

2014

)

Antibody epitopes on g protein-coupled receptors mapped with genetically encoded photoactivatable cross-linkers

.

Biochemistry

53

,

1302

–

1310

[PubMed]

https://doi.org/10.1021/cb300059z

94.

Grunbeck

A.

,

Huber

T.

,

Abrol

R.

,

Trzaskowski

B.

,

Goddard

W.A.

III and

Sakmar

T.P.

(

2012

)

Genetically encoded photo-cross-linkers map the binding site of an allosteric drug on a G protein-coupled receptor

.

ACS Chem. Biol.

7

,

967

–

972

[PubMed]

https://doi.org/10.1038/ncomms11261

95.

Rannversson

H.

,

Andersen

J.

,

Sorensen

L.

,

Bang-Andersen

B.

,

Park

M.

,

Huber

T.

et al.

(

2016

)

Genetically encoded photocrosslinkers locate the high-affinity binding site of antidepressant drugs in the human serotonin transporter

.

Nat. Commun.

7

,

11261

[PubMed]

https://doi.org/10.1016/j.chembiol.2015.09.014

96.

Park

M.

,

Sivertsen

B.B.

,

Els-Heindl

S.

,

Huber

T.

,

Holst

B.

,

Beck-Sickinger

A.G.

et al.

(

2015

)

Bioorthogonal labeling of ghrelin receptor to facilitate studies of ligand-dependent conformational dynamics

.

Chem. Biol.

22

,

1431

–

1436

[PubMed]

https://doi.org/10.1021/ja408011q

97.

Chen

Z.-j.

,

Ren

W.

,

Wright

Q.E.

and

Ai

H.-W.

(

2013

)

Genetically encoded fluorescent probe for the selective detection of peroxynitrite

.

J. Am. Chem. Soc

135

,

14940

–

14943

[PubMed]

https://doi.org/10.1021/cb400917a

98.

Tharp

J.M.

,

Wang

Y.S.

,

Lee

Y.J.

,

Yang

Y.

and

Liu

W.R.

(

2014

)

Genetic incorporation of seven ortho-substituted phenylalanine derivatives

.

ACS Chem. Biol.

9

,

884

–

890

[PubMed]

https://doi.org/10.1074/jbc.M707355200

99.

Ye

S.

,

Köhrer

C.

,

Huber

T.

,

Kazmi

M.

,

Sachdev

P.

,

Yan

E.C.

et al.

(

2008

)

Site-specific incorporation of keto amino acids into functional G protein-coupled receptors using unnatural amino acid mutagenesis

.

J. Biol. Chem.

283

,

1525

–

1533

[PubMed]

https://doi.org/10.1073/pnas.1211023109

100.

Axup

J.Y.

,

Bajjuri

K.M.

,

Ritland

M.

,

Hutchins

B.M.

,

Kim

C.H.

,

Kazane

S.A.

et al.

(

2012

)

Synthesis of site-specific antibody-drug conjugates using unnatural amino acids

.

Proc. Natl. Acad. Sci. U.S.A.

109

,

16101

–

16106

https://doi.org/10.1038/nmeth.2595

101.

Xiang

Z.

,

Ren

H.

,

Hu

Y.S.

,

Coin

I.

,

Wei

J.

,

Cang

H.

et al.

(

2013

)

Adding an unnatural covalent bond to proteins through proximity-enhanced bioreactivity

.

Nat. Methods

10

,

885

–

888

[PubMed]

https://doi.org/10.1038/nmeth739

102.

Hino

N.

,

Okazaki

Y.

,

Kobayashi

T.

,

Hayashi

A.

,

Sakamoto

K.

and

Yokoyama

S.

(

2005

)

Protein photo-cross-linking in mammalian cells by site-specific incorporation of a photoreactive amino acid

.

Nat. Methods

2

,

201

–

206

[PubMed]

https://doi.org/10.1002/stem.679

103.

Shen

B.

,

Xiang

Z.

,

Miller

B.

,

Louie

G.

,

Wang

W.

,

Noel

J.P.

et al.

(

2011

)

Genetically encoding unnatural amino acids in neural stem cells and optically reporting voltage-sensitive domain changes in differentiated neurons

.

Stem Cells

29

,

1231

–

1240

[PubMed]

https://doi.org/10.7554/eLife.11815

104.

Murray

C.I.

,

Westhoff

M.

,

Eldstrom

J.

,

Thompson

E.

,

Emes

R.

and

Fedida

D.

(

2016

)

Unnatural amino acid photo-crosslinking of the IKs channel complex demonstrates a KCNE1:KCNQ1 stoichiometry of up to 4:4

.

Elife

5

,

e11815

[PubMed]

https://doi.org/10.1074/jbc.M113.527085

105.

Valentin-Hansen

L.

,

Park

M.

,

Huber

T.

,

Grunbeck

A.

,

Naganathan

S.

,

Schwartz

T.W.

et al.

(

2014

)

Mapping substance P binding sites on the neurokinin-1 receptor using genetic incorporation of a photoreactive amino acid

.

J. Biol. Chem.

289

,

18045

–

18054

[PubMed]

https://doi.org/10.1002/cbic.201300400

106.

Lacey

V.K.

,

Louie

G.V.

,

Noel

J.P.

and

Wang

L.

(

2013

)

Expanding the library and substrate diversity of the pyrrolysyl-tRNA synthetase to incorporate unnatural amino acids containing conjugated rings

.

ChemBioChem

14

,

2100

–

2105

[PubMed]

https://doi.org/10.1021/ja4059553

107.

Chatterjee

A.

,

Guo

J.

,

Lee

H.S.

and

Schultz

P.G.

(

2013

)

A genetically encoded fluorescent probe in mammalian cells

.

J. Am. Chem. Soc.

135

,

12540

–

12543

[PubMed]

https://doi.org/10.1002/cbic.201600668

108.

Mitchell

A.L.

,

Addy

P.S.

,

Chin

M.A.

and

Chatterjee

A.

(

2017

)

A unique genetically encoded FRET pair in mammalian cells

.

ChemBioChem

18

,

511

–

514

[PubMed]

https://doi.org/10.1021/jacs.8b10098

109.

Park

S.-H.

,

Ko

W.

,

Lee

H.S.

and

Shin

I.

(

2019

)

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.

141

,

4273

–

4281

,

[PubMed]

https://doi.org/10.1021/jacs.8b05814

110.

Fu

C.

,

Kobayashi

T.

,

Wang

N.

,

Hoppmann

C.

,

Yang

B.

,

Irannejad

R.

et al.

(

2018

)

Genetically encoding quinoline reverses chromophore charge and enables fluorescent protein brightening in acidic vesicles

.

J. Am. Chem. Soc.

140

,

11058

–

11066

[PubMed]

https://doi.org/10.1002/cbic.201800226

111.

Luo

J.

,

Samanta

S.

,

Convertino

M.

,

Dokholyan

N.V.

and

Deiters

A.

(

2018

)

Reversible and tunable photoswitching of protein function through genetic encoding of azobenzene amino acids in mammalian cells

.

ChemBioChem

19

,

2178

–

2185

[PubMed]

https://doi.org/10.1002/anie.201400001

112.

Hoppmann

C.

,

Lacey

V.K.

,

Louie

G.V.

,

Wei

J.

,

Noel

J.P.

and

Wang

L.

(

2014

)

Genetically encoding photoswitchable click amino acids in Escherichia coli and mammalian cells

.

Angew. Chem. Int. Ed.

53

,

3932

–

3936

https://doi.org/10.1021/cb500032c

113.

Xiao

H.

,

Peters

F.B.

,

Yang

P.Y.

,

Reed

S.

,

Chittuluru

J.R.

and

Schultz

P.G.

(

2014

)

Genetic incorporation of histidine derivatives using an engineered pyrrolysyl-tRNA synthetase

.

ACS Chem. Biol.

9

,

1092

–

1096

[PubMed]

https://doi.org/10.1002/cbic.201500170

114.

Wang

T.

,

Zhou

Q.

,

Li

F.

,

Yu

Y.

,

Yin

X.

and

Wang

J.

(

2015

)

Genetic incorporation of N(epsilon)-Formyllysine, a new histone post-translational modification

.

ChemBioChem

16

,

1440

–

1442

[PubMed]

https://doi.org/10.1038/nmeth.3701

115.

Elsässer

S.J.

,

Ernst

R.J.

,

Walker

O.S.

and

Chin

J.W.

(

2016

)

Genetic code expansion in stable cell lines enables encoded chromatin modification

.

Nat. Methods

13

,

158

–

164

[PubMed]

https://doi.org/10.1021/jacs.7b05725

116.

Xuan

W.

,

Yao

A.

and

Schultz

P.G.

(

2017

)

Genetically encoded fluorescent probe for detecting sirtuins in living cells

.

J. Am. Chem. Soc.

139

,

12350

–

12353

[PubMed]

https://doi.org/10.1002/cbic.201500681

117.

Cohen

S.

and

Arbely

E.

(

2016

)

Single-plasmid-based system for efficient noncanonical amino acid mutagenesis in cultured mammalian cells

.

ChemBioChem

17

,

1008

–

1011

[PubMed]

https://doi.org/10.1016/j.bbrc.2008.04.164

118.

Mukai

T.

,

Kobayashi

T.

,

Hino

N.

,

Yanagisawa

T.

,

Sakamoto

K.

and

Yokoyama

S.

(

2008

)

Adding l-lysine derivatives to the genetic code of mammalian cells with engineered pyrrolysyl-tRNA synthetases

.

Biochem. Biophys. Res. Commun.

371

,

818

–

822

[PubMed]

https://doi.org/10.1002/anie.201303477

119.

Li

F.

,

Zhang

H.

,

Sun

Y.

,

Pan

Y.

,

Zhou

J.

and

Wang

J.

(

2013

)

Expanding the genetic code for photoclick chemistry in E. coli, mammalian cells, and A. thaliana

.

Angew. Chem. Int. Ed.

52

,

9700

–

9704

https://doi.org/10.1002/anie.201203349

120.

Kim

C.H.

,

Kang

M.

,

Kim

H.J.

,

Chatterjee

A.

and

Schultz

P.G.

(

2012

)

Site-specific incorporation of epsilon-N-crotonyllysine into histones

.

Angew. Chem. Int. Ed.

51

,

7246

–

7249

https://doi.org/10.1002/anie.201706927

121.

Cigler

M.

,

Müller

T.G.

,

Horn-Ghetko

D.

,

von Wrisberg

M.K.

,

Fottner

M.

,

Goody

R.S.

et al.

(

2017

)

Proximity-triggered covalent stabilization of low-affinity protein complexes in vitro and in vivo

.

Angew. Chem. Int. Ed.

56

,

15737

–

15741

https://doi.org/10.1021/jacs.7b02615

122.

Tian

Y.

,

Jacinto

M.P.

,

Zeng

Y.

,

Yu

Z.

,

Qu

J.

,

Liu

W.R.

et al.

(

2017

)

Genetically encoded 2-aryl-5-carboxytetrazoles for site-selective protein photo-cross-linking

.

J. Am. Chem. Soc.

139

,

6078

–

6081

[PubMed]

https://doi.org/10.1021/jacs.6b08733

123.

Peng

T.

and

Hang

H.C.

(

2016

)

Site-specific bioorthogonal labeling for fluorescence imaging of intracellular proteins in living cells

.

J. Am. Chem. Soc.

138

,

14423

–

14433

[PubMed]

https://doi.org/10.1021/jacs.7b05674

124.

Ramil

C.P.

,

Dong

M.

,

An

P.

,

Lewandowski

T.M.

,

Yu

Z.

,

Miller

L.J.

et al.

(

2017

)

Spirohexene-tetrazine ligation enables bioorthogonal labeling of class B G protein-coupled receptors in live cells

.

J. Am. Chem. Soc.

139

,

13376

–

13386

[PubMed]

https://doi.org/10.1021/ja910688s

125.

Gautier

A.

,

Nguyen

D.P.

,

Lusic

H.

,

An

W.

,

Deiters

A.

and

Chin

J.W.

(

2010

)

Genetically encoded photocontrol of protein localization in mammalian cells

.

J. Am. Chem. Soc.

132

,

4086

–

4088

[PubMed]

https://doi.org/10.1021/ja3046958

126.

Arbely

E.

,

Torres-Kolbus

J.

,

Deiters

A.

and

Chin

J.W.

(

2012

)

Photocontrol of tyrosine phosphorylation in mammalian cells via genetic encoding of photocaged tyrosine

.

J. Am. Chem. Soc.

134

,

11912

–

11915

[PubMed]

https://doi.org/10.1038/nchem.1250

127.

Lang

K.

,

Davis

L.

,

Torres-Kolbus

J.

,

Chou

C.

,

Deiters

A.

and

Chin

J.W.

(

2012

)

Genetically encoded norbornene directs site-specific cellular protein labelling via a rapid bioorthogonal reaction

.

Nat. Chem.

4

,

298

–

304

[PubMed]

https://doi.org/10.1038/s41598-018-32824-1

128.

Aloush

N.

,

Schvartz

T.

,

König

A.I.

,

Cohen

S.

,

Brozgol

E.

,

Tam

B.

et al.

(

2018

)

Live cell imaging of bioorthogonally labelled proteins generated with a single pyrrolysine tRNA gene

.

Sci. Rep.

8

,

14527

[PubMed]

https://doi.org/10.1021/acschembio.8b00594

129.

Schneider

N.

,

Gabelein

C.

,

Wiener

J.

,

Georgiev

T.

,

Gobet

N.

,

Weber

W.

et al.

(

2018

)

Genetic code expansion method for temporal labeling of endogenously expressed proteins

.

ACS Chem. Biol.

13

,

3049

–

3053

[PubMed]

https://doi.org/10.1021/cb500649q

130.

Li

N.

,

Ramil

C.P.

,

Lim

R.K.

and

Lin

Q.

(

2015

)

A genetically encoded alkyne directs palladium-mediated protein labeling on live mammalian cell surface

.

ACS Chem. Biol.

10

,

379

–

384

[PubMed]

https://doi.org/10.1002/cbic.201400057

131.

Yang

Y.

,

Lin

S.

,

Lin

W.

and

Chen

P.R.

(

2014

)

Ligand-assisted dual-site click labeling of EGFR on living cells

.

ChemBioChem

15

,

1738

–

1743

[PubMed]

https://doi.org/10.1093/nar/gkv202

132.

Zheng

Y.

,

Yu

F.

,

Wu

Y.

,

Si

L.

,

Xu

H.

,

Zhang

C.

et al.

(

2015

)

Broadening the versatility of lentiviral vectors as a tool in nucleic acid research via genetic code expansion

.

Nucleic Acids Res.

43

,

e73

[PubMed]

https://doi.org/10.1016/j.biomaterials.2015.11.066

133.

Zhang

C.

,

Yao

T.

,

Zheng

Y.

,

Li

Z.

,

Zhang

Q.

,

Zhang

L.

et al.

(

2016

)

Development of next generation adeno-associated viral vectors capable of selective tropism and efficient gene delivery

.

Biomaterials

80

,

134

–

145

[PubMed]

https://doi.org/10.1016/j.bbrc.2017.05.178

134.

Zhang

Z.

,

Xu

H.

,

Si

L.

,

Chen

Y.

,

Zhang

B.

,

Wang

Y.

et al.

(

2017

)

Construction of an inducible stable cell line for efficient incorporation of unnatural amino acids in mammalian cells

.

Biochem. Biophys. Res. Commun.

489

,

490

–

496

[PubMed]

https://doi.org/10.3390/molecules22071155

135.

Yao

T.

,

Zhou

X.

,

Zhang

C.

,

Yu

X.

,

Tian

Z.

,

Zhang

L.

et al.

(

2017

)

Site-specific PEGylated adeno-associated viruses with increased serum stability and reduced immunogenicity

.

Molecules

22

,

1155

https://doi.org/10.1038/s41598-018-21742-x

136.

Katrekar

D.

,

Moreno

A.M.

,

Chen

G.

,

Worlikar

A.

and

Mali

P.

(

2018

)

Oligonucleotide conjugated multi-functional adeno-associated viruses

.

Sci. Rep.

8

,

3589

[PubMed]

https://doi.org/10.1002/anie.201604067

137.

Kelemen

R.E.

,

Mukherjee

R.

,

Cao

X.

,

Erickson

S.B.

,

Zheng

Y.

and

Chatterjee

A.

(

2016

)

A precise chemical strategy to alter the receptor specificity of the adeno-associated virus

.

Angew. Chem. Int. Ed.

55

,

10645

–

10649

https://doi.org/10.1021/acs.jpclett.8b01991

138.

Zhang

J.

,

Yan

S.

,

He

Z.

,

Ding

C.

,

Zhai

T.

,

Chen

Y.

et al.

(

2018

)

Small unnatural amino acid carried raman tag for molecular imaging of genetically targeted proteins

.

J. Phys. Chem. Lett.

9

,

4679

–

4685

[PubMed]

https://doi.org/10.1021/acsbiomaterials.5b00236

139.

Lühmann

T.

,

Jones

G.

,

Gutmann

M.

,

Rybak

J.-C.

,

Nickel

J.

,

Rubini

M.

et al.

(

2015

)

Bio-orthogonal immobilization of fibroblast growth factor 2 for spatial controlled cell proliferation

.

ACS Biomater. Sci. Eng.

1

,

740

–

746

https://doi.org/10.1002/anie.201305787

140.

Lin

S.

,

Yan

H.

,

Li

L.

,

Yang

M.

,

Peng

B.

,

Chen

S.

et al.

(

2013

)

Site-specific engineering of chemical functionalities on the surface of live hepatitis D virus

.

Angew. Chem. Int. Ed.

52

,

13970

–

13974

https://doi.org/10.1002/anie.200900683

141.

Chen

P.R.

,

Groff

D.

,

Guo

J.

,

Ou

W.

,

Cellitti

S.

,

Geierstanger

B.H.

et al.

(

2009

)

A facile system for encoding unnatural amino acids in mammalian cells

.

Angew. Chem. Int. Ed.

48

,

4052

–

4055

https://doi.org/10.1039/c2mb05321g

142.

Yanagisawa

T.

,

Hino

N.

,

Iraha

F.

,

Mukai

T.

,

Sakamoto

K.

and

Yokoyama

S.

(

2012

)

Wide-range protein photo-crosslinking achieved by a genetically encoded N(epsilon)-(benzyloxycarbonyl)lysine derivative with a diazirinyl moiety

.

Mol. Biosyst.

8

,

1131

–

1135

[PubMed]

https://doi.org/10.1038/srep36946

143.

Kita

A.

,

Hino

N.

,

Higashi

S.

,

Hirota

K.

,

Narumi

R.

,

Adachi

J.

et al.

(

2016

)

Adenovirus vector-based incorporation of a photo-cross-linkable amino acid into proteins in human primary cells and cancerous cell lines

.

Sci. Rep.

6

,

36946

[PubMed]

https://doi.org/10.1021/ja4013535

144.

Zhao

J.

,

Lin

S.

,

Huang

Y.

,

Zhao

J.

and

Chen

P.R.

(

2013

)

Mechanism-based design of a photoactivatable firefly luciferase

.

J. Am. Chem. Soc.

135

,

7410

–

7413

[PubMed]

https://doi.org/10.1002/cbic.201500204

145.

Ren

W.

,

Ji

A.

,

Wang

M.X.

and

Ai

H.-W.

(

2015

)

Expanding the Genetic Code for a Dinitrophenyl Hapten

.

ChemBioChem

16

,

2007

–

2010

[PubMed]

https://doi.org/10.1002/cbic.200900690

146.

Groff

D.

,

Chen

P.R.

,

Peters

F.B.

and

Schultz

P.G.

(

2010

)

A genetically encoded epsilon-N-methyl lysine in mammalian cells

.

ChemBioChem

11

,

1066

–

1068

[PubMed]

https://doi.org/10.1002/anie.201700683

147.

Erickson

S.B.

,

Mukherjee

R.

,

Kelemen

R.E.

,

Wrobel

C.J.J.

,

Cao

X.

and

Chatterjee

A.

(

2017

)

Precise photoremovable perturbation of a virus–host interaction

.

Angew. Chem. Int. Ed.

56

,

4234

–

4237

https://doi.org/10.1039/C6CC03934K

148.

Luo

J.

,

Arbely

E.

,

Zhang

J.

,

Chou

C.

,

Uprety

R.

,

Chin

J.W.

et al.

(

2016

)

Genetically encoded optical activation of DNA recombination in human cells

.

Chem. Commun.

52

,

8529

–

8532

https://doi.org/10.1021/sb400192a

149.

Engelke

H.

,

Chou

C.

,

Uprety

R.

,

Jess

P.

and

Deiters

A.

(

2014

)

Control of protein function through optochemical translocation

.

ACS Synth. Biol.

3

,

731

–

736

[PubMed]

https://doi.org/10.1021/ja512664v

150.

Hemphill

J.

,

Borchardt

E.K.

,

Brown

K.

,

Asokan

A.

and

Deiters

A.

(

2015

)

Optical control of CRISPR/Cas9 gene editing

.

J. Am. Chem. Soc.

137

,

5642

–

5645

[PubMed]

https://doi.org/10.1021/ja1109979

151.

Gautier

A.

,

Deiters

A.

and

Chin

J.W.

(

2011

)

Light-activated kinases enable temporal dissection of signaling networks in living cells

.

J. Am. Chem. Soc.

133

,

2124

–

2127

[PubMed]

https://doi.org/10.1021/ja4051026

152.

Hemphill

J.

,

Chou

C.

,

Chin

J.W.

and

Deiters

A.

(

2013

)

Genetically encoded light-activated transcription for spatiotemporal control of gene expression and gene silencing in mammalian cells

.

J. Am. Chem. Soc.

135

,

13433

–

13439

[PubMed]

https://doi.org/10.1021/ja5055862

153.

Luo

J.

,

Uprety

R.

,

Naro

Y.

,

Chou

C.

,

Nguyen

D.P.

,

Chin

J.W.

et al.

(

2014

)

Genetically encoded optochemical probes for simultaneous fluorescence reporting and light activation of protein function with two-photon excitation

.

J. Am. Chem. Soc.

136

,

15551

–

15558

[PubMed]

https://doi.org/10.1002/anie.201205352

154.

Yu

Z.

,

Pan

Y.

,

Wang

Z.

,

Wang

J.

and

Lin

Q.

(

2012

)

Genetically encoded cyclopropene directs rapid, photoclick-chemistry-mediated protein labeling in mammalian cells

.

Angew. Chem. Int. Ed.

51

,

10600

–

10604

https://doi.org/10.1021/acschembio.8b00571

155.

Meineke

B.

,

Heimgartner

J.

,

Lafranchi

L.

and

Elsässer

S.J.

(

2018

)

Methanomethylophilus alvus Mx1201 provides basis for mutual orthogonal pyrrolysyl tRNA/Aminoacyl-tRNA synthetase pairs in mammalian cells

.

ACS Chem. Biol.

13

,

3087

–

3096

[PubMed]

https://doi.org/10.1038/nbt.2860

156.

Elliott

T.S.

,

Townsley

F.M.

,

Bianco

A.

,

Ernst

R.J.

,

Sachdeva

A.

,

Elsässer

S.J.

et al.

(

2014

)

Proteome labeling and protein identification in specific tissues and at specific developmental stages in an animal

.

Nat. Biotechnol.

32

,

465

–

472

[PubMed]

https://doi.org/10.1002/cbic.201100194

157.

Ai

H.-W.

,

Shen

W.

,

Sagi

A.

,

Chen

P.R.

and

Schultz

P.G.

(

2011

)

Probing protein-protein interactions with a genetically encoded photo-crosslinking amino acid

.

ChemBioChem

12

,

1854

–

1857

[PubMed]

https://doi.org/10.1039/C0SC00373E

158.

Chou

C.

,

Uprety

R.

,

Davis

L.

,

Chin

J.W.

and

Deiters

A.

(

2011

)

Genetically encoding an aliphatic diazirine for protein photocrosslinking

.

Chem. Sci.

2

,

480

–

483

https://doi.org/10.1021/acs.biochem.8b00397

159.

Hoffmann

J.E.

,

Dziuba

D.

,

Stein

F.

and

Schultz

C.

(

2018

)

A bifunctional noncanonical amino acid: synthesis, expression, and residue-specific proteome-wide incorporation

.

Biochemistry

57

,

4747

–

4752

[PubMed]

https://doi.org/10.1038/srep46569

160.

Zhang

C.

,

Lu

J.

,

Su

H.

,

Yang

J.

and

Zhou

D.

(

2017

)

Fatty acid synthase cooperates with protrudin to facilitate membrane outgrowth of cellular protrusions

.

Sci. Rep.

7

,

46569

[PubMed]

https://doi.org/10.1002/anie.201309847

161.

Nikić

I.

,

Plass

T.

,

Schraidt

O.

,

Szymański

J.

,

Briggs

J.A.

,

Schultz

C.

et al.

(

2014

)

Minimal tags for rapid dual-color live-cell labeling and super-resolution microscopy

.

Angew. Chem. Int. Ed.

53

,

2245

–

2249

https://doi.org/10.1002/anie.201608284

162.

Nikić

I.

,

Estrada Girona

G.

,

Kang

J.H.

,

Paci

G.

,

Mikhaleva

S.

,

Koehler

C.

,

Shymanska

N.V.

et al.

(

2016

)

Debugging eukaryotic genetic code expansion for site-specific Click-PAINT super-resolution microscopy

.

Angew. Chem. Int. Ed.

55

,

16172

–

16176

https://doi.org/10.1002/cbic.201402189

163.

Rutkowska

A.

,

Plass

T.

,

Hoffmann

J.E.

,

Yushchenko

D.A.

,

Feng

S.

and

Schultz

C.

(

2014

)

T-CrAsH: a heterologous chemical crosslinker

.

ChemBioChem

15

,

1765

–

1768

[PubMed]

https://doi.org/10.1002/chem.201501647

164.

Hoffmann

J.E.

,

Plass

T.

,

Nikić

I.

,

Aramburu

I.V.

,

Koehler

C.

,

Gillandt

H.

et al.

(

2015

)

Highly stable trans-cyclooctene amino acids for live-cell labeling

.

Chem. Eur. J.

21

,

12266

–

12270

https://doi.org/10.1002/cbic.201600284

165.

Kozma

E.

,

Nikić

I.

,

Varga

B.R.

,

Aramburu

I.V.

,

Kang

J.H.

,

Fackler

O.T.

et al.

(

2016

)

Hydrophilic trans-cyclooctenylated noncanonical amino acids for fast intracellular protein labeling

.

ChemBioChem

17

,

1518

–

1524

[PubMed]

https://doi.org/10.1016/j.chembiol.2017.04.007

166.

Sakin

V.

,

Hanne

J.

,

Dunder

J.

,

Anders-Össwein

M.

,

Laketa

V.

,

Nikic

I.

et al.

(

2017

)

A versatile tool for live-cell imaging and super-resolution nanoscopy studies of hiv-1 env distribution and mobility

.

Cell Chem. Biol.

24

,

635

–

645

[PubMed]

https://doi.org/10.1021/ja302832g

167.

Lang

K.

,

Davis

L.

,

Wallace

S.

,

Mahesh

M.

,

Cox

D.J.

,

Blackman

M.L.

et al.

(

2012

)

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.

134

,

10317

–

10320

[PubMed]

https://doi.org/10.1021/ja512838z

168.

Uttamapinant

C.

,

Howe

J.D.

,

Lang

K.

,

Beránek

V.

,

Davis

L.

,

Mahesh

M.

et al.

(

2015

)

Genetic code expansion enables live-cell and super-resolution imaging of site-specifically labeled cellular proteins

.

J. Am. Chem. Soc.

137

,

4602

–

4605

[PubMed]

https://doi.org/10.1002/anie.201108231

169.

Plass

T.

,

Milles

S.

,

Koehler

C.

,

Szymański

J.

,

Mueller

R.

,

Wießler

M.

et al.

(

2012

)

Amino acids for Diels-Alder reactions in living cells

.

Angew. Chem. Int. Ed.

51

,

4166

–

4170

https://doi.org/10.1021/jacs.6b03034

170.

Xue

L.

,

Prifti

E.

and

Johnsson

K.

(

2016

)

A general strategy for the semisynthesis of ratiometric fluorescent sensor proteins with increased dynamic range

.

J. Am. Chem. Soc.

138

,

5258

–

5261

[PubMed]

https://doi.org/10.1002/cbic.201200407

171.

Borrmann

A.

,

Milles

S.

,

Plass

T.

,

Dommerholt

J.

,

Verkade

J.M.

,

Wiessler

M.

et al.

(

2012

)

Genetic encoding of a bicyclo[6.1.0]nonyne-charged amino acid enables fast cellular protein imaging by metal-free ligation

.

ChemBioChem

13

,

2094

–

2099

[PubMed]

https://doi.org/10.1091/mbc.e17-03-0161

172.

Schvartz

T.

,

Aloush

N.

,

Goliand

I.

,

Segal

I.

,

Nachmias

D.

,

Arbely

E.

et al.

(

2017

)

Direct fluorescent-dye labeling of alpha-tubulin in mammalian cells for live cell and superresolution imaging

.

Mol. Biol. Cell

28

,

2747

–

2756

[PubMed]

https://doi.org/10.1002/cbic.201700147

173.

Luo

J.

,

Torres-Kolbus

J.

,

Liu

J.

and

Deiters

A.

(

2017

)

Genetic encoding of photocaged tyrosines with improved light-activation properties for the optical control of protease function

.

ChemBioChem

18

,

1442

–

1447

[PubMed]

https://doi.org/10.1021/jacs.8b01087

174.

Wang

N.

,

Yang

B.

,

Fu

C.

,

Zhu

H.

,

Zheng

F.

,

Kobayashi

T.

et al.

(

2018

)

Genetically encoding fluorosulfate-l-tyrosine to react with lysine, histidine, and tyrosine via SuFEx in proteins in vivo

.

J. Am. Chem. Soc.

140

,

4995

–

4999

[PubMed]

https://doi.org/10.1002/cbic.201000436

175.

Takimoto

J.K.

,

Xiang

Z.

,

Kang

J.Y.

and

Wang

L.

(

2010

)

Esterification of an unnatural amino acid structurally deviating from canonical amino acids promotes its uptake and incorporation into proteins in mammalian cells

.

ChemBioChem

11

,

2268

–

2272

[PubMed]

https://doi.org/10.1016/j.chembiol.2018.05.013

176.

Beránek

V.

,

Reinkemeier

C.D.

,

Zhang

M.S.

,

Liang

A.D.

,

Kym

G.

and

Chin

J.W.

(

2018

)

Genetically encoded protein phosphorylation in mammalian cells

.

Cell Chem. Biol.

25

,

1067

–

1074

[PubMed]