Incorporation of Unnatural Amino Acids in Response to the AGG Codon

Lee, Byeong Sung; Shin, Seunggun; Jeon, Jong Yeob; Jang, Kyoung‐Soon; Lee, Bun Yeol; Choi, Sangdun; Yoo, Tae Hyeon

doi:10.1021/acschembio.5b00230

Cited by 40 publications

(50 citation statements)

References 42 publications

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“…The cells were cultured in 2× YT medium at 37 °C until the OD 600 reached ~0.5, and then 1 mM isopropyl β-D-1-thiogalactopyranoside was added to induce expression of BMP2. In the case of BMP2-OpgY, the plasmid for BMP2-OpgY expression (pSPEL117) and the plasmid encoding an orthogonal pair of aminoacyl-tRNA synthetase and tRNA for incorporation of the amino acid analogue into the amber codon (pEVOL) were transformed into E. coli BL21(DE3) 45, 46 . We used a pEVOL plasmid encoding aminoacyl-tRNA synthetase to incorporate p-azido- l -phenylalanine (AzF) into the amber codon, because the enzyme was reported to be active toward OpgY as well as AzF 46 .…”

Section: Methodsmentioning

confidence: 99%

“…In the case of BMP2-OpgY, the plasmid for BMP2-OpgY expression (pSPEL117) and the plasmid encoding an orthogonal pair of aminoacyl-tRNA synthetase and tRNA for incorporation of the amino acid analogue into the amber codon (pEVOL) were transformed into E. coli BL21(DE3) 45, 46 . We used a pEVOL plasmid encoding aminoacyl-tRNA synthetase to incorporate p-azido- l -phenylalanine (AzF) into the amber codon, because the enzyme was reported to be active toward OpgY as well as AzF 46 . The cells were cultured in 2xYT medium at 37 °C until the OD 600 reached ~0.5, and then 0.2 % L-arabinose, 1 mM isopropyl β-D-1-thiogalactopyranoside and 1 mM OpgY were added to the culture to induce expression of aminoacyl-tRNA synthetase and BMP2-OpgY.…”

Section: Methodsmentioning

confidence: 99%

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BMP2-modified injectable hydrogel for osteogenic differentiation of human periodontal ligament stem cells

Park

Kwon

Lee

et al. 2017

Sci Rep

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This is the first report on the development of a covalently bone morphogenetic protein-2 (BMP2)-immobilized hydrogel that is suitable for osteogenic differentiation of human periodontal ligament stem cells (hPLSCs). O-propargyl-tyrosine (OpgY) was site-specifically incorporated into BMP2 to prepare BMP2-OpgY with an alkyne group. The engineered BMP2-OpgY exhibited osteogenic characteristics after in vitro osteogenic differentiation of hPLSCs, indicating the osteogenic ability of BMP2-OpgY. A methoxy polyethylene glycol-(polycaprolactone-(N3)) block copolymer (MC-N3) was prepared as an injectable in situ-forming hydrogel. BMP2 covalently immobilized on an MC hydrogel (MC-BMP2) was prepared quantitatively by a simple biorthogonal reaction between alkyne groups on BMP2-OpgY and azide groups on MC-N3 via a Cu(I)-catalyzed click reaction. The hPLSCs-loaded MC-BMP2 formed a hydrogel almost immediately upon injection into animals. In vivo osteogenic differentiation of hPLSCs in the MC-BMP2 formulation was confirmed by histological staining and gene expression analyses. Histological staining of hPLSC-loaded MC-BMP2 implants showed evidence of mineralized calcium deposits, whereas hPLSC-loaded MC-Cl or BMP2-OpgY mixed with MC-Cl, implants showed no mineral deposits. Additionally, MC-BMP2 induced higher levels of osteogenic gene expression in hPLSCs than in other groups. In conclusion, BMP2-OpgY covalently immobilized on MC-BMP2 induced osteogenic differentiation of hPLSCs as a noninvasive method for bone tissue engineering.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

BMP2-modified injectable hydrogel for osteogenic differentiation of human periodontal ligament stem cells

Park

Kwon

Lee

et al. 2017

Sci Rep

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“…Although the translation function of the AGG codon has been shown to permit efficient suppression with nonstandard amino acids (24)(25)(26), AGG necessarily remains translated as Arg in each of these studies. No study has yet demonstrated that all instances of any sense codon can be removed from essential genes.…”

Section: Significancementioning

confidence: 99%

Emergent rules for codon choice elucidated by editing rare arginine codons in Escherichia coli

Napolitano

Landon

Gregg

et al. 2016

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

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The degeneracy of the genetic code allows nucleic acids to encode amino acid identity as well as noncoding information for gene regulation and genome maintenance. The rare arginine codons AGA and AGG (AGR) present a case study in codon choice, with AGRs encoding important transcriptional and translational properties distinct from the other synonymous alternatives (CGN). We created a strain of Escherichia coli with all 123 instances of AGR codons removed from all essential genes. We readily replaced 110 AGR codons with the synonymous CGU codons, but the remaining 13 "recalcitrant" AGRs required diversification to identify viable alternatives. Successful replacement codons tended to conserve local ribosomal binding site-like motifs and local mRNA secondary structure, sometimes at the expense of amino acid identity. Based on these observations, we empirically defined metrics for a multidimensional "safe replacement zone" (SRZ) within which alternative codons are more likely to be viable. To evaluate synonymous and nonsynonymous alternatives to essential AGRs further, we implemented a CRISPR/Cas9-based method to deplete a diversified population of a wild-type allele, allowing us to evaluate exhaustively the fitness impact of all 64 codon alternatives. Using this method, we confirmed the relevance of the SRZ by tracking codon fitness over time in 14 different genes, finding that codons that fall outside the SRZ are rapidly depleted from a growing population. Our unbiased and systematic strategy for identifying unpredicted design flaws in synthetic genomes and for elucidating rules governing codon choice will be crucial for designing genomes exhibiting radically altered genetic codes.codon choice | genome editing | recoded genomes

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“…Successful Arg AGG codon reassignment in E. coli was subsequently reported with M. mazei PylRS/tRNA Pyl CCU 18 and Methanocaldococcus jannaschii TyrRS/tRNA Tyr CCU , 19 with efficiencies in the 80–83% range 18 and at the quantitative level. 19 In a recent study, complete reassignment of the Arg AGG codon in E. coli was achieved by converting all instances of the Arg AGG codon in essential genes to synonymous codons.…”

mentioning

confidence: 99%

“…19 In a recent study, complete reassignment of the Arg AGG codon in E. coli was achieved by converting all instances of the Arg AGG codon in essential genes to synonymous codons. 20 These levels of reassignment required the deletion or downregulation of competing endogenous tRNA Arg CCU , 18–20 the deletion of genes involved in arginine biosynthesis, 19 or providing a high concentration of the ncAA, 18 which can result in toxicity. These studies achieved efficient reassignment in large part due to the low codon usage of the rare Arg AGG codon (1491 instances in E. coli MG1655) and the fact that Arg AGG has the smallest pool size of endogenous tRNA isoacceptors (0.65% of total tRNAs, Figure 1).…”

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confidence: 99%

Efficient Reassignment of a Frequent Serine Codon in Wild-Type Escherichia coli

Reynolds

Rivera

et al. 2015

ACS Synth. Biol.

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Expansion of the genetic code through engineering the translation machinery has greatly increased the chemical repertoire of the proteome. This has been accomplished mainly by read-through of UAG or UGA stop codons by the noncanonical aminoacyl-tRNA of choice. While stop codon read-through involves competition with the translation release factors, sense codon reassignment entails competition with a large pool of endogenous tRNAs. We used an engineered pyrrolysyl-tRNA synthetase to incorporate 3-iodo-l-phenylalanine (3-I-Phe) at a number of different serine and leucine codons in wild-type Escherichia coli. Quantitative LC-MS/MS measurements of amino acid incorporation yields carried out in a selected reaction monitoring experiment revealed that the 3-I-Phe abundance at the Ser208AGU codon in superfolder GFP was 65 ± 17%. This method also allowed quantification of other amino acids (serine, 33 ± 17%; phenylalanine, 1 ± 1%; threonine, 1 ± 1%) that compete with 3-I-Phe at both the aminoacylation and decoding steps of translation for incorporation at the same codon position. Reassignments of different serine (AGU, AGC, UCG) and leucine (CUG) codons with the matching tRNAPyl anticodon variants were met with varying success, and our findings provide a guideline for the choice of sense codons to be reassigned. Our results indicate that the 3-iodo-l-phenylalanyl-tRNA synthetase (IFRS)/tRNAPyl pair can efficiently outcompete the cellular machinery to reassign select sense codons in wild-type E. coli.

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Incorporation of Unnatural Amino Acids in Response to the AGG Codon

Cited by 40 publications

References 42 publications

BMP2-modified injectable hydrogel for osteogenic differentiation of human periodontal ligament stem cells

BMP2-modified injectable hydrogel for osteogenic differentiation of human periodontal ligament stem cells

Emergent rules for codon choice elucidated by editing rare arginine codons in Escherichia coli

Efficient Reassignment of a Frequent Serine Codon in Wild-Type Escherichia coli

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