An unnatural base pair for incorporating amino acid analogs into proteins

Hirao, Ichiro; Ohtsuki, Takashi; Fujiwara, Tsuyoshi; Mitsui, Tsuneo; Yokogawa, Tomoko; Okuni, Taeko; Nakayama, Hiroshi; Takio, Koji; Yabuki, Takashi; Kigawa, T.; Kodama, Kazuto; Yokogawa, Takashi; Nishikawa, Kazuya; Yokoyama, Shigeyuki

doi:10.1038/nbt0202-177

Cited by 262 publications

(177 citation statements)

References 51 publications

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“…At minimum, an unnatural base synthetically incorporated into a DNA template needs to specify the efficient in vitro transcription of unnatural basecontaining mRNA and tRNA. (This is similar to what Hirao et al have accomplished, although the tRNA preparation was somewhat complicated [94].) It would be considerably more convenient if the unnatural base could be inserted into DNA with PCR and/or plasmid replication, which requires applicable thermophilic and cellular polymerases.…”

Section: Recoding -Beyond Amber Suppressionsupporting

confidence: 50%

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Unnatural Protein Engineering: Producing Proteins with Unnatural Amino Acids

Magliery¹

2005

MCRO

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Less than a decade ago, the ability to generate proteins with unnatural modifications was a Herculean task available only to specialty labs. Recent advances make it possible to generate reasonable quantities of protein with unnatural amino acids both in vitro and in vivo . The combination of solid-phase peptide synthesis and enzymatic or chemoselective ligation now permits construction of entirely-synthetic proteins as large as 25 kD. Incorporation of recombinant fragments (expressed protein ligation) allows unnatural modifications near protein termini in proteins of virtually any size. Site-specific modification of small quantities of protein at any position can be achieved using chemically-acylated tRNA. Microelectroporation now extends this method to cells like mammalian neurons, and combination with RNAdisplay makes unnatural proteins compatible with combinatorial methods. Widespread or residue-specific methods of amino acid replacement are especially suitable for the production of biomaterials, and bacteria have been engineered to expand the repertoire of amino acids available for this technique. Excitingly, the wholesale addition of engineered tRNAs and synthetases to bacteria and yeast now makes site-specific incorporation of unnatural amino acids possible in living cells with no chemical intervention. Methods of expanding the genetic code at the nucleic acid level, including 4-base codons and unnatural base pairs, are becoming useful for the addition of multiple amino acids to the genetic code. These recent advances in unnatural protein engineering are reviewed with an eye toward future challenges, including methods of creating nonpeptidic molecules using templated synthesis.

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Section: Recoding -Beyond Amber Suppressionsupporting

confidence: 50%

“…Interestingly, this pair was used for in vitro site-specific unnatural amino acid incorporation [94]. DNA was synthesized to contain a CTs sequence that could be transcribed into a yUG codon by T7 RNA polymerase.…”

Section: Recoding -Beyond Amber Suppressionmentioning

confidence: 99%

Unnatural Protein Engineering: Producing Proteins with Unnatural Amino Acids

Magliery¹

2005

MCRO

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“…Within 3 days, 0.3-g yields of the ras(Am) products, including 3-iodo-L-tyrosine, were synthesized in a 30-l reaction mixture. The liquid chromatography electrospray mass spectrometry (LC-MS) analysis and the tandem MS sequencing were performed as described (13). The occupancy of iodotyrosine at the amber position was determined for the ras(Am) products on the basis of the relative abundance in the mass spectrometric analysis and the peak areas in liquid chromatogram by UV detection for the IY and Y fragments.…”

Section: In Vitro Analyses Of the Aminoacylation And Pyrophosphate-exmentioning

confidence: 99%

“…By using this feature, unnatural amino acids have been incorporated into proteins at the positions for the corresponding canonical amino acids in vivo (1,3,14,15) and in vitro (6,16). In addition, some nonphysiological conditions have been found to facilitate the aminoacylation of tRNA with unnatural amino acids (13). The site-specific incorporation of an unnatural amino acid has been attempted by using a wild-type aaRS.…”

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confidence: 99%

“…One method of attaching an unnatural amino acid to its adapter tRNA is chemical acylation (7), by which the aminoacylated forms of tRNAs corresponding to a special codon, such as amber codons (2,8,9), four-base codons (10,11), and artificial codons with unnatural bases (12,13), have actually been synthesized before their use in the cell-free translation (2, 4, 8-10, 12, 13) or in Xenopus oocytes (5). This adapter tRNA is engineered so it is not aminoacylated again by any aaRS involved in the translation system, to prevent the misincorporation of a canonical amino acid at the positions for the unnatural amino acid.…”

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confidence: 99%

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An engineered Escherichia coli tyrosyl–tRNA synthetase for site-specific incorporation of an unnatural amino acid into proteins in eukaryotic translation and its application in a wheat germ cell-free system

Kiga

Sakamoto

Kodama

et al. 2002

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

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161

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Tyrosyl-tRNA synthetase (TyrRS) from Escherichia coli was engineered to preferentially recognize 3-iodo-L-tyrosine rather than Ltyrosine for the site-specific incorporation of 3-iodo-L-tyrosine into proteins in eukaryotic translation systems. The wild-type TyrRS does not recognize 3-iodo-L-tyrosine, because of the bulky iodine substitution. On the basis of the reported crystal structure of Bacillus stearothermophilus TyrRS, three residues, Y37, Q179, and Q195, in the L-tyrosine-binding site were chosen for mutagenesis. Thirty-four single amino acid replacements and 16 of their combinations were screened by in vitro biochemical assays. A combination of the Y37V and Q195C mutations changed the amino acid specificity in such a way that the variant TyrRS activates 3-iodo-L-tyrosine 10-fold more efficiently than L-tyrosine. This engineered enzyme, TyrRS(V37C195), was tested for use in the wheat germ cell-free translation system, which has recently been significantly improved, and is now as productive as conventional recombinant systems. During the translation in the wheat germ system, an E. coli suppressor tRNA Tyr was not aminoacylated by the wheat germ enzymes, but was aminoacylated by the E. coli TyrRS(V37C195) variant with 3-iodo-L-tyrosine. After the use of the 3-iodotyrosyl-tRNA in translation, the resultant uncharged tRNA could be aminoacylated again in the system. A mass spectrometric analysis of the produced protein revealed that more than 95% of the amino acids incorporated for an amber codon were iodotyrosine, whose concentration was only twice that of L-tyrosine in the translation. Therefore, the variant enzyme, 3-iodo-L-tyrosine, and the suppressor tRNA can serve as an additional set orthogonal to the 20 endogenous sets in eukaryotic in vitro translation systems.

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Unnatural Nucleosides with Unusual Base Pairing Properties

Bergstrom

2009

CP Nucleic Acid Chemistry

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Synthetic modified nucleosides designed to pair in unusual ways with natural nucleobases have many potential applications in biology and biotechnology. This overview lays the foundation for future protocol units on synthesis and application of unnatural bases, with particular emphasis on unnatural base analogs that mimic natural bases in size, shape, and biochemical processing. Topics covered include base pairs with alternative H-bonding schemes, dimensionally expanded base pairs, hydrophobic base pairs, metal-ligated bases, degenerate bases, universal nucleosides, and triplex constituents.

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An unnatural base pair for incorporating amino acid analogs into proteins

Cited by 262 publications

References 51 publications

Unnatural Protein Engineering: Producing Proteins with Unnatural Amino Acids

Unnatural Protein Engineering: Producing Proteins with Unnatural Amino Acids

An engineered Escherichia coli tyrosyl–tRNA synthetase for site-specific incorporation of an unnatural amino acid into proteins in eukaryotic translation and its application in a wheat germ cell-free system

Unnatural Nucleosides with Unusual Base Pairing Properties

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