Authorship：Corresponding author  

DOI： https://doi.org/10.1002/anie.202206240

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.21769/BioProtoc.4125

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

We developed a cDNA TRAP display for the rapid selection of antibody-like proteins in various conditions. By modifying the original puromycin linker in the TRAP display, a monobody was covalently attached to the cDNA. As a proof-of-concept, we demonstrated a rapid model selection of an anti-EGFR1 monobody in a solution containing ribonuclease.

DOI： 10.1039/d0cc07541h

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society ({ACS})  

Preventing the escape of hazardous genes from genetically modified organisms (GMOs) into the environment is one of the most important issues in biotechnology research. Various strategies were developed to create "genetic firewalls" that prevent the leakage of GMOs; however, they were not specially designed to prevent the escape of genes. To address this issue, we developed amino acid (AA)-swapped genetic codes orthogonal to the standard genetic code, namely SL (Ser and Leu were swapped) and SLA genetic codes (Ser, Leu, and Ala were swapped). From mRNAs encoded by the AA-swapped genetic codes, functional proteins were only synthesized in translation systems featuring the corresponding genetic codes. These results clearly demonstrated the orthogonality of the AA-swapped genetic codes against the standard genetic code and their potential to function as "genetic firewalls for genes". Furthermore, we propose "a codon-bypass strategy" to develop a GMO with an AA-swapped genetic code.

DOI： 10.1021/acssynbio.0c00196

PubMed

Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Flexizymes are tRNA acylation ribozymes that have been successfully used to facilitate genetic code reprogramming. They are capable of charging acid substrates onto various tRNAs and tRNA analogues. However, their minimal RNA substrate has not been investigated. Here we have designed fluorescently labeled short RNAs corresponding to the four, three, and two bases (4bRNA, 3bRNA, 2bRNA) at the tRNA 3'-end and explored the minimal RNA substrate of flexizymes, dFx and eFx. 3bRNA was the observed minimal RNA substrate of the flexizymes, but the efficiency of acylation of this short RNA was two to three times lower than that of 4bRNA. The efficiency of acylation of 4bRNA was comparable with that of the microhelix, a 22-base RNA conventionally used as a tRNA analogue for analyzing acylation efficiency. We also compared the efficiencies of acylation of the microhelix and 4bRNA with various acid substrates. Thanks to the short length of 4bRNA, its acyl-4bRNA products exhibited larger mobility shifts in gel electrophoresis than those exhibited by acyl-microhelix products with every substrate tested. This indicated that 4bRNA was an ideal RNA substrate for analyzing the efficiency of acylation by flexizymes.

DOI： 10.1002/cbic.201900150

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer New York  

Constructing protein-coding genes with desired mutations is a basic step for protein engineering. Herein, we describe a multiple site-directed and saturation mutagenesis method, termed MUPAC. This method has been used to introduce multiple site-directed mutations in the green fluorescent protein gene and in the moloney murine leukemia virus reverse transcriptase gene. Moreover, this method was also successfully used to introduce randomized codons at five desired positions in the green fluorescent protein gene, and for simple DNA assembly for cloning.

DOI： 10.1007/978-1-4939-6472-7_22

Scopus

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1007/s12010-016-2187-4

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media {LLC}  

In ribosomal polypeptide synthesis the library of amino acid building blocks is limited by the manner in which codons are used. Of the proteinogenic amino acids, 18 are coded for by multiple codons and therefore many of the 61 sense codons can be considered redundant. Here we report a method to reduce the redundancy of codons by artificially dividing codon boxes to create vacant codons that can then be reassigned to non-proteinogenic amino acids and thereby expand the library of genetically encoded amino acids. To achieve this, we reconstituted a cell-free translation system with 32 in vitro transcripts of transfer RNA(SNN) (tRNA(SNN)) (S = G or C), assigning the initiator and 20 elongator amino acids. Reassignment of three redundant codons was achieved by replacing redundant tRNA(SNN)s with tRNA(SNN)s pre-charged with non-proteinogenic amino acids. As a demonstration, we expressed a 32-mer linear peptide that consists of 20 proteinogenic and three non-proteinogenic amino acids, and a 14-mer macrocyclic peptide that contains more than four non-proteinogenic amino acids.

DOI： 10.1038/nchem.2446

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society ({ACS})  

The compatibility of beta-amino acids with ribosomal translation was studied for decades, but it has been still unclear whether the ribosome can accept various beta-amino acids, and whether the ribosome can introduce multiple beta-amino acids in a peptide. In the present study, by using the Escherichia coli reconstituted cell-free translation system with a reprogramed genetic code, we screened beta-amino acids that give high single incorporation efficiency and used them to synthesize peptides containing multiple beta-amino acids. The experiments of single beta-amino acid incorporation into a peptide revealed that 13 beta-amino acids are compatible with ribosomal translation. Six of the tested beta-amino acids (beta hGly, L-beta hAla, L-beta hPhg, L-beta hMet, and D-beta hPhg) showed high incorporation efficiencies, and seven (L-beta hLeu, L-beta hIle, L-beta hAsn, L-beta hPhe, L-beta hLys, D-beta hAla, and D-beta hLeu) showed moderate incorporation efficiencies; whereas no full-length peptide was produced using other beta-amino acids (L-beta hPro, L-beta hTrp, and L-beta hGlu). Subsequent double-incorporation experiments using beta-amino acids with high single incorporation efficiency revealed that elongation of peptides with successive beta-amino acids is prohibited. Efficiency of the double-incorporation of the beta-amino acids was restored by the insertion of Tyr or Ile between the two beta-amino acids. On the basis of these experiments, we also designed mRNA sequences of peptides, and demonstrated the ribosomal synthesis of peptides containing different types of beta-amino acids at multiple positions.

DOI： 10.1021/jacs.5b12482

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Wiley  

The potential applications of non-proteinogenic amino acids have increased continuously since the introduction of these molecules into a ribosomal translation system. An increasing number of studies concerning topics, such as the addition of an artificial function to a protein, cellular expression of a protein with an artificial residue, and development of an artificial peptide with a novel function, have been done using these molecules. Here, we describe recent studies that elucidate the compatibility of non-proteinogenic amino acids with ribosomal translation. We also describe the development of a simple and high-speed selection method and its potential application for the creation of a novel functional peptide with non-proteinogenic amino acids. As these studies have expanded the diversity of the artificial peptide library and increased the speed of novel functional peptide selection, they will significantly facilitate the development of new molecules, such as pharmaceutical drug candidates and bioassay probes.

DOI： 10.1002/tcr.201500239

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： https://doi.org/10.1039/C3SC52744A

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Oxford University Press ({OUP})  

<jats:title>Abstract</jats:title>
<jats:p>Low-density lipoprotein receptor (LDLR) mRNA is unstable, but is stabilized upon extracellular signal-regulated kinase (ERK) activation, possibly through the binding of certain proteins to the LDLR mRNA 3′-untranslated region (UTR), although the detailed mechanism underlying this stability control is unclear. Here, using a proteomic approach, we show that proteins ZFP36L1 and ZFP36L2 specifically bind to the 3′-UTR of LDLR mRNA and recruit the CCR4-NOT-deadenylase complex, resulting in mRNA destabilization. We also show that the C-terminal regions of ZFP36L1 and ZFP36L2 are directly phosphorylated by p90 ribosomal S6 kinase, a kinase downstream of ERK, resulting in dissociation of the CCR4-NOT-deadenylase complex and stabilization of LDLR mRNA. We further demonstrate that targeted disruption of the interaction between LDLR mRNA and ZFP36L1 and ZFP36L2 using antisense oligonucleotides results in upregulation of LDLR mRNA and protein. These results indicate that ZFP36L1 and ZFP36L2 regulate LDLR protein levels downstream of ERK. Our results also show the usefulness of our method for identifying critical regulators of specific RNAs and the potency of antisense oligonucleotide-based therapeutics.</jats:p>

DOI： 10.1093/nar/gku652

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Royal Society of Chemistry ({RSC})  

DOI： 10.1039/c3sc52744a

Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society ({ACS})  

We here demonstrate a translation system that is governed by a reprogrammed genetic code consisting of "dual sense codons." A dual sense codon assigns two distinct amino acids for initiation and elongation. Because multiple dual sense codons independently function without cross-readings, this system enables the expansion of the repertoire of initiators as well as elongators that can be used simultaneously.

DOI： 10.1021/cb400549p

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media {LLC}  

<jats:title>Abstract</jats:title>
<jats:sec>
<jats:title>Background</jats:title>
<jats:p>Various DNA manipulation methods have been developed to prepare mutant genes for protein engineering. However, development of more efficient and convenient method is still demanded. Homologous DNA assembly methods, which do not depend on restriction enzymes, have been used as convenient tools for cloning and have been applied to site-directed mutagenesis recently. This study describes an optimized homologous DNA assembly method, termed as multiple patch cloning (MUPAC), for multiple site-directed and saturation mutagenesis.</jats:p>
</jats:sec>
<jats:sec>
<jats:title>Results</jats:title>
<jats:p>To demonstrate MUPAC, we introduced five back mutations to a mutant green fluorescent protein (GFPuv) with five deleterious mutations at specific sites and transformed <jats:italic>Escherichia coli</jats:italic> (<jats:italic>E. coli</jats:italic>) with the plasmids obtained. We observed that the over 90% of resulting colonies possessed the plasmids containing the reverted GFPuv gene and exhibited fluorescence. We extended the test to introduce up to nine mutations in Moloney Murine Leukemia Virus reverse transcriptase (M-MLV RT) by assembling 11 DNA fragments using MUPAC. Analysis of the cloned plasmid by electrophoresis and DNA sequencing revealed that approximately 30% of colonies had the objective mutant M-MLV RT gene. Furthermore, we also utilized this method to prepare a library of mutant GFPuv genes containing saturation mutations at five specific sites, and we found that MUPAC successfully introduced NNK codons at all five sites, whereas other site remained intact.</jats:p>
</jats:sec>
<jats:sec>
<jats:title>Conclusions</jats:title>
<jats:p>MUPAC could efficiently introduce various mutations at multiple specific sites within a gene. Furthermore, it could facilitate the preparation of experimental gene materials important to molecular and synthetic biology research.</jats:p>
</jats:sec>

DOI： 10.1186/1472-6750-13-91

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society ({ACS})  

Cyclic structures can increase the proteolytic stability and conformational rigidity of peptides, and N-alkylation of the peptide backbone can make peptides more cell-permeable and resistant to proteolysis. Therefore, cyclic N-alkyl amino acids are expected to be useful building blocks to increase simultaneously these pharmacological properties of peptides. In this study, we screened various cyclic N-alkyl amino acids for their ribosomal incorporation into peptides and identified cyclic N-alkyl amino acids that can be efficiently and successively incorporated. We also demonstrated genetic code reprogramming for reassigning 16 NNU codons to 16 different cyclic N-alkyl amino acids with high fidelity to synthesize highly N-alkylated polycyclic peptidomimetics and an mRNA-displayed library of completely N-alkylated polycyclic peptidomimetics by using our recently developed TRAP (transcription/translation coupled with association of puromycin linker) display. In vitro selection from a highly diverse library of such completely N-alkylated polycyclic peptidomimetics could become a powerful means to discover small-molecule ligands such as drug candidates that can be targeted to biomolecules inside living cells.

DOI： 10.1021/ja405044k

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society ({ACS})  

We report the in vitro selection of thioether-macrocyclized peptides against vascular endothelial growth factor receptor 2 (VEGFR2) from multiple, highly diverse peptide libraries constructed utilizing genetic code reprogramming. The macrocyclic peptide libraries consisted of combinations of four types of amino acid linkers for cyclization and two types of elongator amino acid compositions, including four backbone-modified non-proteinogenic amino acids. Affinity selection from these libraries, using our recently developed TRAP (Tran scription-translation coupled with Association of Puromycin-linker) display, yielded multiple anti-VEGFR2 macrocydic peptide leads. Further antagonizing activity-based screening of the chemically synthesized lead peptides identified a potent macrocyclic peptide that inhibited VEGF-induced VEGFR2 autophosphorylation, proliferation, and angiogenesis of living vascular endothelial cells. The TRAP display-based selection from multiple, highly diverse peptide libraries followed by activity-based screening of selected peptides is a powerful strategy for discovering biologically active peptides targeted to various biomolecules.

DOI： 10.1021/cb300697h

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society ({ACS})  

Here, we describe a novel method that enables highspeed in vitro selection of functional peptides, peptidomimetics, and proteins via a simple procedure. We first developed a new cell-free translation system, the TRAP system (transcription translation coupled with association of Euromycin linker), which automatically produces a polypeptide library through a series of sequential reactions: transcription, association of puromycin-DNA linker, translation, and conjugation between the nascent polypeptide and puromycin-DNA linker. We then applied the TRAP system for the selection of macrocyclic peptides against human serum albumin. Six rounds of selection using TRAP display were performed in approximately 14 h, yielding macrocyclic peptides with nanomolar affinity to their target protein. Because TRAP display enables high-speed selection of functional polypeptides, it will facilitate the generation of various polypeptides that are useful for biological and therapeutic applications.

DOI： 10.1021/ja312579u

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society ({ACS})  

The compatibility of D-amino acids with peptide elongation during translation has been examined in several studies. However, some of the studies have reported that D-amino acids are incompatible with translation, whereas others have reported that D-amino acids are incorporated into polypeptides. Here, we have reevaluated the incorporation of a series of D-amino acids into the nascent chain of short peptides with a reprogrammed genetic code by using the flexible in vitro translation (FIT) system. The FIT system enables the compatibility of each D-amino acid with elongation to be assessed quantitatively in the absence of potential competitors. The incorporation efficiencies were determined by Tricine-SDS-PAGE and the full-length peptide was detected by MALDI-TOF-MS. The D-amino acids were categorized into three groups based on their incorporation efficiencies relative to the corresponding L-amino acid. The D-isomers in group I showed efficiencies of 40% or higher (Ala, Ser, Cys, Met, Thr, His, Phe, and Tyr), and those in group H showed efficiencies of 10-40% (Asn, Gln, Val, and Leu). The D-amino acids in group III produced truncated peptides or no detectable full-length peptides (Arg, Lys, Asp, Glu, Ile, Trp, and Pro). When group I D-amino acids were used consecutively or were alternated with L-amino acids, this completely inhibited their elongation. However, when two or three L-amino acids were inserted between the D-amino acids, the double-incorporation efficiency was restored. Our results quantitatively reveal the compatibility of D-amino acids with peptide elongation and raise new questions about the mechanism of D-amino acid selection and incorporation by the ribosome.

DOI： 10.1021/ja309570x

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1155/2013/219263

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Hindawi Limited  

<jats:p>The presence of a nonproteinogenic moiety in a nonstandard peptide often improves the biological properties of the peptide. Non-standard peptide libraries are therefore used to obtain valuable molecules for biological, therapeutic, and diagnostic applications. Highly diverse non-standard peptide libraries can be generated by chemically or enzymatically modifying standard peptide libraries synthesized by the ribosomal machinery, using posttranslational modifications. Alternatively, strategies for encoding non-proteinogenic amino acids into the genetic code have been developed for the direct ribosomal synthesis of non-standard peptide libraries. In the strategies for genetic code expansion, non-proteinogenic amino acids are assigned to the nonsense codons or 4-base codons in order to add these amino acids to the universal genetic code. In contrast, in the strategies for genetic code reprogramming, some proteinogenic amino acids are erased from the genetic code and non-proteinogenic amino acids are reassigned to the blank codons. Here, we discuss the generation of genetically encoded non-standard peptide libraries using these strategies and also review recent applications of these libraries to the selection of functional non-standard peptides.</jats:p>

DOI： 10.1155/2012/713510

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media {LLC}  

We report a methodology for the ribosomal synthesis of backbone-cyclized peptides involving genetic code reprogramming to introduce one or more nonproteinogenic amino acids. Expression of linear peptides bearing a cysteine-proline dipeptide sequence followed by glycolic acid results in self-rearrangement to a C-terminal diketopiperadine-thioester, which non-enzymatically generates a cyclized peptide. We demonstrate the ribosomal synthesis of several naturally occurring backbone-cyclized peptides and a library based on a bicyclic scaffold, and we identify bioactive sequences by screening and deconvolution.

DOI： 10.1038/nchembio.259

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier {BV}  

We have developed a new flexizyme (a flexible de novo tRNA acylation ribozyme) system, a pair of amino-derivatized benzyl thioester (ABT) and amino flexizyme (aFx). ABT bearing the ammonium ion was designed to render the acyl-donor substrates better water solubility. Although the previously reported flexizymes (eFx and dFx) did not show acylation activity for the ABT derivatives, a new flexizyme variant aFx, generated by in vitro selection against an amino acid activated ABT, exhibits high selectivity toward those activated ABT. The flexizymes system including aFx, eFx, and dFx enables us to prepare a wide variety of acyl-tRNAs charged with non-proteinogenic amino acids. (C) 2009 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.bmcl.2009.03.114

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Wiley  

DOI： 10.1002/cbic.200900021

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Wiley  

The C terminus of a peptide expressed by the translation apparatus generally ends in a carboxylate group. On the other hand, the C termini of some naturally occuring peptides have amide moieties instead of carboxylates, which are believed to give better biostability. Here, we describe a new strategy for the ribosomal synthesis of peptides featuring C-terminal lactam, thiolactone, and alkylamide units. The method was based on the concept of genetic code reprogramming involving the flexizymes (flexible tRNA acylation ribozymes) and the PURE (peptide synthesis using recombinant elements) system, in which vacant codons are reassingned to nonproteinogenic amino acids, this enabled us to convert the C termini of peptides into the above functionalities. We have also applied this method to the synthesis of a macrocyclic peptide closed by an amide bond formed between a lysine side chain and the peptide C terminus. This method thus offers us new opportunities to express various peptides using the translation apparatus, and potentially to accelerate the discovery of peptide drugs designed for various therapeutic targets.

DOI： 10.1002/cbic.200900058

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media {LLC}  

The bases at positions 32 and 38 in the tRNA anticodon loop are known to have a specific conservation depending upon the anticodon triplets. Here we report that evolutionarily conserved pairs of bases at positions 32 and 38 in tRNA(GGC)(Ala) prevent misreading of a near-cognate valine codon, GUC. The tRNA(GGC)(Ala) molecules with the conserved A32-U38 and C32-G38 pairs do not read GUC, whereas those with three representative nonconserved pairs, U32-U38, U32-A38 and C32-A38, direct the misincorporation of alanine at this valine codon into the peptide chain. Overexpression of the nonconserved tRNA(GGC)(Ala) in Escherichia coli is toxic and prevents cell growth. These results suggested that the bases at positions 32 and 38 in tRNA(GGC)(Ala) evolved to preserve the fidelity of the cognate codon reading.

DOI： 10.1038/nsmb.1580

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Royal Society of Chemistry ({RSC})  

We report here the ribosomal synthesis of methyllanthionine-containing cyclic peptides involving a site-specific incorporation of vinylglycine under the reprogrammed genetic code, followed by the isomerization of the vinylglycine to dehydrobutyrine, and the subsequent intramolecular Michael addition of a cysteine residue placed at a downstream position of the vinylglycine.

DOI： 10.1039/b904314d

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society ({ACS})  

Here we report a new methodology for mRNA-programmed synthesis of peptoids and peptoid-peptide hybrids by means of translation machinery under the reprogrammed genetic code. We initially screened N-substituted glycines (rGly) for their single incorporation into a nascent peptide chain and found translation machinery accepts a variety of rGly for elongation. Moreover, we have shown consecutive elongations of rGly and mRNA-directed synthesis of cyclic peptoid-peptide hybrids. This methodology offers a powerful tool for mRNA-programmed library synthesis of peptoids and peptoid-peptide hybrids with linear and cyclic scaffolds, potentially leading to the discovery of drug candidates with proteolytic stability and membrane permeability.

DOI： 10.1021/ja806998v

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Wiley  

Over 30 years ago, Fahnestock and Rich reported intriguing data showing the capability of the ribosome to polymerize phenyllactic acid. Although the polymerization was initiated and terminated randomly on polyuridic acids, the given data convincingly suggested that the generated polymer was composed of an approximately 7:3 mixture of phenyllactic acid and phenylalanine. Despite the fact that Fahnestock&apos;s conclusion was very likely correct, there have been no reports to follow up the ribosome-catalyzed polymerization of alpha-hydroxy acids until very recently. At the end of 2007, we reported messenger RNA (mRNA)-directed polyester synthesis by using the new emerging method of generic-code reprogramming in which alpha-hydroxy acids with various kinds of side-chains. are assigned to arbitrarily chosen codons. In this work, we have achieved the ribosomal synthesis of polyesters with the sequence composition and length in a fully controlled manner according to the sequence of mRNA. This Concept article describes the background of the method development and its application to the synthesis of polyesters.

DOI： 10.1002/cbic.200800439

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media {LLC}  

In modern organisms, protein enzymes are solely responsible for the aminoacylation of transfer RNA. However, the evolution of protein synthesis in the RNA world required RNAs capable of catalysing this reaction. Ribozymes that aminoacylate RNA by using activated amino acids have been discovered through selection in vitro(1-5). Flexizyme is a 45- nucleotide ribozyme capable of charging tRNA in trans with various activated L- phenylalanine derivatives. In addition to a more than 10(5) rate enhancement and more than 10(4)-fold discrimination against some non- cognate amino acids, this ribozyme achieves good regioselectivity: of all the hydroxyl groups of a tRNA, it exclusively aminoacylates the terminal 3&apos;-OH(5-7). Here we report the 2.8-angstrom resolution structure of flexizyme fused to a substrate RNA. Together with randomization of ribozyme core residues and reselection, this structure shows that very few nucleotides are needed for the aminoacylation of specific tRNAs. Although it primarily recognizes tRNA through base- pairing with the CCA terminus of the tRNA molecule, flexizyme makes numerous local interactions to position the acceptor end of tRNA precisely. A comparison of two crystallographically independent flexizyme conformations, only one of which appears capable of binding activated phenylalanine, suggests that this ribozyme may achieve enhanced specificity by coupling active- site folding to tRNA docking. Such a mechanism would be reminiscent of the mutually induced fit of tRNA and protein employed by some aminoacyl- tRNA synthetases(8,9) to increase specificity.

DOI： 10.1038/nature07033

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Cold Spring Harbor Laboratory  

<jats:p>Here we report experimental evidence that the translation initiation apparatus accepts <jats:italic>D</jats:italic>-amino acids (<jats:italic><jats:sup>D</jats:sup></jats:italic>aa), as opposed to only <jats:italic>L</jats:italic>-methionine, as initiators. Nineteen <jats:italic><jats:sup>D</jats:sup></jats:italic>aa, as the stereoisomers to their natural <jats:italic>L</jats:italic>-amino acids, were charged onto initiator tRNA<jats:sup>fMet</jats:sup><jats:sub>CAU</jats:sub> using flexizyme technology and tested for initiation in a reconstituted <jats:italic>Escherichia coli</jats:italic> translation system lacking methionine, i.e., the initiator was reprogrammed from methionine to <jats:italic><jats:sup>D</jats:sup></jats:italic>aa. Remarkably, all <jats:italic><jats:sup>D</jats:sup></jats:italic>aa could initiate translation while the efficiency of initiation depends upon the type of side chain. The peptide product initiated with <jats:italic><jats:sup>D</jats:sup></jats:italic>aa was generally in a nonformylated form, indicating that methionyl-tRNA formyltransferase poorly formylated the corresponding <jats:italic><jats:sup>D</jats:sup></jats:italic>aa-tRNA<jats:sup>fMet</jats:sup><jats:sub>CAU</jats:sub>. Although the inefficient formylation of <jats:italic><jats:sup>D</jats:sup></jats:italic>aa-tRNA<jats:sup>fMet</jats:sup><jats:sub>CAU</jats:sub> resulted in modest expression of the corresponding peptide, preacetylation of <jats:italic><jats:sup>D</jats:sup></jats:italic>aa-tRNA<jats:sup>fMet</jats:sup><jats:sub>CAU</jats:sub> dramatically increased expression level, implying that the formylation efficiency is one of the critical determinants of initiation efficiency with <jats:italic><jats:sup>D</jats:sup></jats:italic>aa. Our findings provide not only the experimental evidence that translation initiation tolerates <jats:italic><jats:sup>D</jats:sup></jats:italic>aa, but also a new means for the mRNA-directed synthesis of peptides capped with <jats:italic><jats:sup>D</jats:sup></jats:italic>aa or acyl-<jats:italic><jats:sup>D</jats:sup></jats:italic>aa at the N terminus.</jats:p>

DOI： 10.1261/rna.1020708

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society ({ACS})  

Here we report a new methodology for the synthesis of bicyclic peptides by using a reconstituted cell-free translation system under the reprogrammed genetic code. Cysteine (Cys) and three different nonproteinogenic amino acids, Cab, Aha, and PgI, were simultaneously incorporated into a peptide chain. The first cyclization occurred between the chloroacetyl group of Cab and the sulfhydryl group in Cys in situ of translation, and the second cyclization on the side chains of Aha-Pgl via Cu(I)-catalyzed azide-alkyne cycloaddition was performed. This offers us a powerful means of mRNA-programmed synthesis of various peptides with uniform bicyclic scaffolds.

DOI： 10.1021/ja800953c

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society ({ACS})  

Here we report a new enabling technology for the synthesis of peptidase-resistant cyclic peptides by means of genetic code reprogramming involving the flexizyme (a tRNA acylation ribozyme) and PURE (a reconstituted cell-free translation) systems. in this work, we have developed a new nonproteinogenic amino acid bearing a chloroacetyl group in the side chain, which forms a physiologically stable thioether bond by intramolecular reaction with the sulfhydryl group of a Cys residue in the peptide chain upon translation. Significantly, this chemistry takes place spontaneously in situ of the translation solution, giving the corresponding cyclic peptides independent of ring sizes. We have used this method to convert human urotensin 11, known as a potent vasoconstrictor, to its analogue containing a thioether bond, showing that this new analogue retains biological activity. Moreover, this peptide exhibits remarkable resistance against peptidases under reducing conditions. Thus, this technology offers a new means to accelerate the discovery of therapeutic peptidic drugs.

DOI： 10.1021/cb800010p

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

The initiation codon dictates that the translation initiation event exclusively begins with methionine. We report here a new technology to reprogram the initiation event, where various amino acids and those bearing N-alpha-acyl groups can be used as an initiator for peptide synthesis. The technology is built upon the concept of genetic code reprogramming, where methionine is depleted from the translation system and the initiation codon is reassigned to the desired amino acid. We have applied this technology to the synthesis of an antitumor cyclic peptide, G7-18NATE, closed by a physiologically stable bond, and it is also extended to the custom synthesis of its analogues with various ring sizes. Significantly, cyclization occurs spontaneously upon translation of the precursor linear peptides. To demonstrate the practicality of this methodology, we also prepared a small cyclic peptide library designated by 160 distinct mRNAs. Thus, this technology offers a new means to prepare a wide array of in vivo compatible cyclic peptide libraries for the discovery of peptidic drug candidates against various therapeutic targets.

DOI： 10.1021/c6700233t

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CELL PRESS  

Natural peptide products often contain N-methylated backbones, and such a modification plays a crucial role in making natural peptides peptidase resistant and membrane permeable. Here, we demonstrate the ribosomal synthesis of N-methyl-peptides by means of genetic code reprogramming. Two key technologies, a ribozyme-based de novo tRNA acylation (flexizyme) system and an E coli reconstituted cell-free translation (PURE) system, were used in order to reassign arbitrarily chosen codons to N-alpha-methylated amino acids ((Me)aa). Using this combination, we determined the general structural requirement of "accessible" Meaa and demonstrated their multiple incorporations into the nascent peptide chain according to the assignments made on mRNA, giving linear and cyclic N-methyl-peptides in high purities. This platform technology offers a convenient tool for the construction of N-methyl-peptide libraries, potentially leading to the discovery of therapeutic peptides.

DOI： 10.1016/j.chembiol.2007.12.008

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PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier {BV}  

Here we report the ribosomal polymerization of alpha-hydroxy acids by means of genetic code reprogramming. The flexizyme system, a ribozyme-based tRNA acylation tool, was used to reassign individual codons to seven types of a-hydroxy acids, and then polyesters were synthesized under controls of the reprogrammed genetic code using a reconstituted cell-free translation system. The sequence and length of the polyester segments were specified by the mRNA template, indicating that high-fidelity ribosome expression of polyesters was possible. This work opens a door for the mRNA-directed synthesis of backbone-altered biopolymers.

DOI： 10.1016/j.chembiol.2007.10.015

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier {BV}  

Flexizymes are de novo ribozymes capable of charging a wide variety of non-natural amino acids on tRNAs. The flexizyme system enables reprogramming of the genetic code by reassigning the codons that are generally assigned to natural amino acids to non-natural residues, and thus mRNA-directed synthesis of non-natural polypeptides can be achieved. In this review, we comprehensively summarize the history of the flexizyme system and its subsequent development into a practical tool. Furthermore, applications to the synthesis of novel biopolymers via genetic code reprogramming and perspectives for future applications are described.

DOI： 10.1016/j.cbpa.2007.08.011

Web of Science

PubMed

Language：Japanese   Publishing type：Research paper (scientific journal)  

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

The translation apparatus is sophisticated machinery that polymerizes amino acids to polypeptide according to the genetic triplets encoded on messenger RNA (mRNA) sequence. Because of this property, ribosome generally acts as a superb template-directed polymer synthesizer and therefore translated polypeptide shows narrow distribution of molecular weights with complete controlled sequence. Unfortunately, this superior system is limited to polypeptide synthesis because of the lack of genetic code which converts genetic triplets to any monomers except for 20 natural amino acids. To expand the application of ribosome, we utilized the concept of genetic code reprogramming to assign several codons as hydroxy acids and performed mRNA-templated polyester synthesis. We have demonstrated the sequence and length of polyesters can be controlled by the mRNA sequence as designed. This is a novel technology for the synthesis of polyester and polyester-polypeptide copolymer for the investigation of new materials. © 2007, The Society of Polymer Science, Japan. All rights reserved.

DOI： 10.1295/kobunshi.56.196

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1093/nass/nrm058

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1093/nass/nrm181

PubMed

Language：Japanese   Publishing type：Research paper (scientific journal)  

PubMed

Language：Japanese   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Wiley  

The protein no longer terminates here. Non-natural amino acids were attached onto a tRNA and were then transferred with the aid of L/F-tRNA-protein transferase to the N termini of proteins possessing N-terminal Lys or Arg components. By this new technique, 1- and 2-naphthylalanine or 3-nitrotyrosine were coupled to the N termini of several proteins. A novel and convenient method for adding single Lys units at the N termini of various proteins was also developed.

DOI： 10.1002/cbic.200600181

Web of Science

PubMed

Language：Japanese   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media {LLC}  

Here we describe a de novo tRNA acytation system, the flexizyme (Fx) system, for the preparation of acyl tRNAs with nearly unlimited selection of amino and hydroxy acids and tRNAs. The combination of the Fx system with an appropriate cell-free translation system allows us to readily perform mRNA-encoded synthesis of proteins and short polypeptides involving multiple non-natural amino acids.

DOI： 10.1038/nmeth877

Web of Science

PubMed

Language：Japanese   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Here we describe a de novo tRNA acylation system consisting of artificial ribozymes. This system, the flexizyme (Fx) system, allows for the preparation of acyl-tRNAs with almost no limitation on the use of a variety of amino/hydroxy acids and tRNAs. To demonstrate its utility, we have carried out protein synthesis involving site-specific incorporation of nonnatural amino and hydroxy acids via amber or programmed frame-shift suppressions. We have also demonstrated peptide synthesis involving multiple nonnatural amino acids via sense codon suppression. The combination of the Fx system and appropriate cell-free translation is a powerful and flexible tool for mRNA-encoded synthesis of nonnatural polypeptides.

DOI： 10.1093/nass/nrl018

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1093/nass/nrl149

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

Bioactive peptides isolated from natural sources have diverse acyl groups on the N-terminus. It is difficult to synthesize these peptides in vitro translation system because ribosomal peptide synthesis generally limits the N-terminal group to be N-formylmethionine (fMet). To overcome this restriction, we developed a novel methodology for the ribosomal synthesis of peptides having various terminal N-acyl groups with desired amino acids. In this methodology, two technologies, Flexizyme system consisting of artificial ribozymes and a reconstitute E. coli cell-free translation system (PURE system), were used. First, an amino acid carrying a desired N-acyl group was charged onto an initiation tRNA by the Flexizyme system. The addition of this N-acyl-aminoacyl-tRNA (N-acyl-aa-tRNA) to the PURE system allowed us to initiate the peptide synthesis with the designated N-acyl-amino acid. By means of this methodology, the translation was exclusively initiated by various N-terminal acyl groups as well as amino acids without contamination of N-formylmethionine.

DOI： 10.1093/nass/nrl146

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier {BV}  

Here we describe a novel technology that allows users to charge nonnatural amino acids onto any tRNA. This technology is based on a resin-immobilized ribozyme system, called Flexiresin. It enables users to readily and rapidly synthesize misacylated tRNAs with a wide variety of phenylalanine analogs. Since Flexiresin is reusable and little effort is necessary for regeneration, it is economical and convenient. Moreover, it can adapt to virtually any tRNA chosen by the user, and can therefore be applied to not only a single site mutation but also multiple sites with designated nonnatural amino acids when both the amber and programmed frame-shift mutations are utilized. The original ribozyme utilized for Flexiresin was artificially generated in vitro, and thus the technology in principle could be broadened from Phe analogues to essentially any amino acid. (c) 2005 Published by Elsevier Inc.

DOI： 10.1016/j.ymeth.2005.04.001

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society ({ACS})  

DOI： 10.1021/ja046843y

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Wiley  

<jats:p>Novel non‐natural amino acids carrying a dansyl fluorescent group were designed, synthesized, and incorporated into various positions of streptavidin by using a CGGG four‐base codon in an <jats:italic>Escherichia coli</jats:italic> in vitro translation system. 2,6‐Dansyl‐aminophenylalanine (2,6‐dnsAF) was found to be incorporated into the protein more efficiently than 1,5‐dansyl‐lysine, 2,6‐dansyl‐lysine, and 1,5‐dansyl‐aminophenylalanine. Fluorescence measurements indicate that the position‐specific incorporation of the 2,6‐dnsAF is a useful technique to probe protein structures. These results also indicate that well‐designed non‐natural amino acids carrying relatively large side chains can be accepted as substrates of the translation system.</jats:p>

DOI： 10.1016/s0014-5793(04)00099-7

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier {BV}  

Here, we report a simple and economical tRNA aminoacylation system based upon a resin-immobilized ribozyme, referred to as Flexiresin. This catalytic system features a broad spectrum of activities toward various phenylalanine (Phe) analogs and suppressor tRNAs. Most importantly, it allows users to perform the tRNA aminoacylation reaction and isolate the aminoacylated tRNAs in a few hours. We coupled the Flexiresin system with a high-performance cell-free translation system and demonstrated protein mutagenesis with seven different Phe analogs in parallel. Thus, the technology developed herein provides a new tool that significantly simplifies the procedures for the synthesis of aminoacyl-tRNAs charged with nonnatural amino acids, which makes the nonnatural amino acid mutagenesis of proteins more user accessible.

DOI： 10.1016/j.chembiol.2003.10.010

Web of Science

PubMed

Publishing type：Research paper (scientific journal)  

PubMed

Language：Japanese   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier {BV}  

Aminoacyl-tRNA synthetase (ARS) ribozymes have potential to develop a novel genetic coding system. Although we have previously isolated such a ribozyme that recognized aromatic amino acids, it could not be used as a versatile catalyst due to its limited ability of aminoacylation to a particular tRNA used for the selection. To overcome this limitation, we used a combination of evolutionary and engineering approaches to generate an optimized ribozyme. The ribozyme, consisting of 45 nucleotides, displays a broad spectrum of activity toward various tRNAs. Most significantly, this ribozyme is able to exhibit multiple turnover activity and charge parasubstituted Phe analogs onto an engineered suppressor tRNA (tRNA(CCCG)(Asn)). Thus, it provides a useful and flexible tool for the custom synthesis of mischarged tRNAs with natural and non-natural amino acids.

DOI： 10.1016/s1074-5521(03)00145-5

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1385/1-59259-395-X:53

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society ({ACS})  

Four-base codon strategy was applied to incorporate a fluorophore-quencher pair into specific positions on a single protein; beta-anthraniloyl-L-alpha,beta-diaminopropionic acid (atnDap) was employed as a fluorophore and p-nitrophenylalanine (ntrPhe) as a quencher. Their positions were directed by the CGGG/CCCG and GGGC/CCCG four-base codon/anticoclon pairs and two doubly mutated streptavidins, i.e., ((52)atnDap, (84)ntrPhe) and ((54)ntrPhe, (84)atnDap) mutants were synthesized through Escherichia coli in vitro protein synthesizing systems. Intramolecular photoinduced electron transfer (ET) was observed as the decrease of intensity in steady-state fluorescence spectroscopy and as the shortening of fluorescence decaytimes. The quenching data indicated that the ET rate reflects the detailed structure of the protein.

DOI： 10.1021/ja017714+

DOI： 10.1021/ja017714

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society ({ACS})  

DOI： 10.1021/ja025872a

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media {LLC}  

A general method was developed for the construction of a library of mutant genes. The method, termed random insertion/deletion (RID) mutagenesis, enables deletion of an arbitrary number of consecutive bases at random positions and, at the same time, insertion of a specific sequence or random sequences of an arbitrary number into the same position. The applicability of the RID mutagenesis was demonstrated by replacing three randomly selected consecutive bases by the Bg/ll recognition sequence (AGATCT) in the GFP(uv) gene. In addition, the randomly selected three bases were replaced by a mixture of 20 codons. These mutants were expressed in Escherichia coli, and those that showed fluorescence properties different from the wild-type GFP were selected. A yellow fluorescent protein and an enhanced green fluorescent protein, neither of which could be obtained by error-prone PCR mutagenesis, were found among the six mutants selected. Several mutants of the DsRed protein that show different fluorescence properties were also obtained.

DOI： 10.1038/nbt0102-76

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society ({ACS})  

DOI： 10.1021/bi0108204

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society ({ACS})  

Fluorescent nonnatural amino acids were incorporated into specific positions of streptavidin. The positions of the nonnatural amino acids were directed by a CGGG/CCCG four-base codon/anticodon pair. The nonnatural mutants with a single 2-anthrylalanine at the 22nd, 43rd, 54th, and 120th positions, respectively, were found to bind biotin, indicating that the mutants retained active conformation. The fluorescence intensities of the anthryl groups were relatively insensitive to the positions and the biotin binding when excited at 265 nm. When the anthryl group at the 120th position was excited through energy transfer from tryptophan units, the fluorescence intensity markedly decreased with biotin binding, because of a suppression of the energy transfer. Amino acids carrying 7-methoxycoumarine fluorophore were also incorporated at the 120th position Their fluorescence quantum yields were very sensitive to the biotin binding. The high sensitivity of the coumarine-labeled streptavidin exemplifies potential applications of fluorescent nonnatural mutants for detecting specific molecules at very low concentrations.

DOI： 10.1021/bm990012g

Scopus

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Royal Society of Chemistry ({RSC})  

DOI： 10.1039/b001908i

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：Japanese   Publishing type：Rapid communication, short report, research note, etc. (scientific journal)   Publisher：共立出版  

CiNii Books

Other Link： http://search.jamas.or.jp/link/ui/2007116796

Publishing type：Rapid communication, short report, research note, etc. (scientific journal)  

PubMed

Language：Japanese   Publishing type：Rapid communication, short report, research note, etc. (scientific journal)   Publisher：羊土社  

CiNii Books

Language：Japanese  

J-GLOBAL

Language：Japanese   Publishing type：Rapid communication, short report, research note, etc. (scientific journal)   Publisher：オーム社  

CiNii Books

Language：Japanese   Publishing type：Rapid communication, short report, research note, etc. (scientific journal)   Publisher：共立出版  

CiNii Books

Publishing type：Rapid communication, short report, research note, etc. (scientific journal)  

PubMed

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Venue：タワーホール船堀  

Applicant：東海国立大学機構

Application no：63/026,802  Date applied：2020.5

Application no：2015-19173  Date applied：2015.9

Country of applicant：Domestic  

Applicant：国立大学法人東京大学、ペプチドリーム株式会社

Application no：特願2013-162440  Date applied：2014.8

Country of applicant：Domestic  

Applicant：国立大学法人東京大学、ペプチドリーム株式会社

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Country of applicant：Domestic  

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Country of applicant：Domestic  

Applicant：国立大学法人東京大学、ペプチドリーム株式会社

Application no：特願2010-270958  Date applied：2011.8

Country of applicant：Domestic  

Applicant：国立大学法人東京大学

Application no：特許第5119444号  Date applied：2005.12

Country of applicant：Domestic  

Applicant：THE RESEARCH FOUNDATION OF STATE UNIVERSITY OF NEW YORK

Application no：米国US Patent 7,622,248 B2  Date applied：2003.2

Country of applicant：Domestic