Updated on 2022/03/29

写真a

 
MURAKAMI Hiroshi
 
Organization
Graduate School of Engineering Biomolecular Engineering 1 Professor
Graduate School
Graduate School of Engineering
Undergraduate School
School of Engineering Chemistry and Biotechnology
Title
Professor
Contact information
メールアドレス

Degree 1

  1. 博士(工学) ( 2000.3   岡山大学 ) 

Research Interests 5

  1. Antibody-like protein

  2. タンパク質

  3. リボソーム

  4. 非天然アミノ酸

  5. ペプチド

Research Areas 1

  1. Life Science / Applied biochemistry

Current Research Project and SDGs 3

  1. Development of in vitro selection method for obtaining antibody-like proteins

  2. Development of anti-SARS-CoV-2 antibody-like proteins

  3. Development of mutant ribosomes for incorporating various non-natural amino acids

Research History 4

  1. Nagoya University   Institute of Innovation for Future Society

    2019.4

  2. Nagoya University   Graduate School of Engineering   Professor

    2015.4

  3. The University of Tokyo   Associate professor

    2009.4 - 2015.3

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    Country:Japan

  4. The University of Tokyo   Assistant Professor

    2003.4 - 2009.3

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    Country:Japan

Education 2

  1. Okayama University   Graduate School, Division of Science and Technology

    1997.4 - 2000.3

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    Country: Japan

  2. Okayama University   Graduate School, Division of Engineering

    1995.4 - 1997.3

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    Country: Japan

Professional Memberships 5

  1. 日本化学会

  2. 日本ペプチド学会

  3. 日本分子生物学会

  4. 日本RNA学会

  5. アメリカ化学会

Committee Memberships 3

  1. ChemBioChem   Editorial Advisory Board  

    2021.9   

  2. ACS Biochemistry   Editorial Advisory Board  

    2021.3   

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    Committee type:Academic society

  3. 日本化学会 生体機能関連部会   幹事  

    2017.9   

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    Committee type:Academic society

Awards 1

  1. 第13回(平成28年度)日本学術振興会賞

    2016.2   独立行政法人日本学術振興会   非天然アミノ酸を含むペプチドの翻訳合成

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    Country:Japan

 

Papers 77

  1. Monobodies with potent neutralizing activity against SARS-CoV-2 Delta and other variants of concern Reviewed

    Kondo, T.; Matsuoka, K.; Umemoto, S.;Fujino, T.; Hayashi, G.; Iwatani, Y.*; Murakami, H.*

    Life Science Alliance   Vol. 5 ( 6 ) page: e202101322   2022.3

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

    DOI: http://doi.org/10.26508/lsa.202101322

  2. Antibody-like proteins that capture and neutralize SARS-CoV-2. Reviewed

    Kondo, T.; Iwatani, Y.; Matsuoka, K.; Fujino, T.; Umemoto, S.; Yokomaku, Y.; Ishizaki, K.; Kito, S.; Sezaki, T.; Hayashi, G.; Murakami, H.*

      Vol. 6 ( 42 ) page: eabd3916   2020.10

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

  3. An amino acid-swapped genetic code. Reviewed

    Fujino, T.; Tozaki, M.; Murakami, H.*

    ACS Synthetic Biology   Vol. 9 ( 10 ) page: 2703 - 2713   2020.9

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

  4. Construction of a Highly Diverse mRNA Library for in vitro Selection of Monobodies. Invited Reviewed

    Kondo T, Eguchi M, Tsuzuki N, Murata N, Fujino T, Hayashi G, Murakami H

      Vol. 11 ( 16 ) page: e4125   2021.8

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

    DOI: 10.21769/BioProtoc.4125

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  5. Organoruthenium-Catalyzed Chemical Protein Synthesis to Elucidate the Functions of Epigenetic Modifications on Heterochromatin Factors. Invited Reviewed

    Kamo, N.; Kujirai, T.; Kurumizaka, H.; Murakami, H.; Hayashi, G.* Okamoto, A.*

    Chemical Science   Vol. 12   page: 5926 - 5937   2021.3

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

  6. cDNA TRAP display for rapid and stable in vitro selection of antibody-like proteins. International journal

    Taishi Kondo, Minori Eguchi, Seita Kito, Tomoshige Fujino, Gosuke Hayashi, Hiroshi Murakami

    Chemical communications (Cambridge, England)   Vol. 57 ( 19 ) page: 2416 - 2419   2021.3

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    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

  7. An Amino Acid-Swapped Genetic Code. International journal

    Tomoshige Fujino, Masahiro Tozaki, Hiroshi Murakami

    ACS synthetic biology   Vol. 9 ( 10 ) page: 2703 - 2713   2020.10

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

    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

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  8. Antibody-like proteins that capture and neutralize SARS-CoV-2 International journal

    T. Kondo, Y. Iwatani, K. Matsuoka, T. Fujino, S. Umemoto, Y. Yokomaku, K. Ishizaki, S. Kito, T. Sezaki, G. Hayashi, H. Murakami

    SCIENCE ADVANCES   Vol. 6 ( 42 )   2020.10

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:AMER ASSOC ADVANCEMENT SCIENCE  

    To combat severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) and any unknown emerging pathogens in the future, the development of a rapid and effective method to generate high-affinity antibodies or antibody-like proteins is of critical importance. We here report high-speed in vitro selection of multiple high-affinity antibody-like proteins against various targets including the SARS-CoV-2 spike protein. The sequences of monobodies against the SARS-CoV-2 spike protein were successfully procured within only 4 days. Furthermore, the obtained monobody efficiently captured SARS-CoV-2 particles from the nasal swab samples of patients and exhibited a high neutralizing activity against SARS-CoV-2 infection (half-maximal inhibitory concentration, 0.5 nanomolar). High-speed in vitro selection of antibody-like proteins is a promising method for rapid development of a detection method for, and of a neutralizing protein against, a virus responsible for an ongoing, and possibly a future, pandemic.

    DOI: 10.1126/sciadv.abd3916

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  9. Exploring the Minimal RNA Substrate of Flexizymes. Reviewed International journal

    Tomoshige Fujino, Taishi Kondo, Hiroaki Suga, Hiroshi Murakami

    Chembiochem : a European journal of chemical biology   Vol. 20 ( 15 ) page: 1959 - 1965   2019.8

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    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

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  10. Multiple Site-Directed and Saturation Mutagenesis by the Patch Cloning Method. Reviewed International journal

    Naohiro Taniguchi, Hiroshi Murakami

    Methods in molecular biology (Clifton, N.J.)   Vol. 1498   page: 339 - 347   2017

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:Humana Press Inc.  

    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

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  11. Transient Tcf3 Gene Repression by TALE-Transcription Factor Targeting.

    Masuda J, Kawamoto H, Strober W, Takayama E, Mizutani A, Murakami H, Ikawa T, Kitani A, Maeno N, Shigehiro T, Satoh A, Seno A, Arun V, Kasai T, Fuss IJ, Katsura Y, Seno M

    Applied biochemistry and biotechnology   Vol. 180 ( 8 ) page: 1559 - 1573   2016.12

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    DOI: 10.1007/s12010-016-2187-4

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  12. Expanding the amino acid repertoire of ribosomal polypeptide synthesis via the artificial division of codon boxes Reviewed

    Yoshihiko Iwane, Azusa Hitomi, Hiroshi Murakami, Takayuki Katoh, Yuki Goto, Hiroaki Suga

    NATURE CHEMISTRY   Vol. 8 ( 4 ) page: 317 - 325   2016.4

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:NATURE PUBLISHING GROUP  

    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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  13. In Vitro Selection Combined with Ribosomal Translation Containing Non-proteinogenic Amino Acids Reviewed

    Tomoshige Fujino, Hiroshi Murakami

    CHEMICAL RECORD   Vol. 16 ( 1 ) page: 365 - 377   2016.2

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:WILEY-V C H VERLAG GMBH  

    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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  14. Ribosomal Synthesis of Peptides with Multiple beta-Amino Acids Reviewed

    Tomoshige Fujino, Yuki Goto, Hiroaki Suga, Hiroshi Murakami

    JOURNAL OF THE AMERICAN CHEMICAL SOCIETY   Vol. 138 ( 6 ) page: 1962 - 1969   2016.2

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

    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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  15. Incorporation of electrically charged N-alkyl amino acids into ribosomally synthesized peptides via post-translational conversion. Reviewed

    Kawakami, T.*; Sasaki, T; Reid, P. C.; Murakami, H.

    Chemical Science   Vol. 5   page: 887-893   2014.11

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

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

  16. ZFP36L1 and ZFP36L2 control LDLR mRNA stability via the ERK-RSK pathway Reviewed

    Shungo Adachi, Masae Homoto, Rikou Tanaka, Yusaku Hioki, Hiroshi Murakami, Hiroaki Suga, Masaki Matsumoto, Keiichi I. Nakayama, Tomohisa Hatta, Shun-ichiro Iemura, Tohru Natsume

    NUCLEIC ACIDS RESEARCH   Vol. 42 ( 15 ) page: 10037 - 10049   2014

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

    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 inmRNA 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.

    DOI: 10.1093/nar/gku652

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  17. Nonstandard Peptide Expression under the Genetic Code Consisting of Reprogrammed Dual Sense Codons Reviewed

    Yuki Goto, Megumi Iseki, Azusa Hitomi, Hiroshi Murakami, Hiroaki Suga

    ACS CHEMICAL BIOLOGY   Vol. 8 ( 12 ) page: 2630 - 2634   2013.12

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

    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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  18. Patch cloning method for multiple site-directed and saturation mutagenesis Reviewed

    Naohiro Taniguchi, Sayumi Nakayama, Takashi Kawakami, Hiroshi Murakami

    BMC BIOTECHNOLOGY   Vol. 13   page: 91   2013.10

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:BIOMED CENTRAL LTD  

    Background: 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.
    Results: To demonstrate MUPAC, we introduced five back mutations to a mutant green fluorescent protein (GFPuv) with five deleterious mutations at specific sites and transformed Escherichia coli (E. coli) 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.
    Conclusions: 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.

    DOI: 10.1186/1472-6750-13-91

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  19. Extensive Reprogramming of the Genetic Code for Genetically Encoded Synthesis of Highly N-Alkylated Polycyclic Peptidomimetics Reviewed

    Takashi Kawakami, Takahiro Ishizawa, Hiroshi Murakami

    JOURNAL OF THE AMERICAN CHEMICAL SOCIETY   Vol. 135 ( 33 ) page: 12297 - 12304   2013.8

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

    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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  20. In Vitro Selection of Multiple Libraries Created by Genetic Code Reprogramming To Discover Macrocyclic Peptides That Antagonize VEGFR2 Activity in Living Cells Reviewed

    Takashi Kawakami, Takahiro Ishizawa, Tomoshige Fujino, Patrick C. Reid, Hiroaki Suga, Hiroshi Murakami

    ACS CHEMICAL BIOLOGY   Vol. 8 ( 6 ) page: 1205 - 1214   2013.6

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    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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  21. TRAP Display: A High-Speed Selection Method for the Generation of Functional Polypeptides Reviewed

    Takahiro Ishizawa, Takashi Kawakami, Patrick C. Reid, Hiroshi Murakami

    JOURNAL OF THE AMERICAN CHEMICAL SOCIETY   Vol. 135 ( 14 ) page: 5433 - 5440   2013.4

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

    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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  22. Reevaluation of the D-Amino Acid Compatibility with the Elongation Event in Translation Reviewed

    Tomoshige Fujino, Yuki Goto, Hiroaki Suga, Hiroshi Murakami

    JOURNAL OF THE AMERICAN CHEMICAL SOCIETY   Vol. 135 ( 5 ) page: 1830 - 1837   2013.2

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

    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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  23. Patch cloning method for multiple site-directed and saturation mutagenesis Reviewed

    Taniguchi, N; Nakayama, S; Kawakami, T.; Murakami, H.

    BMC Biotechnology     page: 13:91   2013

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

  24. Extensive reprogramming of the genetic code for genetically encoded synthesis of highly N-alkylated polycyclic peptidomimetics. Reviewed

    Kawakami, T.; Ishizawa, T.; Murakami, H

    Journal of the American Chemical Society   Vol. 135   page: 12297-304   2013

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  25. Artificially created nucleic acids and peptides/proteins in chemical biology.

    Kuwahara M, Li Y, Rozners E, Murakami H

    Journal of nucleic acids   Vol. 2013   page: 219263   2013

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    DOI: 10.1155/2013/219263

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  26. Genetically encoded libraries of nonstandard peptides Reviewed

    Takashi Kawakami, Hiroshi Murakami

    Journal of Nucleic Acids   Vol. 2012   page: 713510   2012

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    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. © 2012 Takashi Kawakami and Hiroshi Murakami.

    DOI: 10.1155/2012/713510

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  27. Diverse backbone-cyclized peptides via codon reprogramming Reviewed

    Takashi Kawakami, Atsushi Ohta, Masaki Ohuchi, Hiroshi Ashigai, Hiroshi Murakami, Hiroaki Suga

    NATURE CHEMICAL BIOLOGY   Vol. 5 ( 12 ) page: 888 - 890   2009.12

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:NATURE PUBLISHING GROUP  

    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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  28. A flexizyme that selectively charges amino acids activated by a water-friendly leaving group Reviewed

    Nobuyoshi Niwa, Yusuke Yamagishi, Hiroshi Murakami, Hiroaki Suga

    BIOORGANIC & MEDICINAL CHEMISTRY LETTERS   Vol. 19 ( 14 ) page: 3892 - 3894   2009.7

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:PERGAMON-ELSEVIER SCIENCE LTD  

    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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  29. Ribosomal Synthesis of Cyclic Peptides with a Fluorogenic Oxidative Coupling Reaction Reviewed

    Yusuke Yamagishi, Hiroshi Ashigai, Yuki Goto, Hiroshi Murakami, Hiroaki Suga

    CHEMBIOCHEM   Vol. 10 ( 9 ) page: 1469 - 1472   2009.6

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    DOI: 10.1002/cbic.200900021

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  30. Ribosomal Synthesis of Peptides with C-Terminal Lactams, Thiolactones, and Alkylamides Reviewed

    Eiji Nakajima, Yuki Goto, Yusuke Sako, Hiroshi Murakami, Hiroaki Suga

    CHEMBIOCHEM   Vol. 10 ( 7 ) page: 1186 - 1192   2009.5

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    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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  31. Bases in the anticodon loop of tRNA(GGC)(Ala) prevent misreading Reviewed

    Hiroshi Murakami, Atsushi Ohta, Hiroaki Suga

    NATURE STRUCTURAL & MOLECULAR BIOLOGY   Vol. 16 ( 4 ) page: 353 - 358   2009.4

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    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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  32. Ribosomal synthesis of dehydrobutyrine- and methyllanthionine-containing peptides Reviewed

    Yuki Goto, Kazuhiro Iwasaki, Kohei Torikai, Hiroshi Murakami, Hiroaki Suga

    CHEMICAL COMMUNICATIONS   ( 23 ) page: 3419 - 3421   2009

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    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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  33. Ribosomal Synthesis of Polypeptoids and Peptoid-Peptide Hybrids Reviewed

    Takashi Kawakami, Hiroshi Murakami, Hiroaki Suga

    JOURNAL OF THE AMERICAN CHEMICAL SOCIETY   Vol. 130 ( 50 ) page: 16861 - +   2008.12

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    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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  34. Polymerization of alpha-Hydroxy Acids by Ribosomes Reviewed

    Atsushi Ohta, Hiroshi Murakami, Hiroaki Suga

    CHEMBIOCHEM   Vol. 9 ( 17 ) page: 2773 - 2778   2008.11

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    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'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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  35. Initiating translation with D-amino acids Reviewed

    Yuki Goto, Hiroshi Murakami, Hiroaki Suga

    RNA-A PUBLICATION OF THE RNA SOCIETY   Vol. 14 ( 7 ) page: 1390 - 1398   2008.7

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

    DOI: 10.1261/rna.1020708

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  36. Structural basis of specific tRNA aminoacylation by a small in vitro selected ribozyme Reviewed

    Hong Xiao, Hiroshi Murakami, Hiroaki Suga, Adrian R. Ferre-D'Amare

    NATURE   Vol. 454 ( 7202 ) page: 358 - U60   2008.7

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    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'-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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  37. Ribosomal synthesis of bicyclic peptides via two orthogonal inter-side-chain reactions Reviewed

    Yusuke Sako, Jurnpei Morimoto, Hiroshi Murakami, Hiroaki Suga

    JOURNAL OF THE AMERICAN CHEMICAL SOCIETY   Vol. 130 ( 23 ) page: 7232 - +   2008.6

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    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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  38. Ribosomal synthesis of peptidase-resistant peptides closed by a nonreducible inter-side-chain bond Reviewed

    Yusuke Sako, Yuki Goto, Hiroshi Murakami, Hiroaki Suga

    ACS CHEMICAL BIOLOGY   Vol. 3 ( 4 ) page: 241 - 249   2008.4

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    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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  39. Reprogramming the translation initiation for the synthesis of physiologically stable cyclic peptides

    Yuki Goto, Atsushi Ohta, Yusuke Sako, Yusuke Yamagishi, Hiroshi Murakami, Hiroaki Suga

    ACS CHEMICAL BIOLOGY   Vol. 3 ( 2 ) page: 120 - 9   2008.2

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    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/cb700233t

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  40. Messenger RNA-Programmed incorporation of multiple N-methyl-amino acids into linear and cyclic peptides Reviewed

    Takashi Kawakami, Hiroshi Murakami, Hiroaki Suga

    CHEMISTRY & BIOLOGY   Vol. 15 ( 1 ) page: 32 - 42   2008.1

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    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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  41. Synthesis of polyester by means of genetic code reprogramming Reviewed

    Atsushi Ohta, Hiroshi Murakami, Ed Higashimura, Hiroaki Suga

    CHEMISTRY & BIOLOGY   Vol. 14 ( 12 ) page: 1315 - 1322   2007.12

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    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

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  42. The flexizyme system: a highly flexible tRNA aminoacylation tool for the translation apparatus Reviewed

    Masaki Ohuchi, Hiroshi Murakami, Hiroaki Suga

    CURRENT OPINION IN CHEMICAL BIOLOGY   Vol. 11 ( 5 ) page: 537 - 542   2007.10

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    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

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  43. [Reprogramming the genetic code using flexizyme].

    Sako Y, Murakami H, Suga H

    Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme   Vol. 52 ( 13 Suppl ) page: 1643 - 8   2007.10

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  44. Ribosomal Polyester Synthesis Using Reprogrammed Genetic Code Reviewed

    Atsushi Ohta, Hiroshi Murakami, Hiroaki Suga

    Kobunshi   Vol. 56 ( 4 ) page: 196 - 199   2007

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    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

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  45. In situ generation of aminoacyl-tRNAs assisted by ribozymes in translation apparatus. Reviewed

    Ohuchi M, Murakami H, Suga H

    Nucleic acids symposium series (2004)   ( 51 ) page: 115-116 - 6   2007

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    DOI: 10.1093/nass/nrm058

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  46. Exploration of incorporation of Nalpha-methylated amino acids into peptides by sense-suppression method. Reviewed

    Kawakami T, Murakami H, Suga H

    Nucleic acids symposium series (2004)   ( 51 ) page: 361-362 - 2   2007

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    DOI: 10.1093/nass/nrm181

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  47. [Expansion and reprogramming of genetic code by Flexizyme].

    Murakami H, Suga H

    Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme   Vol. 51 ( 16 Suppl ) page: 2496 - 501   2006.12

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  48. フレキシザイムを用いた遺伝暗号の拡張とリプログラミング (RNAと生命) -- (RNAテクノロジーと創薬)

    村上 裕, 菅 裕明

    蛋白質核酸酵素   Vol. 51 ( 16 ) page: 2496-2501   2006.12

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  49. Leucyl/phenylalanyl-tRNA-protein transferase-mediated chemoenzymatic coupling of N-terminal arg/lys units in posttranslationally processed proteins with non-natural amino acids Reviewed

    Masumi Taki, Atsushi Kuno, Shinsuke Matoba, Yuki Kobayashi, Junichiro Futami, Hiroshi Murakami, Hiroaki Suga, Kazunari Taira, Tsunemi Hasegawa, Masahiko Sisido

    CHEMBIOCHEM   Vol. 7 ( 11 ) page: 1676 - +   2006.11

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    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

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  50. テクノ・トレンド フレキシザイム--遺伝暗号の拡張とリプログラミングへの応用

    村上 裕, 菅 裕明

    バイオテクノロジージャーナル   Vol. 6 ( 6 ) page: 734-737   2006.11

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  51. A highly flexible tRNA acylation method for non-natural potypeptide synthesis Reviewed

    H Murakami, A Ohta, H Ashigai, H Suga

    NATURE METHODS   Vol. 3 ( 5 ) page: 357 - 359   2006.5

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    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

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  52. セントラルドグマをつくりかえる (特集 生命をデザインする合成生物学)

    村上 裕, 菅 裕明, 平尾 一郎

    バイオニクス   Vol. 3 ( 3 ) page: 34-39   2006.3

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  53. Flexizyme as a versatile tRNA acylation catalyst and the application for translation. Reviewed International journal

    Murakami H, Ohta A, Goto Y, Sako Y, Suga H

    Nucleic acids symposium series (2004)   ( 50 ) page: 35-36 - 6   2006

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    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

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  54. In vitro evolution of flexizymes that function under the conditions in translation system. Reviewed

    Ohuchi M, Murakami H, Suga H

    Nucleic acids symposium series (2004)   ( 50 ) page: 299-300   2006

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    DOI: 10.1093/nass/nrl149

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  55. Translation initiation by using various N-acylaminoacyl tRNAs. Reviewed International journal

    Goto Y, Ashigai H, Sako Y, Murakami H, Suga H

    Nucleic acids symposium series (2004)   ( 50 ) page: 293-294 - 4   2006

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    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

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  56. Programmable ribozymes for mischarging tRNA with nonnatural amino acids and their applications to translation Reviewed

    D Kourouklis, H Murakami, H Suga

    METHODS   Vol. 36 ( 3 ) page: 239 - 244   2005.7

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    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

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  57. Designer ribozymes: Programming the tRNA specificity into flexizyme Reviewed

    K Ramaswamy, H Saito, H Murakami, K Shiba, H Suga

    JOURNAL OF THE AMERICAN CHEMICAL SOCIETY   Vol. 126 ( 37 ) page: 11454 - 11455   2004.9

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    DOI: 10.1021/ja046843y

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  58. Position-specific incorporation of dansylated non-natural amino acids into streptavidin by using a four-base codon Reviewed

    T Hohsaka, N Muranaka, C Komiyama, K Matsui, S Takaura, R Abe, H Murakami, M Sisido

    FEBS LETTERS   Vol. 560 ( 1-3 ) page: 173 - 177   2004.2

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    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 Escherichia coli in vitro translation system. 2,6-Dansyl-aminophenylalanine (2,6-dusAF) 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. (C) 2004 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

    DOI: 10.1016/S0014-5793(04)00099-7

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  59. Using a solid-phase ribozyme aminoacylation system to reprogram the genetic code Reviewed

    H Murakami, D Kourouklis, H Suga

    CHEMISTRY & BIOLOGY   Vol. 10 ( 11 ) page: 1077 - 1084   2003.11

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    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

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  60. [Artificial ribozymes: selection and application for nonnatural amino acid mutagenesis]. Reviewed

    Murakami H, Suga H

    Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme   Vol. 48 ( 11 Suppl ) page: 1511-1518   2003.8

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    Publishing type:Research paper (scientific journal)  

    PubMed

  61. 人工リボザイム--試験管内分子進化と非天然アミノ酸変異法への応用 (化学と生物学の接点がつくるNewバイオテクノロジー) -- (新規遺伝子と蛋白質を創製する)

    村上 裕, 菅 裕明

    蛋白質核酸酵素   Vol. 48 ( 11 ) page: 1511-1518   2003.8

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

  62. A versatile tRNA aminoacylation catalyst based on RNA Reviewed

    H Murakami, H Saito, H Suga

    CHEMISTRY & BIOLOGY   Vol. 10 ( 7 ) page: 655 - 662   2003.7

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

    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

  63. Random insertion and deletion mutagenesis. Reviewed

    Murakami H, Hohsaka T, Sisido M

    Methods in molecular biology (Clifton, N.J.)   Vol. 231   page: 53-64   2003

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    DOI: 10.1385/1-59259-395-X:53

    PubMed

  64. Position-specific incorporation of a fluorophore-quencher pair into a single streptavidin through orthogonal four-base codon/anticodon pairs Reviewed

    M Taki, T Hohsaka, H Murakami, K Taira, M Sisido

    JOURNAL OF THE AMERICAN CHEMICAL SOCIETY   Vol. 124 ( 49 ) page: 14586 - 14590   2002.12

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    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+

    Web of Science

    PubMed

  65. Aminoacyl-tRNA synthesis by a resin-immobilized ribozyme Reviewed

    H Murakami, NJ Bonzagni, H Suga

    JOURNAL OF THE AMERICAN CHEMICAL SOCIETY   Vol. 124 ( 24 ) page: 6834 - 6835   2002.6

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

    DOI: 10.1021/ja025872a

    Web of Science

    PubMed

  66. Random insertion and deletion of arbitrary number of bases for codon-based random mutation of DNAs Reviewed

    H Murakami, T Hohsaka, M Sisido

    NATURE BIOTECHNOLOGY   Vol. 20 ( 1 ) page: 76 - 81   2002.1

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:NATURE AMERICA INC  

    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

  67. A novel fluorescent nonnatural amino acid that can be incorporated into a specific position of streptavidin. Reviewed

    Taki M, Hohsaka T, Murakami H, Kuno A, Hasegawa T, Taira K, Sisido M

    Nucleic acids research. Supplement (2001)   ( 2 ) page: 203-204   2002

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    PubMed

  68. A non-natural amino acid for efficient incorporation into proteins as a sensitive fluorescent probe

    Taki, M. and Hohsaka, T. and Murakami, H. and Taira, K. and Sisido, M.

    FEBS Letters   Vol. 507 ( 1 ) page: 35-38   2001

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  69. Five-base codons for incorporation of nonnatural amino acids into proteins

    Hohsaka, T. and Ashizuka, Y. and Murakami, H. and Sisido, M.

    Nucleic Acids Res.   Vol. 29 ( 17 ) page: 3646-3651   2001

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  70. Incorporation of nonnatural amino acids into proteins by using various four-base codons in an Escherichia coli in vitro translation system

    Hohsaka, T. and Ashizuka, Y. and Taira, H. and Murakami, H. and Sisido, M.

    Biochemistry   Vol. 40 ( 37 ) page: 11060-11064   2001

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  71. Five-base codons for incorporation of nonnatural amino acids into proteins

    Hohsaka, T, Ashizuka, Y, Murakami, H, Sisido, M

    Nucleic Acids Res.   Vol. 29 ( 17 ) page: 3646-3651   2001

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

  72. Site-directed incorporation of fluorescent nonnatural amino acids into streptavidin for highly sensitive detection of biotin

    Hiroshi Murakami, Takahiro Hohsaka, Yuki Ashizuka, Kimiko Hashimoto, Masahiko Sisido

    Biomacromolecules   Vol. 1 ( 1 ) page: 118 - 125   2000

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

    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

  73. A chiral Eu3+-thienoyltrifluoroacetone complex on an avidin tetramer: luminescence and CD studies on the supramolecular protein-metal chelate complex

    Taki, M. and Murakami, H. and Sisido, M.

    Chem. Commun.   ( 13 ) page: 1199-1200   2000

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  74. Efficient incorporation of nonnatural amino acids with large aromatic groups into streptavidin in in vitro protein synthesizing systems

    Hohsaka, T. and Kajihara, D. and Ashizuka, Y. and Murakami, H. and Sisido, M.

    J. Am. Chem. Soc.   Vol. 121 ( 1 ) page: 34-40   1999

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  75. Incorporation of two different nonnatural amino acids independently into a single protein through extension of the genetic code

    Hohsaka, T. and Ashizuka, Y. and Sasaki, H. and Murakami, H. and Sisido, M.

    J. Am. Chem. Soc.   Vol. 121 ( 51 ) page: 12194-12195   1999

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  76. Site-directed incorporation of p-nitrophenylalanine into streptavidin and site-to-site photoinduced electron transfer from a pyrenyl group to a nitrophenyl group on the protein framework

    Murakami, H. and Hohsaka, T. and Ashizuka, Y. and Sisido, M.

    J. Am. Chem. Soc.   Vol. 120 ( 30 ) page: 7520-7529   1998

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  77. Incorporation of nonnatural amino acids into streptavidin through in vitro frame-shift suppression

    Hohsaka, T. and Ashizuka, Y. and Murakami, H. and Sisido, M.

    J. Am. Chem. Soc.   Vol. 118 ( 40 ) page: 9778-9779   1996

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Books 1

  1. 創薬研究者がこれだけは知っておきたい最新のウイルス学

    近藤太志、梅本駿、藤野公茂、林剛介、村上裕( Role: Contributor ,  新型コロナウイルスに対する迅速な人工抗体創製)

    技術情報協会  2021.8  ( ISBN:978-4-86104-855-5

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    Total pages:601   Responsible for pages:10  

MISC 9

  1. 質量分析計によるRNA制御因子の同定法の開発とその応用

    足達俊吾, 穂本真佐江, 田中利好, 日置雄策, 村上裕, 菅裕明, 松本雅記, 中山敬一, 堀本勝久, 家村俊一郎, 夏目徹

    日本分子生物学会年会プログラム・要旨集(Web)   Vol. 35th   page: 3W12III-1 (WEB ONLY)   2012

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    Language:Japanese  

    J-GLOBAL

  2. [Reprogramming the genetic code using flexizyme]. Reviewed

    Sako Y, Murakami H, Suga H

    Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme   Vol. 52 ( 13 Suppl ) page: 1643-1648   2007.10

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

    PubMed

  3. フレキシザイムを用いた遺伝暗号の拡張とリプログラミング (RNAと生命) -- (RNAテクノロジーと創薬)

    村上 裕, 菅 裕明

    蛋白質核酸酵素   Vol. 51 ( 16 ) page: 2496-2501   2006.12

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

  4. [Expansion and reprogramming of genetic code by Flexizyme]. Reviewed

    Murakami H, Suga H

    Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme   Vol. 51 ( 16 Suppl ) page: 2496-2501   2006.12

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

    PubMed

  5. テクノ・トレンド フレキシザイム--遺伝暗号の拡張とリプログラミングへの応用

    村上 裕, 菅 裕明

    バイオテクノロジージャーナル   Vol. 6 ( 6 ) page: 734-737   2006.11

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

  6. Novel N-terminal specific labelling method of proteins by using L/F-tRNA-protein transferase

    瀧真清, 久野敦, 的場進介, 小林由紀, 村上裕, 菅裕明, 多比良和誠, 長谷川典巳, 宍戸昌彦

    日本化学会講演予稿集   Vol. 86th ( 2 ) page: 877   2006.3

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    Language:Japanese  

    J-GLOBAL

  7. セントラルドグマをつくりかえる (特集 生命をデザインする合成生物学)

    村上 裕, 菅 裕明, 平尾 一郎

    バイオニクス   Vol. 3 ( 3 ) page: 34-39   2006.3

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

  8. 人工リボザイム--試験管内分子進化と非天然アミノ酸変異法への応用 (化学と生物学の接点がつくるNewバイオテクノロジー) -- (新規遺伝子と蛋白質を創製する)

    村上 裕, 菅 裕明

    蛋白質核酸酵素   Vol. 48 ( 11 ) page: 1511-1518   2003.8

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

  9. [Artificial ribozymes: selection and application for nonnatural amino acid mutagenesis]. Reviewed

    Murakami H, Suga H

    Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme   Vol. 48 ( 11 Suppl ) page: 1511-1518   2003.8

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

    PubMed

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Presentations 33

  1. 幅広い結合親和性を網羅する低分子リガンド−Monobody結合ペアの開発

    梅本 駿,都築 成晃,吉井 達之,近藤 太志,藤野 公茂,林 剛介,築地 真也,村上 裕

    日本化学会 第102春季年会  2022.3.23  日本化学会

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    Event date: 2022.3

    Language:Japanese   Presentation type:Oral presentation (general)  

    Venue:On line  

  2. ワンポットペプチド連結反応による人工抗体モノボディの化学合成と機能評価

    小澤英知,林剛介,村上 裕

    日本化学会 第102春季年会  2022.3.23  日本化学会

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    Event date: 2022.3

    Language:Japanese   Presentation type:Oral presentation (general)  

    Venue:On line  

  3. ジケトピペラジン形成を伴うペプチドセレノエステルの合成とタンパク質化学合成への応用

    橋本 雅也, 林 剛介,村上 裕

    日本化学会 第102春季年会  2022.3.23  日本化学会

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    Event date: 2022.3

    Language:Japanese   Presentation type:Oral presentation (general)  

    Venue:On line  

  4. Tyr, Trp, Pro-rich環状ペプチドライブラリの構築とSARS-CoV2に対する環状ペプチドの選択

    藤野 公茂,園田 凌吾,鷲見 大河,村上 裕

    日本化学会 第102春季年会  2022.3.23  日本化学会

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    Event date: 2022.3

    Language:Japanese   Presentation type:Oral presentation (general)  

    Venue:On line  

  5. ワンポットペプチド連結反応を駆使したジユビキチン化PAF15タンパク質の化学合成

    高橋侑也,郡聡実,有田恭平,林剛介,村上 裕

    日本化学会 第102春季年会  2022.3.23  日本化学会

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    Event date: 2022.3

    Language:Japanese   Presentation type:Oral presentation (general)  

    Venue:On line  

  6. 新型コロナウイルスに対する人工抗体Monobodyの親和性成熟による中和活性の向上

    近藤太志、松岡和弘、藤野公茂、梅本駿、林剛介、岩谷靖雅 、村上裕

    日本化学会 第102春季年会  2022.3.23  日本化学会

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    Event date: 2022.3

    Language:Japanese   Presentation type:Oral presentation (general)  

    Venue:On line  

  7. 転写・抽出tRNAを用いたSer/Leu交換翻訳系の構築

    園田凌吾,藤野公茂,村上裕

    第44回日本分子生物学会年会  2021.12.1  日本分子生物学会年会

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    Event date: 2021.12

    Language:Japanese   Presentation type:Poster presentation  

    Venue:幕張メッセ&On line  

  8. アミノ酸交換遺伝暗号の構築

    藤野公茂,戸崎将弘,村上裕

    第44回日本分子生物学会年会  2021.12.1  日本分子生物学会

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    Event date: 2021.12

    Language:Japanese   Presentation type:Poster presentation  

    Venue:幕張メッセ&On line  

  9. SARS-CoV-2を捕捉する人工抗体の迅速創製 Invited

    近藤太志,岩谷靖雅,松岡和弘,藤野公茂,梅本駿,横幕能行,林剛介,村上裕

    第31回基礎及び最新の分析化学講習会  2021.11.9 

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    Event date: 2021.11

    Language:Japanese   Presentation type:Oral presentation (invited, special)  

  10. 新型コロナウイルスを捕まえる人工抗体をつくる Invited

    近藤太志,岩谷靖雅,松岡和弘,藤野公茂,梅本駿,横幕能行,林剛介,村上裕

    第11回 CSJ化学フェスタ  2021.10.20 

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    Event date: 2021.10

    Language:Japanese   Presentation type:Oral presentation (invited, special)  

  11. 新型コロナウイルスに対する人工抗体の迅速創製 Invited

    近藤太志,岩谷靖雅,松岡和弘,藤野公茂,梅本駿,横幕能行,林剛介,村上裕

    化学工学会第52回秋季大会  2021.9.24 

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    Event date: 2021.9

    Language:Japanese   Presentation type:Oral presentation (invited, special)  

  12. SARS-CoV-2 に対する Monobody の親和性成熟による従来株及び 変異株に対する中和活性の向上

    近藤太志,松岡和弘,藤野公茂,梅本駿,林剛介,岩谷靖雅,村上裕

    第15回バイオ関連化学シンポジウム  2021.9.10 

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    Event date: 2021.9

    Language:Japanese   Presentation type:Oral presentation (general)  

  13. 維持メチル化機構解明を指向したジユビキチン化PAF15タンパク質の化学合成

    高橋侑也,郡聡美,有田恭平,林剛介,村上裕

    第15回バイオ関連化学シンポジウム  2021.9.9 

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    Event date: 2021.9

    Language:Japanese   Presentation type:Poster presentation  

  14. 高効率ペプチドライゲーションのための新規セレノエステル合成法の開発

    橋本雅也,林剛介,村上裕

    第15回バイオ関連化学シンポジウム  2021.9.9 

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    Event date: 2021.9

    Language:Japanese   Presentation type:Poster presentation  

  15. パラジウム錯体を利用したコロナウイルス結合人工抗体の効率的全合成

    小澤英知,林剛介,村上裕

    第15回バイオ関連化学シンポジウム  2021.9.9 

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    Event date: 2021.9

    Language:Japanese   Presentation type:Poster presentation  

  16. C末端配列改変による人工抗体Monobodyの可溶性向上 –SARS-CoV-2中和抗体医薬への応用を指向して–

    梅本駿,齊木輝,近藤太志,藤野公茂,林剛介,村上裕

    第15回バイオ関連化学シンポジウム  2021.9.8 

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    Event date: 2021.9

    Language:Japanese   Presentation type:Poster presentation  

    Venue:On line  

  17. 天然・転写tRNAによるSer/Leu交換遺伝暗号の翻訳高効率化

    藤野公茂,園田凌吾,村上裕

    第15回バイオ関連化学シンポジウム  2021.9.8 

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    Event date: 2021.9

    Language:Japanese   Presentation type:Poster presentation  

  18. 2-aminobenzamide誘導体を用いたN末端チアゾリジン脱保護による高効率one-potペプチド連結法

    中津幸輝,村上裕,林剛介,岡本晃充

    第15回バイオ関連化学シンポジウム  2021.9.8 

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    Event date: 2021.9

    Language:Japanese   Presentation type:Poster presentation  

    Venue:On line  

  19. 人工抗体迅速創製法によるSARS-CoV-2変異株中和抗体の創製 Invited

    近藤太志,岩谷靖雅,松岡和弘,藤野公茂,梅本駿,林剛介,村上裕

    第8回バイオ関連化学シンポジウム若手フォーラム  2021.9.3 

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    Event date: 2021.9

    Language:Japanese   Presentation type:Oral presentation (invited, special)  

    Venue:On line  

  20. SARS-CoV-2 由来スパイクタンパク質に対する迅速な人工抗体創製 Invited

    近藤太志,岩谷靖雅,松岡和弘,藤野公茂,梅本駿,横幕能行,林剛介,村上裕

    第34回 日本動物細胞工学会2021年度大会  2021.7.28 

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    Event date: 2021.7

    Language:Japanese   Presentation type:Oral presentation (invited, special)  

    Venue:On line  

  21. 新型コロナウイルスに対する迅速な人工抗体創製 Invited

    近藤太志,岩谷靖雅,松岡和弘,藤野公茂,梅本駿,横幕能行,林剛介,村上裕

    第68回構造生物応用研究会  2021.7.15 

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    Event date: 2021.7

    Language:Japanese   Presentation type:Oral presentation (invited, special)  

    Venue:On line  

  22. SARS-CoV-2に対する人工抗体の高速創製 Invited

    近藤太志,岩谷靖雅,松岡和弘,藤野公茂,梅本駿,横幕能行,林剛介,村上裕

    第21回日本蛋白質科学会年会  2021.6.18 

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    Event date: 2021.6

    Language:Japanese   Presentation type:Oral presentation (invited, special)  

    Venue:On line  

  23. 新型コロナウイルスに対する迅速な人工抗体創製 Invited

    近藤大志,岩谷靖雅,松岡和弘,藤野公茂,梅本駿,横幕能行,林剛介,村上裕

    構造生物学・化学・計算科学を融合させたウイルス・パンデミックに対する取り組み  2021.4.2 

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    Event date: 2021.4

    Language:Japanese  

  24. Ru触媒を用いたリンカーヒストンH1.2とHP1αの化学合成とその翻訳後修飾の機能解析

    加茂 直己、鯨井 智也、胡桃坂 仁志、村上 裕、林 剛介、岡本 晃充

    日本化学会 第101回春期年会  2021.3.22 

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    Event date: 2021.3

    Language:Japanese   Presentation type:Oral presentation (general)  

  25. セレノシステインを用いたセレノエステル合成とペプチド連結反応への応用

    橋本 雅也、林 剛介、村上 裕

    日本化学会 第101回春期年会  2021.3.21 

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    Event date: 2021.3

    Language:Japanese   Presentation type:Oral presentation (general)  

  26. High-speed selection of antibody-like proteins that capture and neutralize SARS-CoV-2 International conference

    2021.3.8 

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    Event date: 2021.3

    Language:English   Presentation type:Oral presentation (general)  

    Country:Japan  

  27. 高速人工抗体創製法の開発とSARS-CoV-2中和抗体作製への応用 Invited

    村上裕

    生物化学的測定研究会 第25回学術シンポジウム  2020.11.6 

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    Event date: 2020.11

    Language:Japanese   Presentation type:Oral presentation (invited, special)  

    Country:Japan  

  28. SARS-CoV-2を結合・中和する人工抗体の開発

    村上裕

    第14回バイオ関連化学シンポジウム  2020.9.8 

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    Event date: 2020.9

    Language:Japanese   Presentation type:Oral presentation (general)  

    Country:Japan  

  29. SARS-CoV-2由来スパイクタンパク質に対する高親和性人工抗体の高速創製

    近藤 太志・岩谷 靖雅・松岡 和弘・藤野 公茂・梅本 駿・横幕 能行・林 剛介・村上 裕

    第14回バイオ関連化学シンポジウム  2020.9.7 

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    Event date: 2020.9

    Language:Japanese   Presentation type:Poster presentation  

  30. 低分子リガンドに特異的に結合する人工抗体の創製

    都築 成晃・吉井 達之・近藤 大志・藤野 公茂・林 剛介・築地 真也・村上 裕

    第14回バイオ関連化学シンポジウム  2020.9.7 

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    Event date: 2020.9

    Language:Japanese   Presentation type:Poster presentation  

  31. アミノ酸入れ替え遺伝暗号の開発

    藤野 公茂・戸崎 将弘・村上 裕

    第14回バイオ関連化学シンポジウム  2020.9.7 

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    Event date: 2020.9

    Language:Japanese   Presentation type:Poster presentation  

  32. TRAP提示法を用いた機能性ペプチド•人工抗体選択 Invited

    村上裕

    化学工学会第49回秋季大会 

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    Event date: 2017.9

    Language:Japanese   Presentation type:Oral presentation (invited, special)  

    Country:Japan  

  33. 翻訳合成系を用いた進化分子工学・合成生物学 Invited

    村上裕

    ChemBio講演会2017-1  2017.1.17 

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    Event date: 2017.1

    Language:Japanese   Presentation type:Oral presentation (invited, special)  

    Country:Japan  

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Research Project for Joint Research, Competitive Funding, etc. 3

  1. SARS-CoV-2に対する抗体ミメティックを活用した治療薬開発

    Grant number:20fk0108293s0101  2020.10 - 2022.3

    新興・再興感染症に対する革新的医薬品等開発推進研究事業 

    村上裕

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    Authorship:Coinvestigator(s)  Grant type:Competitive

    Grant amount:\16900000 ( Direct Cost: \13000000 、 Indirect Cost:\3900000 )

  2. パンデミックウイルスに迅速に対応する高速人工抗体創製プラットホームの開発

    Grant number:20he0622010h0001  2020.9 - 2021.3

    ウイルス等感染症対策技術開発事業 基礎研究支援 

    村上裕

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    Authorship:Principal investigator  Grant type:Competitive

    Grant amount:\34580000 ( Direct Cost: \26600000 、 Indirect Cost:\7980000 )

  3. 人工RNA触媒を用いた革新的な創薬技術の開発

    2005.4 - 2008.3

    分野横断的公募事業 若手研究グラント 3年型 

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    Grant type:Competitive

KAKENHI (Grants-in-Aid for Scientific Research) 8

  1. 抗体医薬を真に置き換える小タンパク質医薬の開発

    Grant number:21H02061  2021.4 - 2025.3

    科学研究費助成事業  基盤研究(B)

    森 健, 村上 裕

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    Authorship:Coinvestigator(s) 

    本研究では、二つの目的を達成する小タンパク質医薬を創製する。一つは抗体医薬の問題である高い薬価を克服する安価なタンパク質医薬でありながら、抗体と同等の細胞傷害活性を持つFc-ARMである。もう一つは、抗がん薬に対するがんの耐性獲得の問題を克服する小タンパク質医薬pH-ARMである。Fc-ARMの概念は申請者が報告してきたものであるが、本研究では、Fc親和性の高い小タンパク質を大規模な無細胞系RNAライブラリーよりスクリーニングして見いだし、用いることで抗体医薬に匹敵する性能を発揮すると期待される。また、pH-ARMは新しい概念であり、耐性がんの克服は、未解決の課題であり、本法は原理的にその問題を解決しうるものである。

  2. High-speed method for selection of synthetic binding proteins and the application for quantitative detection of a target protein at single-molecule resolution

    Grant number:15H02006  2015.4 - 2019.3

    Grants-in-Aid for Scientific Research  Grant-in-Aid for Scientific Research (A)

    MURAKAMI HIROSHI

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    Authorship:Principal investigator 

    Grant amount:\41990000 ( Direct Cost: \32300000 、 Indirect Cost:\9690000 )

    The purpose of this research was to develop a high-speed method for selection of synthetic binding proteins (SBPs) and quantitative detection of the corresponding target proteins at single-molecule resolution. First, we modified our previous high-speed method of screening functional polypeptides (transcription-translation coupled with association of puromycin linker [TRAP] display; JACS, 2013) for SBPs selection. Then, using the improved TRAP display, we demonstrated successful selection of high-affinity (nM level) SBPs against model target proteins, EGFR1 and HER2. Finally, we applied the modified method to obtain several SBPs against low-copy-number proteins and to detect a target protein immobilized on a glass plate using a fluorescently labeled SBP. The improved version of the TRAP display for SBP selection and single-molecule detection of target proteins with the obtained SBPs could become a basic high-precision technology for protein quantification at single-molecule resolution.

  3. Creating neo-genetic code

    Grant number:15K12741  2015.4 - 2016.3

    Grants-in-Aid for Scientific Research  Grant-in-Aid for Challenging Exploratory Research

    MURAKAMI Hiroshi

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    Authorship:Principal investigator 

    Grant amount:\3640000 ( Direct Cost: \2800000 、 Indirect Cost:\840000 )

    A translation system employing a neo-genetic code could be a safe and useful tool, because it has a genetic code orthogonal to that of organisms on earth. Here, we developed a cell-free in vitro translation system employing a neo-genetic code. We designed a streptavidin gene based on the neo-genetic code, and expressed it in a translation system employing either a neo-genetic code or the universal genetic code. The protein produced in the translation system employing a neo-genetic code showed biotin binding activity, whereas that produced in the translation system employing the universal genetic code showed no activity. This result indicates that the former protein had the correct amino acid sequence of streptavidin, whereas the latter had a meaningless amino acid sequence, although both proteins were produced from the same gene. The new translation system could be used for safe expression of proteins whose genes are harmful to natural environments and life.

  4. 広汎用リボソームの開発

    Grant number:23350076  2011

    科学研究費助成事業  基盤研究(B)

    村上 裕

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    Authorship:Principal investigator 

    Grant amount:\11050000 ( Direct Cost: \8500000 、 Indirect Cost:\2550000 )

  5. JSPS 特殊ペプチド増幅法の開発と創薬への応

    2010.4 - 2014.3

    科学研究費助成事業  最先端・次世代研究開発支援プログラ

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    Grant type:Competitive

  6. D-amino acids and beta-amino acids : a new frontier in Ribosomal synthesis of non-standard polypeptides

    Grant number:20681022  2008 - 2010

    Grants-in-Aid for Scientific Research  Grant-in-Aid for Young Scientists (A)

    MURAKAMI Hiroshi

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    Authorship:Principal investigator 

    Grant amount:\24960000 ( Direct Cost: \19200000 、 Indirect Cost:\5760000 )

    It is believed that the ribosomal translation machinery does not accept D-amino acids and beta-amino acids. In this study, we investigated the incorporation of these non-proteinogenic amino acids into peptides, and found that the translation machinery actually accepts several kinds of D-amino acids as well as beta-amino acids. These results open the way for the ribosomal synthesis of novel non-standard peptides with main-chain modified groups, and would facilitate the use of these unique building blocks in peptides for drug discovery.

  7. 新規アミノアシルtRNA合成リボザイムの創製

    Grant number:17750151  2005 - 2006

    科学研究費助成事業  若手研究(B)

    村上 裕

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    Authorship:Principal investigator 

    Grant amount:\3600000 ( Direct Cost: \3600000 )

    非天然アミノ酸変異法は、人工的な官能基を蛋白質に部位特異的に導入する優れた方法である。この変異法にはアシルtRNAの合成が必須であり、我々はリボザイムを人工的に進化させることでアシルtRNAの合成を簡便化することを提案してきた。これまでに試験管内分子進化により創製したRNA触媒を用いて、Phe誘導体でアシル化されたtRNAの合成に成功している。本課題では、RNA触媒を進化させ、本方法をPhe誘導体以外の非天然アミノ酸に拡張することを目的とした。
    RNA触媒の基質となるアミノ酸は、カルボン酸をエステル化して弱く活性化したものを用いている。これまでの研究から、RNA触媒は基質の脱離基を認識せず、側鎖の芳香環を認識するため、側鎖に芳香環を持つアミノ酸のみがRNA触媒の基質となることが分かっていた。そこで、もしRNA触媒の基質認識部位を、側鎖から脱離基に変更できれば、様々な側鎖をもつアミノ酸をtRNAに連結することが可能になると考えた。この設計指針によって、脱離基に芳香環(認識部位)をもつ新しい基質を合成した。この基質は当初の考え通り、既存のRNA触媒の基質となったが、そのアシル化効率は低かった。そこで新しい基質に対してRNA触媒を最適化するため、試験管内進化を行なった。得られたRNA触媒は進化を行なう前のものに比べ、5倍以上の活性を持っていた。このRNA触媒と、脱離基に芳香環(認識部位)をもつ基質を用いることで、これまでの基質の制限を大幅に取り払ったtRNAアシル化法が完成した(Nat.Methods 2006)。今後、本方法によりアシルtRNAの合成が簡略化され、非天然アミノ酸を用いた蛋白質の改変が容易になると考えられる。さらには、RNA触媒を遺伝暗号のリプログラミングと組み合わせ、非天然型ペプチドを調整することで、革新的な創薬技術の開発に応用できると期待される。

  8. Generation of ribozymes that catalyze fatty acid biosynthesis

    Grant number:16101007  2004 - 2008

    Grants-in-Aid for Scientific Research  Grant-in-Aid for Scientific Research (S)

    SUGA Hiroaki

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    Authorship:Coinvestigator(s) 

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Industrial property rights 12

  1. ポリヌクレオチド提示法のディスプレイ効率を調節する配列をスクリーニングする方法

    村上裕、梅本駿、近藤太志、林剛介、藤野公茂

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    Applicant:東海国立大学機構

    Application no:2022-025979  Date applied:2022.2

  2. ユビキチンに対する人工抗体

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    Applicant:東海国立大学機構

    Application no:2022-010877  Date applied:2022.1

  3. フィブロネクチンIII型ドメイン骨格を含むポリペプチド

    村上裕、都築成晃、近藤太志、林剛介、藤野公茂、築地真也、吉井達之

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    Applicant:東海国立大学機構、名古屋工業大学

    Application no:PCT/JP2021/031961  Date applied:2021.8

    Announcement no:WO2022/045365   Date announced:2022.3

    Country of applicant:Foreign country   Country of acquisition:Foreign country

  4. SARS-CoV-2結合性分子

    東海国立大学機構,国立病院機構

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    Application no:PCT/JP2021/018668  Date applied:2021.5

    Announcement no:WO2021/235404   Date announced:2021.11

    Country of applicant:Foreign country   Country of acquisition:Foreign country

  5. SARS-CoV-2のSタンパク質に結合する人工抗体

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    Applicant:東海国立大学機構

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

  6. ポリペプチドを製造するための新遺伝暗号と翻訳系、及びポリペプチドの製造方法

    村上 裕、石沢尭大

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    Application no:2015-19173  Date applied:2015.9

    Country of applicant:Domestic  

  7. 荷電性非タンパク質性アミノ酸含有ペプチドの製造方法

    村上 裕、川上 隆史、パトリック・リード、佐々木 亨

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    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  

  8. ペプチドライブラリの製造方法、ペプチドライブラリ、及びスクリーニング方法

    村上 裕、川上隆史、石沢尭大、パトリック・リード、佐々木亨

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    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2013-100661  Date applied:2013.5

    Country of applicant:Domestic  

  9. 血管内皮細胞増殖因子受容体阻害ペプチド

    村上 裕、川上隆史、石沢尭大、菅裕明、パトリック・リード

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    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2012-193453  Date applied:2012.9

    Country of applicant:Domestic  

  10. 新規人工翻訳合成系

    菅 裕明、村上 裕、後藤 佑樹

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    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  

  11. 多目的アシル化触媒とその用途

    菅裕明、村上裕

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    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  

  12. RIBOZYMES WITH BROAD tRNA AMINOACYLATION ACTIVITY

    SUGA, Hiroaki, MURAKAMI, Hiroshi, SAITO, Hirohide

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    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  

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Teaching Experience (On-campus) 1

  1. 分析化学3

    2021

 

Media Coverage 3

  1. 新型コロナに対する人工抗体 TV or radio program

    NHK、CBC放送  2020.9

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    Author:Other 

  2. 新型コロナ捕まえる「人工抗体」 短期間で作る手法開発 Newspaper, magazine

    読売新聞、中日新聞、朝日新聞、共同通信、等その他12社  2020.9

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    Author:Other 

  3. Vol.7 かつてない速さで抗体をつくる〜新型コロナウイルスとその先へ〜 Internet

    名古屋大学産学連携  2020.11