Faculty Profiles - MURAKAMI Hiroshi

Papers - MURAKAMI Hiroshi

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De novo chemo-optogenetics through the rational design of photoresponsive molecules and selection of their artificial protein binding pairs. Reviewed

Tomoki Miyazaki, Tomoshige Fujino, Tatsuyuki Yoshii, Haruto Kosugi, Mamoru Funane, Naoya Murata, Kim Chung Nguyen, Satoru Nagatoishi, Kouhei Tsumoto, Gosuke Hayashi, Hiroshi Murakami, Shinya Tsukiji

Nature Chemistry Vol. - ( - ) page： - - - 2026.4

Generating a mirror-image monobody targeting MCP-1 via TRAP display and chemical protein synthesis. International journal Open Access

Gosuke Hayashi, Toshinori Naito, Sayaka Miura, Naoya Iwamoto, Yusuke Usui, Mika Bando-Shimizu, Sae Suzuki, Katsuaki Higashi, Motohiro Nonaka, Shinya Oishi, Hiroshi Murakami

Nature communications Vol. 15 ( 1 ) page： 10723 - 10723 2024.12

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

Biologically produced protein drugs are generally susceptible to degradation by proteases and often exhibit immunogenicity. To address this issue, mirror-image peptide/protein binders consisting of D-amino acids have been developed so far through the mirror-image phage display technique. Here, we develop a mirror-image protein binder derived from a monobody, one of the promising protein scaffolds, utilizing two notable technologies: chemical protein synthesis and TRAP display, an improved version of mRNA display. A sequential workflow of initial screening followed by affinity maturation, facilitated by TRAP display, generates an L-monobody with high affinity (KD = 1.3 nM) against monocyte chemoattractant protein-1 (MCP-1) D-enantiomer. The chemically synthesized D-monobody demonstrates strong and specific binding to L-MCP-1 and exhibits pharmaceutically favorable properties such as proteolytic resistance, minimal immune response, and a potent inhibitory effect on MCP-1-induced cell migration. This study elevates the value of mirror-image peptide/protein binders as an alternative modality in drug discovery.

DOI： 10.1038/s41467-024-54902-x

Open Access

PubMed

Ser/Leu-swapped cell-free translation system constructed with natural/in vitro transcribed-hybrid tRNA set. International journal Open Access

Tomoshige Fujino, Ryogo Sonoda, Taito Higashinagata, Emi Mishiro-Sato, Keiko Kano, Hiroshi Murakami

Nature communications Vol. 15 ( 1 ) page： 4143 - 4143 2024.5

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

The Ser/Leu-swapped genetic code can act as a genetic firewall, mitigating biohazard risks arising from horizontal gene transfer in genetically modified organisms. Our prior work demonstrated the orthogonality of this swapped code to the standard genetic code using a cell-free translation system comprised of 21 in vitro transcribed tRNAs. In this study, to advance this system for protein engineering, we introduce a natural/in vitro transcribed-hybrid tRNA set. This set combines natural tRNAs from Escherichia coli (excluding Ser, Leu, and Tyr) and in vitro transcribed tRNAs, encompassing anticodon-swapped tRNASerGAG and tRNALeuGGA. This approach reduces the number of in vitro transcribed tRNAs required from 21 to only 4. In this optimized system, the production of a model protein, superfolder green fluorescent protein, increases to 3.5-fold. With this hybrid tRNA set, the Ser/Leu-swapped cell-free translation system will stand as a potent tool for protein production with reduced biohazard concerns in future biological endeavors.

DOI： 10.1038/s41467-024-48056-z

Open Access

PubMed

Large-scale analysis of mRNA sequences localized near the start and amber codons and their impact on the diversity of mRNA display libraries Reviewed Open Access

Umemoto, S.; Kondo, T.; Fujino, T.; Hayashi, G.; Murakami, H.*

Nucleic Acids Research page： gkad555 2023.7

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

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

Science Advances Vol. 6 ( 42 ) page： eabd3916 2020.10

Crystallization and X-ray structure of a highly aggregation-prone monobody engineered for high-affinity small-molecule recognition. Reviewed Open Access

Koushirou Endo, Shun Umemoto, Nariaki Tsuzuki, Hideo Okumura, Yusuke Sato, Tatsuyuki Yoshii, Shinya Tsukiji, Shingo Nagano, Hiroshi Murakami, Tomoya Hino

Acta crystallographica. Section F, Structural biology communications Vol. 82 ( Pt 3 ) page： 75 - 82 2026.3

Chemical Protein Synthesis via Direction-Switching One-Pot Peptide Ligation Enabled by Orthogonal Cysteinyl Protection. Invited Reviewed Open Access

Koki Nakatsu, Fumika Yoshitomi, Hiroki Onoda, Reia Shikimachi, Kyohei Arita, Akimitsu Okamoto, Hiroshi Murakami, Gosuke Hayashi

Journal of the American Chemical Society Vol. 148 ( 4 ) page： 4622 - 4634 2026.2

Design and Evaluation of Stable Cysteine-Modified Monobody Scaffolds for Mirror-Image Synthesis. Invited Reviewed

Vol. 36 ( 7 ) page： 1504 - 1515 2025.7

The effect of Fc region affinity of protein-based antibody-recruiting molecules on antibody-dependent cellular cytotoxicity. International journal Open Access

Hiroshi Tagawa, Riku Saeki, Chihaya Yamamoto, Kenta Tanito, Chihiro Tanaka, Shoki Munekawa, Teruki Nii, Akihiro Kishimura, Hiroshi Murakami, Takeshi Mori, Yoshiki Katayama

RSC advances Vol. 14 ( 32 ) page： 22860 - 22866 2024.7

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

Previously, we reported anticancer molecules, Fc-binding antibody-recruiting molecules (Fc-ARMs), which crosslink proteins on cancer cells with endogenous immunoglobulin Gs (IgGs) via their Fc region. The mobilized IgGs on cancer cells can accommodate natural killer cells to induce antibody-dependent cellular cytotoxicity (ADCC). Because previous Fc-ARMs utilized Fc-binding peptides, their affinity to IgGs is weak, which resulted in the limited induction capability of ADCC. Previous Fc-ARMs also unitized small molecular ligands to cancer cells, which limited their universal applicability to any cancer cells. A recent study reported that protein-based Fc-ARMs might overcome the issues associated with non-proteinous Fc-ARMs. Here, we examined the universality of a protein-based Fc-ARM by replacing its tumor-binding domain with a human epidermal growth factor receptor 2 (HER2)-specific affibody (ZHER2:342). We also examined the requirement of its Fc-binding domain affinity. We found that the Fc-ARMs accepted an affibody as a tumor-binding domain to induce ADCC. Furthermore, the required residence time of the complex between Fc-ARM and IgG was ∼102 min, which was comparable to that when monoclonal antibodies bind to their specific antigens. However, we found that the extent of ADCC induced by Fc-ARM was lower than that of conventional IgG-mediated ADCC, indicating that further enhancement of the affinity of the antibody-binding terminus and tumor-binding terminus of Fc-ARM may be needed to achieve ADCC equivalent to that of conventional IgG-mediated ADCC.

DOI： 10.1039/d4ra03391d

Open Access

PubMed

Optineurin provides a mitophagy contact site for TBK1 activation Reviewed

Koji Yamano, Momoha Sawada, Reika Kikuchi, Kafu Nagataki, Waka Kojima, Ryu Endo, Hiroki Kinefuchi, Atsushi Sugihara, Tomoshige Fujino, Aiko Watanabe, Keiji Tanaka, Gosuke Hayashi, Hiroshi Murakami, and Noriyuki Matsuda

EMBO J ( 43 ) page： 754 2024.1

Thiocholine-Mediated One-Pot Peptide Ligation and Desulfurization Open Access

Sae Suzuki, Yuya Nakajima, Naoki Kamo, Akihisa Osakabe, Akimitsu Okamoto, Gosuke Hayashi, Hiroshi Murakami

Molecules Vol. 28 ( 9 ) page： 3655 - 3655 2023.4

Language：English Publishing type：Research paper (scientific journal) Publisher：{MDPI} {AG}

<jats:p>Thiol catalysts are essential in native chemical ligation (NCL) to increase the reaction efficiency. In this paper, we report the use of thiocholine in chemical protein synthesis, including NCL-based peptide ligation and metal-free desulfurization. Evaluation of thiocholine peptide thioester in terms of NCL and hydrolysis kinetics revealed its practical utility, which was comparable to that of other alkyl thioesters. Importantly, thiocholine showed better reactivity as a thiol additive in desulfurization, which is often used in chemical protein synthesis to convert Cys residues to more abundant Ala residues. Finally, we achieved chemical synthesis of two differently methylated histone H3 proteins via one-pot NCL and desulfurization with thiocholine.</jats:p>

DOI： 10.3390/molecules28093655

Open Access

PubMed

Repetitive Thiazolidine Deprotection Using a Thioester-Compatible Aldehyde Scavenger for One-Pot Multiple Peptide Ligation. Invited Reviewed

Koki Nakatsu, Prof. Dr. Akimitsu Okamoto, Prof. Dr. Gosuke Hayashi, Prof. Dr. Hiroshi Murakami

Angewandte Chemie International Edition Vol. 61 ( 39 ) page： e202206240 2022.7

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

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

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

Construction of a highly diverse mRNA library for in vitro selection of monobodies Open Access

Taishi Kondo, Minori Eguchi, Nariaki Tsuzuki, Naoya Murata, Tomoshige Fujino, Gosuke Hayashi, Hiroshi Murakami

Bio-protocol Vol. 11 ( 16 ) page： e4125 2021.8

Language：English Publishing type：Research paper (scientific journal) Publisher：Bio-protocol LLC

Recently, we developed transcription/translation coupled with the association of puromycin linker (TRAP) display as a quick in vitro selection method to obtain antibody-like proteins. For the in vitro selection, it is important to prepare mRNA libraries among which the diversity is high. Here, we describe a method for the preparation of monobody mRNA libraries with greater than 1013 theoretical diversity. First, we synthesized two long single-stranded DNAs that corresponded to fragments of monobody DNA, with random codons in the BC and FG loops. These oligonucleotides were ligated by T4 DNA ligase with the support of guide oligonucleotides containing 3′ ends that were protected by a modification. After amplifying the product DNAs by PCR, one end of each DNA fragment was digested with the type II restriction enzyme BsaI, and the resulting DNA fragments were ligated using T4 DNA ligase. After amplification of the DNA product, mRNAs were synthesized by T7 RNA polymerase. This method is simple and could be used for the preparation of mRNA libraries for various antibody-like proteins.

DOI： 10.21769/BioProtoc.4125

Scopus

PubMed

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

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

Chemical Science Vol. 12 page： 5926 - 5937 2021.3

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

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

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

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

Open Access

PubMed

An Amino Acid-Swapped Genetic Code International journal Open Access

Tomoshige Fujino, Masahiro Tozaki, Hiroshi Murakami

ACS Synthetic Biology Vol. 9 ( 10 ) page： 2703 - 2713 2020.10

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

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

DOI： 10.1021/acssynbio.0c00196

Open Access

PubMed

Antibody-like proteins that capture and neutralize SARS-CoV-2 Open Access

Taishi Kondo, Y. Iwatani, Kazuhiro Matsuoka, Tomoshige Fujino, S. Umemoto, Y. Yokomaku, Keigo Ishizaki, 清太鬼頭, Sezaki Takahiro, G. Hayashi, Hiroshi Murakami

Science Advances Vol. 6 ( 42 ) 2020.10

An amino acid-swapped genetic code. Reviewed Open Access

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

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

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

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

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

DOI： 10.1002/cbic.201900150

PubMed

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Papers - MURAKAMI Hiroshi