Updated on 2024/06/06

写真a

 
OHTSUKI Takashi
 
Organization
Faculty of Interdisciplinary Science and Engineering in Health Systems Professor
Position
Professor
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Degree

  • 学士 ( 東京工業大学 )

  • Doctor(Engineering) ( The University of Tokyo )

  • 修士 ( 東京大学 )

Research Interests

  • アポトーシス

  • miRNA

  • 光制御

  • RNAキャリア

  • 超音波

  • エンドソーム脱出

  • ナノキャリア

  • 拡張翻訳系

  • tRNA

  • EF-Tu

  • RNA delivery

  • siRNA

  • 光増感剤

  • RNA工学

  • RNAi

  • ケージド化合物

  • タンパク質生合成系

  • 非天然アミノ酸

  • mRNA

  • tRNA

  • siRNA

  • Biochemistry

  • Molecular Biology

  • translation

  • nonnatural amino acid

  • RNAi

  • EF-Tu

  • messenger RNA

Research Areas

  • Nanotechnology/Materials / Bio chemistry

  • Life Science / Structural biochemistry

  • Manufacturing Technology (Mechanical Engineering, Electrical and Electronic Engineering, Chemical Engineering) / Biofunction and bioprocess engineering

  • Nanotechnology/Materials / Chemistry and chemical methodology of biomolecules

  • Life Science / Biomedical engineering

  • Life Science / Biomaterials

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Education

  • The University of Tokyo   工学研究科   化学生命工学専攻 博士課程

    1995 - 1998

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  • The University of Tokyo   工学研究科   化学生命工学専攻 修士課程

    1993 - 1995

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    Country: Japan

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  • Tokyo Institute of Technology   工学部   生体分子工学科

    1989 - 1993

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    Country: Japan

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Research History

  • Okayama University   Graduate School of Interdisciplinary Science and Engineering in Health Systems   Professor

    2018.4

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  • 岡山大学自然科学研究科 教授

    2010.6 - 2018.3

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  • 岡山大学自然科学研究科 准教授

    2007.10 - 2010.5

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  • Okayama University   Faculty of Engineering, Department of Bioscience and Biotechnology

    2003.11 - 2007.9

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  • The University of Tokyo   Graduate School of Frontier Sciences

    2002.4 - 2003.10

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  • The University of Tokyo   The Graduate School of Engineering

    2000.3 - 2002.3

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  • 理化学研究所ゲノム化学総合研究センター リサーチアソシエイト

    1999.1 - 2000.3

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  • 日本学術振興会 特別研究員 日本学術振興会特別研究員

    1997.4 - 1998.12

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Professional Memberships

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Committee Memberships

  • 日本光医学・光生物学会   評議員  

    2023.4   

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    Committee type:Academic society

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  • 日本化学会中国四国支部   役員  

    2021.4 - 2023.3   

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  • 日本核酸化学会   評議員  

    2016   

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    Committee type:Academic society

    日本核酸化学会

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  • 日本生化学会   代議員  

    2014.4 - 2018.3   

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    Committee type:Academic society

    日本生化学会

  • 日本化学会 生体機能関連化学部会   ニュースレター編集委員  

    2011.4 - 2014.3   

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    Committee type:Academic society

    日本化学会 生体機能関連化学部会

  • 日本生化学会   評議員  

    2011   

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    Committee type:Academic society

    日本生化学会

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  • 日本化学会 生体機能関連化学部会   幹事  

    2010   

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    日本化学会 生体機能関連化学部会

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Papers

  • In Vitro Study of Tumor-Homing Peptide-Modified Magnetic Nanoparticles for Magnetic Hyperthermia

    Shengli Zhou, Kaname Tsutsumiuchi, Ritsuko Imai, Yukiko Miki, Anna Kondo, Hiroshi Nakagawa, Kazunori Watanabe, Takashi Ohtsuki

    Molecules   29 ( 11 )   2632 - 2632   2024.6

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    Publishing type:Research paper (scientific journal)   Publisher:MDPI AG  

    Cancer cells have higher heat sensitivity compared to normal cells; therefore, hyperthermia is a promising approach for cancer therapy because of its ability to selectively kill cancer cells by heating them. However, the specific and rapid heating of tumor tissues remains challenging. This study investigated the potential of magnetic nanoparticles (MNPs) modified with tumor-homing peptides (THPs), specifically PL1 and PL3, for tumor-specific magnetic hyperthermia therapy. The synthesis of THP-modified MNPs involved the attachment of PL1 and PL3 peptides to the surface of the MNPs, which facilitated enhanced tumor cell binding and internalization. Cell specificity studies revealed an increased uptake of PL1- and PL3-MNPs by tumor cells compared to unmodified MNPs, indicating their potential for targeted delivery. In vitro hyperthermia experiments demonstrated the efficacy of PL3-MNPs in inducing tumor cell death when exposed to an alternating magnetic field (AMF). Even without exposure to an AMF, an additional ferroptotic pathway was suggested to be mediated by the nanoparticles. Thus, this study suggests that THP-modified MNPs, particularly PL3-MNPs, hold promise as a targeted approach for tumor-specific magnetic hyperthermia therapy.

    DOI: 10.3390/molecules29112632

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  • Self-assembly of Peptide Amphiphiles with Alkyl Groups for siRNA Delivery

    Taufik F.N. Hakim, Kazunori Watanabe, Shomu Fujimoto, Mizuki Kitamatsu, Takashi Ohtsuki

    Chemistry Letters   2023.9

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    Publishing type:Research paper (scientific journal)   Publisher:The Chemical Society of Japan  

    DOI: 10.1246/cl.230302

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  • Photo-dependent cytosolic delivery of shRNA into a single blastomere in a mouse embryo. Reviewed International journal

    Yuka Ikawa, Takuya Wakai, Hiroaki Funahashi, Tet Htut Soe, Kazunori Watanabe, Takashi Ohtsuki

    Scientific reports   13 ( 1 )   13050 - 13050   2023.8

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

    Single-cell-specific delivery of small RNAs, such as short hairpin RNA (shRNA) and small noncoding RNAs, allows us to elucidate the roles of specific upregulation of RNA expression and RNAi-mediated gene suppression in early embryo development. The photoinduced cytosolic dispersion of RNA (PCDR) method that we previously reported can introduce small RNAs into the cytosol of photoirradiated cells and enable RNA delivery into a single-cell in a spatiotemporally specific manner. However, the PCDR method has only been applied to planer cultured cells and not to embryos. This study demonstrated that the PCDR method can be utilized for photo-dependent cytosolic shRNA delivery into a single blastomere and for single blastomere-specific RNA interference in mouse embryos. Our results indicate that PCDR is a promising approach for studying the developmental process of early embryogenesis.

    DOI: 10.1038/s41598-023-40361-9

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  • FRET probe for detecting two mutations in one EGFR mRNA. International journal

    Myat Thu, Kouta Yanai, Hajime Shigeto, Shohei Yamamura, Kazunori Watanabe, Takashi Ohtsuki

    Analyst   148 ( 11 )   2626 - 2632   2023.5

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

    Technologies for visualizing and tracking RNA are essential in molecular biology, including in disease-related fields. In this study, we propose a novel probe set (DAt-probe and T-probe) that simultaneously detects two mutations in the same RNA using fluorescence resonance energy transfer (FRET). The DAt-probe carrying the fluorophore Atto488 and the quencher Dabcyl were used to detect a cancer mutation (exon19del), and the T-probe carrying the fluorophore Tamra was used to detect drug resistance mutations (T790M) in epidermal growth factor receptor (EGFR) mRNA. These probes were designed to induce FRET when both mutations were present in the mRNA. Gel electrophoresis confirmed that the two probes could efficiently bind to the mutant mRNA. We measured the FRET ratios using wild-type and double-mutant RNAs and found a significant difference between them. Even in living cells, the FRET probe could visualize mutant RNA. As a result, we conclude that this probe set provides a method for detecting two mutations in the single EGFR mRNA via FRET.

    DOI: 10.1039/d3an00554b

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  • Photochemical internalizationに基づく光依存的細胞質内RNA導入法の副作用低減

    Akinari Bando, Kazunori Watanabe, Takashi Ohtsuki

    The Journal of Japan Society for Laser Surgery and Medicine   44 ( 1 )   62 - 68   2023.4

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    Publishing type:Research paper (scientific journal)   Publisher:Japan Society for Laser Surgery and Medicine  

    DOI: 10.2530/jslsm.jslsm-44_0004

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  • Reduction of the side effects of photoinduced cytosolic dispersion of RNA (PCDR) based on photochemical internalization Invited Reviewed International journal

    Akinari Bando, Kazunori Watanabe, Takashi Ohtsuki

    2023.4

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

    BNCT is a non-invasive cancer therapy that allows for cancer cell death without harming adjacent cells. However, the application is limited, owing to the challenges of working with clinically approved boron (B) compounds and drug delivery systems (DDS). To address the issues, we developed self-forming nanoparticles consisting of a biodegradable polymer, namely, "AB-type Lactosome (AB-Lac)" loaded with B compounds. Three carborane isomers (o-, m-, and p-carborane) and three related alkylated derivatives, i.e., 1,2-dimethy-o-carborane (diC1-Carb), 1,2-dihexyl-o-carborane (diC6-Carb), and 1,2-didodecyl-o-carborane (diC12-Carb), were separately loaded. diC6-Carb was highly loaded with AB-Lac particles, and their stability indicated the "molecular glue" effect. The efficiency of in vitro B uptake of diC6-Carb for BNCT was confirmed at non-cytotoxic concentration in several cancer cell lines. In vivo/ex vivo biodistribution studies indicated that the AB-Lac particles were remarkably accumulated within 72 h post-injection in the tumor lesions of mice bearing syngeneic breast cancer (4T1) cells, but the maximum accumulation was reached at 12 h. In ex vivo B biodistribution, the ratios of tumor/normal tissue (T/N) and tumor/blood (T/Bl) of the diC6-Carb-loaded particles remained stably high up to 72 h. Therefore, we propose the diC6-Carb-loaded AB-Lac particles as a promising candidate medicine for BNCT.

  • Adjusting Heterodimeric Coiled-Coils (K/E Zipper) to Connect Autophagy-Inducing Peptide with Cell-Penetrating Peptide. International journal

    Yoshiyuki Hakata, Kazuma Yamashita, Sonoko Hashimoto, Takashi Ohtsuki, Masaaki Miyazawa, Mizuki Kitamatsu

    Pharmaceutics   15 ( 4 )   2023.3

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

    A connection of a functional peptide with a cell-penetrating peptide (CPP) used a heterodimeric coiled-coil as a molecular zipper can improve the intracellular delivery and activity of the functional peptide. However, the chain length of the coiled coil required for functioning as the molecular zipper is unknown at present. To solve the problem, we prepared an autophagy-inducing peptide (AIP) that conjugates with the CPP via heterodimeric coiled-coils consisting of 1 to 4 repeating units (K/E zipper; AIP-Kn and En-CPP), and we investigated the optimum length of the K/E zipper for effective intracellular delivery and autophagy induction. Fluorescence spectroscopy showed that K/E zippers with n = 3 and 4 formed a stable 1:1 hybrid (AIP-K3/E3-CPP and AIP-K4/E4-CPP, respectively). Both AIP-K3 and AIP-K4 were successfully delivered into cells by the corresponding hybrid formation with K3-CPP and K4-CPP, respectively. Interestingly, autophagy was also induced by the K/E zippers with n = 3 and 4, more intensively by the former than by the latter. The peptides and K/E zippers used in this study did not show significant cytotoxicity. These results indicate that the effective induction of autophagy occurs via an exquisite balance of the association and dissociation of the K/E zipper in this system.

    DOI: 10.3390/pharmaceutics15041048

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  • Novel Self-Forming Nanosized DDS Particles for BNCT: Utilizing A Hydrophobic Boron Cluster and Its Molecular Glue Effect. International journal

    Abdul Basith Fithroni, Kazuko Kobayashi, Hirotaka Uji, Manabu Ishimoto, Masaru Akehi, Takashi Ohtsuki, Eiji Matsuura

    Cells   11 ( 20 )   2022.10

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

    BNCT is a non-invasive cancer therapy that allows for cancer cell death without harming adjacent cells. However, the application is limited, owing to the challenges of working with clinically approved boron (B) compounds and drug delivery systems (DDS). To address the issues, we developed self-forming nanoparticles consisting of a biodegradable polymer, namely, "AB-type Lactosome (AB-Lac)" loaded with B compounds. Three carborane isomers (o-, m-, and p-carborane) and three related alkylated derivatives, i.e., 1,2-dimethy-o-carborane (diC1-Carb), 1,2-dihexyl-o-carborane (diC6-Carb), and 1,2-didodecyl-o-carborane (diC12-Carb), were separately loaded. diC6-Carb was highly loaded with AB-Lac particles, and their stability indicated the "molecular glue" effect. The efficiency of in vitro B uptake of diC6-Carb for BNCT was confirmed at non-cytotoxic concentration in several cancer cell lines. In vivo/ex vivo biodistribution studies indicated that the AB-Lac particles were remarkably accumulated within 72 h post-injection in the tumor lesions of mice bearing syngeneic breast cancer (4T1) cells, but the maximum accumulation was reached at 12 h. In ex vivo B biodistribution, the ratios of tumor/normal tissue (T/N) and tumor/blood (T/Bl) of the diC6-Carb-loaded particles remained stably high up to 72 h. Therefore, we propose the diC6-Carb-loaded AB-Lac particles as a promising candidate medicine for BNCT.

    DOI: 10.3390/cells11203307

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  • Fluorescence ratiometric DNA detection by peptide nucleic acid-pyrene binary probes. International journal

    Koki Ishii, Sakura Tsuchitani, Miyu Toyama, Hajime Shigeto, Shohei Yamamura, Takashi Ohtsuki, Yoshitane Imai, Mizuki Kitamatsu

    Bioorganic & medicinal chemistry letters   71   128838 - 128838   2022.9

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

    We developed a method for detecting DNA by excimer fluorescence from two peptide nucleic acids (PNAs) modified with a pyrene (Pyr). The two PNA-Pyr probes were prepared by solid-phase peptide synthesis, and we assessed fluorescence from the mixture of probes with DNA. From the results, excimer fluorescence derived from the two PNA-Pyr probes forming hybrids with the complementary DNA was observed, and the two probes showed the maximum excimer/monomer ratio when the probes and DNA were hybridized at a 1:1:1 ratio, indicating that the PNA-Pyr probes can detect target DNA. Furthermore, we adjusted the spatial arrangement between the two PNA-Pyr hybrids formed on the DNA to promote optimal excimer formation. As a result, optimal excimer formation was achieved by spacing the two nucleobases between the formed two hybrids and further inserting a hexamethylene linker (C6) between the PNA and Pyr of the PNA-Pyr probe on one side.

    DOI: 10.1016/j.bmcl.2022.128838

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  • Ultrasound-dependent RNAi using TatU1A-rose bengal conjugate. Reviewed International journal

    Nanako Sumi, Shota Nagahiro, Eiji Nakata, Kazunori Watanabe, Takashi Ohtsuki

    Bioorganic & medicinal chemistry letters   68   128767 - 128767   2022.7

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

    Tat-U1A-rose bengal conjugate (TatU1A-RB) was prepared as an ultrasound-sensitive RNA carrier molecule. This molecule consists of Tat cell-penetrating peptide, U1A RNA-binding protein, and rose bengal as a sonosensitizer. We demonstrated that TatU1A-RB delivered RNA via the endocytosis pathway, which was followed by ultrasound-dependent endosomal escape and cytosolic dispersion of the RNA. A short hairpin RNA (shRNA) delivered by TatU1A-RB mediated RNA interference (RNAi) ultrasound-dependently. Even by ultrasound irradiation through blood cells, RNAi could be induced with TatU1A-RB and the shRNA. This ultrasound-dependent cytosolic RNA delivery method will serve as the basis for a new approach to nucleic acid therapeutics.

    DOI: 10.1016/j.bmcl.2022.128767

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  • Adjusting the Structure of a Peptide Nucleic Acid (PNA) Molecular Beacon and Promoting Its DNA Detection by a Hybrid with Quencher-Modified DNA Invited Reviewed

    Hajime Shigeto, Takashi Ohtsuki, Shohei Yamamura, Mizuki Kitamatsu

    PROCESSES   10 ( 4 )   2022.4

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    In this study, we performed an elaborate adjustment of the structure of peptide nucleic acid (PNA) molecular beacons as probes for detecting nucleic acids. We synthesized the PNA beacons with various numbers of Glu, Lys, and dabcyl (Dab) quenchers in them, and we investigated their fluorescence changes (F-1/1/F-0) with and without full-match DNA. As the numbers of Glu/Lys or Dab increased, the F-1/1/F-0 tended to decrease. Among the different beacons, the PNA beacon with one Glu and one Lys (P1Q1) showed the largest F-1/1/F-0. On the other hand, a relatively large F-1/1/F-0 was obtained when the number of Glu/Lys and the number of Dab were the same, and the balance between the numbers of Glu/Lys and Dab seemed to affect the F-1/1/F-0. We also investigated the DNA detection by the prehybrid of P1Q1, which consists of the T790M base sequence, [P1Q1(T790M)], with quencher-modified DNA (Q-DNA). We examined the DNA detection with single-base mismatch by P1Q1(T790M), and we clarified that there was difficulty in detecting the sequence with P1Q1 alone, but that the sequence was successfully detected by the prehybrid of P1Q1 with the Q-DNA.

    DOI: 10.3390/pr10040722

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  • Fluorophore-PNA-Quencher/Quencher-DNA probe for miRNA detection. Reviewed International journal

    Kentaro Tabara, Kazunori Watanabe, Hajime Shigeto, Shohei Yamamura, Takamasa Kishi, Mizuki Kitamatsu, Takashi Ohtsuki

    Bioorganic & medicinal chemistry letters   in press   128359 - 128359   2021.9

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    Micro RNAs (miRNAs) are involved in a variety of biological functions and are attracting attention as diagnostic and prognostic markers for various diseases. Highly sensitive RNA detection methods are required to determine miRNA expression levels and intracellular localization. In this study, we designed new double-stranded peptide nucleic acid (PNA)/DNA probes consisting of a fluorophore-PNA-quencher (fPq) and a quencher-DNA (qD) for miR-221 detection. We optimized the fPq structure, PNA-DNA hybrid length, and hybrid position. The resultant fPq-2/qD-6b probe was a 6-bp hybrid probe with a 10-base fPq and a 6-base qD. The signal-to-background ratios of the probes showed that fPq-2/qD-6b had a higher target sensitivity than fPq (PNA beacon)-type and fP/qD-type probes. The results of the detection limit and target specificity indicate that the fPq/qD probe is promising for RNA detection in both cells and cell extracts as well as for miRNA diagnosis.

    DOI: 10.1016/j.bmcl.2021.128359

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  • Photocontrolled apoptosis induction using precursor miR-664a and an RNA carrier-conjugated with photosensitizer. Reviewed International journal

    Kazunori Watanabe, Tomoko Nawachi, Ruriko Okutani, Takashi Ohtsuki

    Scientific reports   11 ( 1 )   14936 - 14936   2021.7

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

    Methods to spatially induce apoptosis are useful for cancer therapy. To control the induction of apoptosis, methods using light, such as photochemical internalization (PCI), have been developed. We hypothesized that photoinduced delivery of microRNAs (miRNAs) that regulate apoptosis could spatially induce apoptosis. In this study, we identified pre-miR-664a as a novel apoptosis-inducing miRNA via mitochondrial apoptotic pathway. Further, we demonstrated the utility of photoinduced cytosolic dispersion of RNA (PCDR), which is an intracellular RNA delivery method based on PCI. Indeed, apoptosis is spatially regulated by pre-miR-664a and PCDR. In addition, we found that apoptosis induced by pre-miR-664a delivered by PCDR was more rapid than that by lipofection. These results suggest that pre-miR-664a is a nucleic acid drug candidate for cancer therapy and PCDR and pre-miR-664a-based strategies have potential therapeutic uses for diseases affecting various cell types.

    DOI: 10.1038/s41598-021-94249-7.

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  • Lactosome-Conjugated siRNA Nanoparticles for Photo-Enhanced Gene Silencing in Cancer Cells. Reviewed International journal

    Melissa Siaw Han Lim, Yuki Nishiyama, Takashi Ohtsuki, Kazunori Watanabe, Hirotsugu Kobuchi, Kazuko Kobayashi, Eiji Matsuura

    Journal of pharmaceutical sciences   110 ( 4 )   1788 - 1798   2021.4

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:ELSEVIER SCIENCE INC  

    The A3B-type Lactosome comprised of poly(sarcosine)3-block-poly(l-lactic acid), a biocompatible and biodegradable polymeric nanomicelle, was reported to accumulate in tumors in vivo via the enhanced permeability and retention (EPR) effect. Recently, the cellular uptake of Lactosome particles was enhanced through the incorporation of a cell-penetrating peptide (CPP), L7EB1. However, the ability of Lactosome as a drug delivery carrier has not been established. Herein, we have developed a method to conjugate the A3B-type Lactosome with ATP-binding cassette transporter G2 (ABCG2) siRNA for inducing in vitro apoptosis in the cancer cell lines PANC-1 and NCI-H226. The L7EB1 peptide facilitates the cellular uptake efficiency of Lactosome but does not deliver siRNA into cytosol. To establish the photoinduced cytosolic dispersion of siRNA, a photosensitizer loaded L7EB1-Lactosome was prepared, and the photosensitizer 5,10,15,20-tetra-kis(pentafluorophenyl)porphyrin (TPFPP) showed superiority in photoinduced cytosolic dispersion. We exploited the combined effects of enhanced cellular uptake by L7EB1 and photoinduced endosomal escape by TPFPP to efficiently deliver ABCG2 siRNA into the cytosol for gene silencing. Moreover, the silencing of ABCG2, a protoporphyrin IX (PpIX) transporter, also mediated photoinduced cell death via 5-aminolevulinic acid (ALA)-mediated PpIX accumulated photodynamic therapy (PDT). The synergistic capability of the L7EB1/TPFPP/siRNA-Lactosome complex enabled both gene silencing and PDT.

    DOI: 10.1016/j.xphs.2021.01.026

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  • Nucleic Acids Chemistry and Engineering: Special Issue on Nucleic Acid Conjugates for Biotechnological Applications

    Tamaki Endoh, Eriks Rozners, Takashi Ohtsuki

    Applied Sciences   11 ( 8 )   2021.4

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    DOI: 10.3390/app11083594

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  • Minimization of apoptosis-inducing CPP-Bim peptide. Reviewed International journal

    Shengli Zhou, Kazunori Watanabe, Seiichiro Koide, Mizuki Kitamatsu, Takashi Ohtsuki

    Bioorganic & medicinal chemistry letters   36   127811 - 127811   2021.3

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

    Pro-apoptotic peptides may be promising agents for cancer therapy owing to their ability to induce apoptosis in cancer cells. TatBim, a fusion peptide of Tat cell-penetrating peptide (CPP) and the BH3 domain derived from Bim apoptosis-inducing protein, is a pro-apoptotic peptide. In this study, based on the TatBim sequence, we attempted to minimize the CPP-Bim peptide while retaining apoptosis-inducing activity. The CPP and Bim parts were systematically shortened, and the pro-apoptotic activities of the shortened peptides were examined. We obtained TatBim-N1C2 and R8Bim-N1C2 as minimized peptides with efficient apoptotic activity. These peptides may have potential applications in future biomedical studies, such as cancer therapeutics.

    DOI: 10.1016/j.bmcl.2021.127811

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  • Inhibition of HSF1 and SAFB Granule Formation Enhances Apoptosis Induced by Heat Stress Reviewed

    Watanabe K, Ohtsuki T.

    Int J Mol Sci.   22 ( 9 )   4982 - 4982   2021.3

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    DOI: 10.3390/ijms22094982.

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  • A Novel 89Zr-labeled DDS Device Utilizing Human IgG Variant (scFv): "Lactosome" Nanoparticle-Based Theranostics for PET Imaging and Targeted Therapy. International journal

    Melissa Siaw Han Lim, Takashi Ohtsuki, Fumiaki Takenaka, Kazuko Kobayashi, Masaru Akehi, Hirotaka Uji, Hirotsugu Kobuchi, Takanori Sasaki, Eiichi Ozeki, Eiji Matsuura

    Life   11 ( 2 )   2021.2

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    "Theranostics," a new concept of medical advances featuring a fusion of therapeutic and diagnostic systems, provides promising prospects in personalized medicine, especially cancer. The theranostics system comprises a novel 89Zr-labeled drug delivery system (DDS), derived from the novel biodegradable polymeric micelle, "Lactosome" nanoparticles conjugated with specific shortened IgG variant, and aims to successfully deliver therapeutically effective molecules, such as the apoptosis-inducing small interfering RNA (siRNA) intracellularly while offering simultaneous tumor visualization via PET imaging. A 27 kDa-human single chain variable fragment (scFv) of IgG to establish clinically applicable PET imaging and theranostics in cancer medicine was fabricated to target mesothelin (MSLN), a 40 kDa-differentiation-related cell surface glycoprotein antigen, which is frequently and highly expressed by malignant tumors. This system coupled with the cell penetrating peptide (CPP)-modified and photosensitizer (e.g., 5, 10, 15, 20-tetrakis (4-aminophenyl) porphyrin (TPP))-loaded Lactosome particles for photochemical internalized (PCI) driven intracellular siRNA delivery and the combination of 5-aminolevulinic acid (ALA) photodynamic therapy (PDT) offers a promising nano-theranostic-based cancer therapy via its targeted apoptosis-inducing feature. This review focuses on the combined advances in nanotechnology and material sciences utilizing the "89Zr-labeled CPP and TPP-loaded Lactosome particles" and future directions based on important milestones and recent developments in this platform.

    DOI: 10.3390/life11020158

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  • Complementary leucine zippering system for effective intracellular delivery of proteins by cell-penetrating peptides. Reviewed International journal

    Mizuki Kitamatsu, Hiroki Yuasa, Takashi Ohtsuki, Hiroyuki Michiue

    Bioorganic & medicinal chemistry   33   116036 - 116036   2021.1

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

    A heterodimeric leucine zipper composed of a pair of leucine zipper peptides containing acidic or basic amino acid residues at appropriate positions in each peptide was used as a molecular glue to connect protein cargos to a cell-penetrating peptide (CPP) carrier. To investigate the hybridization properties by fluorescence experiments, we prepared an enhanced green fluorescent protein (EGFP) fused with an acidic leucine zipper (LzK), EGFP-LzK, and a basic leucine zipper (LzE) modified with a CPP, LzE-CPP. The LzK and LzE formed a 1:1 hybrid when EGFP-LzK and LzE-CPP were mixed in phosphate buffer saline, thereby conjugating the EGFP with the CPP. The formation of the 1:1 hybrid was confirmed by fluorescence spectra and fluorescence titration curves. Results from fluorescence microscopy experiments showed that EGFP was successfully delivered into cells by conjugating with the CPP via formation of the LzK/LzE hybrid. We also fused the apoptotic protein p53 with LzK (p53-LzK) and investigated the inhibition of cell proliferation of various cell lines by incubation with the p53-LzK/LzE-CPP hybrid. This hybrid was found to localize in nuclei and successfully inhibited cell-specific proliferation. The LzE/LzK zipper system inhibited cell proliferation more efficiently than the directly fused conjugate, p53-CPP. Our method will be a useful drug delivery system for delivering bioactive proteins to treat various diseases.

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  • Fluorescence lifetime probes for detection of RNA degradation Reviewed International journal

    Riku Hirata, Kazutaka Hirakawa, Naotaka Shimada, Kazunori Watanabe, Takashi Ohtsuki

    Analyst   146 ( 1 )   277 - 282   2021.1

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    <p>To investigate RNA degradation in live cells, detection methods that do not require RNA extraction from cells are necessary. In this study, we examined the utility of fluorescence lifetime measurements...</p>

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  • Photoinduced Endosomal Escape Mechanism: A View from Photochemical Internalization Mediated by CPP-Photosensitizer Conjugates. International journal

    Tet Htut Soe, Kazunori Watanabe, Takashi Ohtsuki

    Molecules   26 ( 1 )   2020.12

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    Endosomal escape in cell-penetrating peptide (CPP)-based drug/macromolecule delivery systems is frequently insufficient. The CPP-fused molecules tend to remain trapped inside endosomes and end up being degraded rather than delivered into the cytosol. One of the methods for endosomal escape of CPP-fused molecules is photochemical internalization (PCI), which is based on the use of light and a photosensitizer and relies on photoinduced endosomal membrane destabilization to release the cargo molecule. Currently, it remains unclear how this delivery strategy behaves after photostimulation. Recent findings, including our studies using CPP-cargo-photosensitizer conjugates, have shed light on the photoinduced endosomal escape mechanism. In this review, we discuss the structural design of CPP-photosensitizer and CPP-cargo-photosensitizer conjugates, and the PCI mechanism underlying their application.

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  • Cell cycle dependence of apoptosis photo-triggered using peptide-photosensitizer conjugate. Reviewed International journal

    Hyungjin Kim, Sho Watanabe, Mizuki Kitamatsu, Kazunori Watanabe, Takashi Ohtsuki

    Scientific reports   10 ( 1 )   19087 - 19087   2020.11

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    Investigation of the relevance between cell cycle status and the bioactivity of exogenously delivered biomacromolecules is hindered by their time-consuming cell internalization and the cytotoxicity of transfection methods. In this study, we addressed these problems by utilizing the photochemical internalization (PCI) method using a peptide/protein-photosensitizer conjugate, which enables immediate cytoplasmic internalization of the bioactive peptides/proteins in a light-dependent manner with low cytotoxicity. To identify the cell-cycle dependent apoptosis, a TatBim peptide-photosensitizer conjugate (TatBim-PS) with apoptotic activity was photo-dependently internalized into HeLa cells expressing a fluorescent ubiquitination-based cell cycle indicator (Fucci2). Upon irradiation, cytoplasmic TatBim-PS internalization exceeded 95% for all cells classified in the G1, S, and G2/M cell cycle phases with no significant differences between groups. TatBim-PS-mediated apoptosis was more efficiently triggered by photoirradiation in the G1/S transition than in the G1 and S/G2/M phases, suggesting high sensitivity of the former phase to Bim-induced apoptosis. Thus, the cell cycle dependence of Bim peptide-induced apoptosis was successfully investigated using Fucci2 indicator and the PCI method. Since PCI-mediated cytoplasmic internalization of peptides is rapid and does not span multiple cell cycle phases, the Fucci-PCI method constitutes a promising tool for analyzing the cell cycle dependence of peptides/protein functions.

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  • Analysis of Single Nucleotide-Mutated Single-Cancer Cells Using the Combined Technologies of Single-Cell Microarray Chips and Peptide Nucleic Acid-DNA Probes. Reviewed International journal

    Hajime Shigeto, Eriko Yamada, Mizuki Kitamatsu, Takashi Ohtsuki, Akira Iizuka, Yasuto Akiyama, Shohei Yamamura

    Micromachines   11 ( 7 )   E628 - E628   2020.6

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    Research into cancer cells that harbor gene mutations relating to anticancer drug-resistance at the single-cell level has focused on the diagnosis of, or treatment for, cancer. Several methods have been reported for detecting gene-mutated cells within a large number of non-mutated cells; however, target single nucleotide-mutated cells within a large number of cell samples, such as cancer tissue, are still difficult to analyze. In this study, a new system is developed to detect and isolate single-cancer cells expressing the T790M-mutated epidermal growth factor receptor (EGFR) mRNA from multiple non-mutated cancer cells by combining single-cell microarray chips and peptide nucleic acid (PNA)-DNA probes. The single-cell microarray chip is made of polystyrene with 62,410 microchambers (31-40 µm diameter). The T790M-mutated lung cancer cell line, NCI-H1975, and non-mutated lung cancer cell line, A549, were successfully separated into single cells in each microchambers on the chip. Only NCI-H1975 cell was stained on the chip with a fluorescein isothiocyanate (FITC)-conjugated PNA probe for specifically detecting T790M mutation. Of the NCI-H1975 cells that spiked into A549 cells, 0-20% were quantitatively analyzed within 1 h, depending on the spike concentration. Therefore, our system could be useful in analyzing cancer tissue that contains a few anticancer drug-resistant cells.

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  • Endosomal Escape of Peptide-Photosensitizer Conjugates Is Affected by Amino Acid Sequences near the Photosensitizer. Reviewed International journal

    Yuichi Miyoshi, Maho Kadono, Shigetoshi Okazaki, Ayano Nishimura, Mizuki Kitamatsu, Kazunori Watanabe, Takashi Ohtsuki

    Bioconjugate Chemistry   31 ( 3 )   916 - 922   2020.3

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    Cell-penetrating peptides (CPPs) are widely used for the intracellular delivery of peptides and proteins, but CPP fusion peptides and proteins are often transported by endocytosis and trapped in endosomes. Photochemical internalization (PCI) is a method for the endosomal escape of the trapped peptide or protein and release into the cytosol using light and photosensitizers. In PCI, endosomal membranes are thought to be destabilized by singlet oxygen (1O2) photogenerated from photosensitizers localized in endosomes. We previously developed CPP-cargo-photosensitizer (PS) conjugates able to photodependently enter the cytosol via the PCI mechanism. For example, TatU1A-PS (a covalent complex of Tat [CPP], U1A RNA-binding protein [cargo], and PS) can photodependently deliver RNAs into the cytosol, and TatBim-PS (a covalent complex of Tat, Bim [cargo], and PS) can photoinduce apoptosis in mammalian cells. However, for many newly created conjugates, the induction of PCI has been insufficient. We hypothesized that the amino acid linker sequence (XX) adjacent to the photosensitizer is an important determinant of PCI efficiency. In this study, using CPP-cargo-XX-PS platforms, we examined the relationship between PCI efficiency and the linker amino acid sequence near the photosensitizer. We found that hydrophobic FF and LL linkers enhanced the PCI efficiencies of both TatBim-XX-PS and TatU1A-XX-PS. The effectiveness of the linker depended, in part, on both the cargo moiety and the photosensitizer. These results may guide the design of CPP-cargo-PS conjugates conferring broad target functions for PCI and photodynamic therapy.

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  • Intracellular delivery of a peptide nucleic acid-based hybrid of an autophagy inducing peptide with a cell-penetrating peptide Reviewed

    Yoshiyuki Hakata, Suzuka Ishikawa, Takashi Ohtsuki, Masaaki Miyazawa, Mizuki Kitamatsu

    Organic & Biomolecular Chemistry   18 ( 10 )   1978 - 1986   2020.3

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    Development of an intracellular delivery method for functional peptides via cell-penetrating peptides (CPPs) expands peptide use in basic research and therapeutic applications. Although direct conjugation of a functional peptide with a CPP is the simplest method for delivery, this method has not always been reliable. CPPs usually contain several positively charged amino acids that potentially interact non-specifically with negatively charged molecules in cells and subsequently interfere with conjugated functional peptide function. Here we demonstrate a new intracellular delivery method for peptides in which a functional peptide is released from a positively charged CPP via peptide nucleic acids (PNAs). We prepared an 8-mer PNA conjugated to octa-arginine in tandem (PNA1-CPP) and linked its complementary PNA to an autophagy inducing peptide (PNA2-AIP) by solid-phase peptide synthesis. PNA1-CPP and PNA2-AIP formed a 1 : 1 hybrid via PNA1/PNA2 interaction, thereby indirectly but stably connecting the AIP to the CPP. PNA2-AIP was successfully delivered into cells in a hybrid formation-dependent manner and at least some portion of the PNA1-CPP/PNA2-AIP hybrids dissociated into PNA2-AIP and PNA1-CPP inside the cells. Notably, PNA2-AIP delivered to cells induced more autophagy than AIP directly conjugated to CPP (CPP-AIP). Further, the PNA hybrid did not induce significant cell death. These findings indicate that the PNA1/PNA2 hybrid can function as a molecular glue enabling the delivery of functional peptides into cells.

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  • Relation of Photochemical Internalization to Heat, pH and Ca2+ Ions. International journal

    Tet Htut Soe, Tomotaka Nanjo, Kazunori Watanabe, Takashi Ohtsuki

    Photochemistry and photobiology   95 ( 6 )   1395 - 1402   2019.11

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    The inefficient endosomal escape of drugs or macromolecules is a major obstacle to achieving successful delivery to therapeutic targets. An efficient approach to circumvent this barrier is photochemical internalization (PCI), which uses light and photosensitizers for endosomal escape of the delivered macromolecules. The PCI mechanism is related to photogenerated singlet oxygen, but the mechanism is still unclear. In this study, we examined the relation of PCI to heat, pH and Ca2+ ions using cell penetrating peptide (CPP)-cargo-photosensitizer (Alexa546 or Alexa633) conjugates. A cell temperature changing experiment demonstrated that heat (thermal mechanism) does not significantly contribute to the photoinduced endosomal escape. Inhibition of V-ATPase proton pump activity and endosomal pH upregulation indicated that PCI-mediated endosomal escape needs endosomal acidification prior to photoirradiation. Imaging of the CPP-cargo-photosensitizer and Ca2+ ions during photostimulation showed that intracellular calcium increase is not the cause of the endosomal escape of the complex. The increment is mainly due to Ca2+ influx. These findings show the importance of extra- and intracellular milieu conditions in the PCI mechanism and enrich our understanding of PCI-related changes in cell.

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  • Imaging analysis of EGFR mutated cancer cells using peptide nucleic acid (PNA)-DNA probes. Reviewed

    Shigeto H, Ohtsuki T, Iizuka A, Akiyama Y, Yamamura S

    Analyst   144 ( 15 )   4613 - 4621   2019.8

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    Lung cancer cells harbor various gene mutations in the mRNA sequence of the Epidermal Growth Factor Receptor (EGFR), especially the mutations of exon19del E746-A750, T790M, and L858R. This results in cancer progression and resistance to anticancer drugs (tyrosine kinase inhibitor; TKI). Therefore, the imaging analysis of EGFR mutations is required for the treatment planning for non-small cell lung cancers. This study focused on the imaging analysis of a single nucleotide substitute in EGFR mutated cancer cells. We developed three novel peptide nucleic acid (PNA)-DNA probes for recognizing and detecting the following three gene mutations in EGFR gene mutations. The PNA-DNA probes consist of fluorescein isothiocyanate (FITC) conjugated PNA as a detection probe and Dabcyl conjugated DNA as a quencher probe. The PNA-DNA probes were used to validate the feasibility for detecting three EGFR mutated sequences: exon19del E746-A750, T790M, and L858R. The three probes emitted fluorescent dose-dependent signals against three target DNA and RNA. Using the three PNA-DNA probes, we succeeded in distinguishing three kinds of lung-cancer cell lines (H1975, PC-9, and A549) which have different EGFR mutations by the fluorescence in situ hybridization (FISH) method.

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  • A leucine zipper-based peptide hybrid delivers functional Nanog protein inside the cell nucleus. Reviewed International journal

    Yoshiyuki Hakata, Hiroyuki Michiue, Takashi Ohtsuki, Masaaki Miyazawa, Mizuki Kitamatsu

    Bioorganic & medicinal chemistry letters   29 ( 7 )   878 - 881   2019.4

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    We synthesized a pair of compounds containing leucine zipper peptides to deliver protein cargo into cells. One is a cell-penetrating peptide (CPP) with Lz(E), a leucine zipper peptide containing negatively charged amino acids, and the other is a Nanog protein with Lz(K), a leucine zipper peptide containing positively charged amino acids. When cells were treated with these equimolar mixtures, Nanog-Lz(K) hybridized with Lz(E)-CPP was successfully delivered into the cells. Furthermore, Nanog-Lz(K) exerted its proper function after nuclear transport.

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  • Combined apoptotic effects of peptide and miRNA in a peptide/miRNA nanocomplex Reviewed

    Hyungjin Kim, Mizuki Kitamatsu, Takashi Ohtsuki

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   128 ( 1 )   110 - 116   2019.1

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    The present study investigated combined biological effects of peptide and miRNA in a peptide/miRNA nanocomplex. We utilized TatBim peptide as a cell-penetrating peptide-based RNA carrier with apoptotic activity. miRNA with apoptotic activity (miR-34a) was used for complex formation to investigate the additional effects of the combination with TatBim peptide. TatBim peptide and the miRNA formed nanocomplexes (approximately 250 nm in diameter), and these complexes were efficiently internalized by cells. Despite its efficient cell internalization, apoptotic activity of the nanocomplex decreased with increasing RNA content. However, photosensitizer-attachment to TatBim and photo irradiation significantly improved the apoptotic activity of the nanocomplex by facilitating dispersion of the peptide and RNA in the cytoplasm. Combined apoptotic activity of both TatBim peptide and miR-34a in the nanocomplex was demonstrated by substituting TatBim with Lipofectamine and by substituting miR-34a with scrambled siRNA. (C) 2019, The Society for Biotechnology, Japan. All rights reserved.

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  • In-stem molecular beacon targeted to a 5'-region of tRNA inclusive of the D arm that detects mature tRNA with high sensitivity. Reviewed International journal

    Miyoshi Y, Ohtsuki T, Kashida H, Asanuma H, Watanabe K

    PloS one   14 ( 1 )   e0211505   2019

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    Cellular functions are regulated by the up- and down-regulation and localization of RNA molecules. Therefore, many RNA detection methods have been developed to analyze RNA levels and localization. Molecular beacon (MB) is one of the major methods for quantitative RNA detection and analysis of RNA localization. Most oligonucleotide-based probes, including MB, are designed to target a long flexible region on the target RNA molecule, e.g., a single-stranded region. Recently, analyses of tRNA localization and levels became important, as it has been shown that environmental stresses and chemical reagents induce nuclear accumulation of tRNA and tRNA degradation in mammalian cells. However, tRNA is highly structured and does not harbor any long flexible regions. Hence, only a few methods are currently available for detecting tRNA. In the present study, we attempted to detect elongator tRNAMet (eMet) and initiator tRNAMet (iMet) by using an in-stem molecular beacon (ISMB), characterized by more effective quenching and significantly higher sensitivity than those of conventional MB. We found that ISMB1 targeted a 5'- region that includes the D arm of tRNA and that it detected eMet and iMet transcripts as well as mature eMet with high sensitivity. Moreover, the analysis revealed that the formation of the ISMB/tRNA transcript complex required more time than the formation of an ISMB/unstructured short RNA complex. These results suggest that ISMB-based tRNA detection can be a useful tool for various biological and medical studies.

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  • Cytoplasmic delivery of a peptide modified with a sonosensitizer by using ultrasound

    Mizuki Kitanciatsu, Takashi Ohtsuki

    JOURNAL OF PEPTIDE SCIENCE   24   S147 - S147   2018.9

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  • Red and Near-Infrared Light-Directed Cytosolic Delivery of Two Different RNAs Using Photosensitive RNA Carriers. Reviewed International journal

    Kaori Shiraga, Tet Htut Soe, Sho Matsumoto, Kazunori Watanabe, Takashi Ohtsuki

    Bioconjugate chemistry   29 ( 9 )   3174 - 3179   2018.8

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    Many cellular events are thought to be controlled by the temporal upregulation of multiple RNAs; the timing of the upregulation of these RNAs is not always the same. In this study, we first show that our light-directed intracellular RNA delivery method induced high concentrations of RNA in a short period. This effect was beneficial for the temporal control of cellular events by functional RNAs. Next, we stimulated the short-term upregulation of two different RNAs at different time points. Cytosolic delivery of a first RNA was induced by red light; thereafter, cytosolic delivery of a second RNA was induced by near-infrared light. The time difference between the introduction of the first and second RNA can be short (0.5-4 h) or long (>8 h). This strategy shows the potential for future applications of the deliberate control of time-dependent RNA concentration to guide various cellular functions by multiple RNAs.

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  • MicroRNA-664a-5p promotes neuronal differentiation of SH-SY5Y cells Reviewed

    Kazunori Watanabe, Ryuhei Yamaji, Takashi Ohtsuki

    Genes to Cells   23 ( 3 )   225 - 233   2018.3

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    MicroRNAs (miRNAs) belong to a class of small noncoding RNAs that play important roles in the translational regulation of gene expression. A number of miRNAs are known to act as key regulators of diverse processes such as neuronal differentiation. In this study, we have attempted to identify novel miRNAs related to neuronal differentiation via microarray analysis in the human neuronal differentiation model neuroblastoma SH-SY5Y cells. We identified 15 up-regulated and eight down-regulated miRNAs in SH-SY5Y cells treated with all-trans retinoic acid to induce differentiation. We further showed that one of the up-regulated miRNAs, miR-664a-5p, promoted neuronal differentiation of SH-SY5Y cells. These findings enhance our understanding of the miRNAs involved in the process of neurogenesis and, in particular, highlight an important role of miR-664a-5p in SH-SY5Y cell neuronal differentiation. Further studies will be required to confirm the function of miR-664-5p in neuronal development and disease and to identify its relevant target genes.

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  • Enhanced intracellular peptide delivery by multivalent cell-penetrating peptide with bioreducible linkage Reviewed

    Hyungjin Kim, Mizuki Kitamatsu, Takashi Ohtsuki

    Bioorganic & Medicinal Chemistry Letters   28 ( 3 )   378 - 381   2018.2

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    Multivalent cell-penetrating peptides (CPPs) have been reported to show enhancement in cellular uptake and endosomolytic activity. However, its application was limited to trans-delivery of cargo which is lower in cellular uptake efficiency of cargo than cis-delivery. Here, we tried the cis-delivery of cargo with multivalent CPP by preparing bioreducible dimeric CPP–cargo with apoptotic activity using TatBim peptide, a fusion of Tat CPP and Bim peptide derived from Bim apoptosis-inducing protein. Dimeric TatBim was almost twice as highly internalized by cells and significantly induced apoptosis compared to monomeric TatBim. Contribution of bioreducible linkage of dimeric TatBim towards apoptotic activity was also confirmed.

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  • Ultrasound-dependent cytoplasmic internalization of a peptidesonosensitizer conjugate Reviewed

    Yuki Inaba, Kazunori Watanabe, Mizuki Kitamatsu, Eiji Nakata, Atsushi Harada, Takashi Ohtsuki

    Bioorganic & Medicinal Chemistry   25 ( 15 )   4212 - 4217   2017.8

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    A method to induce cytoplasmic peptide delivery, using ultrasound, was demonstrated using a molecular conjugate of a cell-penetrating peptide (CPP), a functional peptide, and a sonosensitizer. As a model of such molecular conjugates, TatBim-RB, consisting of the Tat CPP, the Bim apoptosis inducing peptide, and the sonosensitizer rose bengal was synthesized. CPPs have been widely used for intracellular delivery of various cargos; however, CPP-fused molecules tend to become entrapped in endosomes, as was observed for TatBim-RB molecules applied to cells. To promote escape of the entrapped TatBim-RB molecules, cells were irradiated with ultrasound, which successfully induced endosomal escape and cytoplasmic dispersion of TatBim-RB, and subsequently apoptosis. Our results suggest that this peptidesonosensitizer conjugate strategy may facilitate numerous kinds of medicinal chemistry studies, and furthermore, this specific conjugate may exhibit potential as a novel therapeutic agent for the promotion of apoptosis. (C) 2017 Elsevier Ltd. All rights reserved.

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  • Detection of small, highly structured RNAs using molecular beacons Reviewed

    J. Li, C. Xu, N. Shimada, Y. Miyoshi, K. Watanabe, W. Cong, T. Ohtsuki

    Analytical Methods   9 ( 20 )   2971 - 2976   2017.5

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    The use of molecular beacons is a versatile method to detect RNAs. Typically, a single-stranded region of RNA is selected as a target sequence for molecular beacons. Therefore, the detection of highly structured short RNAs, such as tRNAs, seems to be difficult. In this study, as an example of highly structured target RNA, we used human tRNA(Lys3), which is known to have functions in protein synthesis and the reverse transcription of human immunodeficiency virus (HIV)-1. No long single-stranded region more than 8 nt is present in this tRNA, which is much shorter than the standard target length of molecular beacons (similar to 20 nt). This study showed that sensitive detection of highly structured RNAs using molecular beacons was much more difficult than that of unstructured or less structured RNAs. However, efficient detection of the tRNA(Lys3) was achieved by selecting the best target region, i.e., the region around the D arm, probably due to the ease of unfolding of this arm. Accordingly, our findings suggested that molecular beacons may have applications in the detection of highly structured RNAs related to various biological functions and diseases.

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  • Duplication of Drosophila melanogaster mitochondrial EF-Tu: pre-adaptation to T-arm truncation and exclusion of bulky aminoacyl residues Reviewed

    Aya Sato, Takuma Suematsu, Koh-Ki Aihara, Kiyoshi Kita, Tsutomu Suzuki, Kimitsuna Watanabe, Takashi Ohtsuki, Yoh-ichi Watanabe

    Biochemical Journal   474   957 - 969   2017.3

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    Translation elongation factor Tu (EF-Tu) delivers aminoacyl-tRNA (aa-tRNA) to ribosomes in protein synthesis. EF-Tu generally recognizes aminoacyl moieties and acceptor-and T-stems of aa-tRNAs. However, nematode mitochondrial (mt) tRNAs frequently lack all or part of the T-arm that is recognized by canonical EF-Tu. We previously reported that two distinct EF-Tu species, EF-Tu1 and EF-Tu2, respectively, recognize mt tRNAs lacking T-arms and D-arms in the mitochondria of the chromadorean nematode Caenorhabditis elegans. C. elegans EF-Tu2 specifically recognizes the seryl moiety of serylated D-armless tRNAs. Mitochondria of the enoplean nematode Trichinella possess three structural types of tRNAs: T-armless tRNAs, D-armless tRNAs, and cloverleaf tRNAs with a short T-arm. Trichinella mt EF-Tu1 binds to all three types and EF-Tu2 binds only to D-armless Ser-tRNAs, showing an evolutionary intermediate state from canonical EF-Tu to chromadorean nematode (e. g. C. elegans) EF-Tu species. We report here that two EF-Tu species also participate in Drosophila melanogaster mitochondria. Both D. melanogaster EF-Tu1 and EF-Tu2 bound to cloverleaf and D-armless tRNAs. D. melanogaster EF-Tu1 has the ability to recognize T-armless tRNAs that do not evidently exist in D. melanogaster mitochondria, but do exist in related arthropod species. In addition, D. melanogaster EF-Tu2 preferentially bound to aatRNAs carrying small amino acids, but not to aa-tRNAs carrying bulky amino acids. These results suggest that the Drosophila mt translation system could be another intermediate state between the canonical and nematode mitochondria-type translation systems.

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  • Phototriggered protein syntheses by using (7-diethylaminocoumarin-4-yl)methoxycarbonyl-caged aminoacyl tRNAs Reviewed

    Takashi Ohtsuki, Shigeto Kanzaki, Sae Nishimura, Yoshio Kunihiro, Masahiko Sisido, Kazunori Watanabe

    Nature Communications   7   12501 - 12501   2016.8

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    The possibility of spatiotemporally photocontrolling translation holds considerable promise for studies on the biological roles of local translation in cells and tissues. Here we report caged aminoacyl-tRNAs (aa-tRNAs) synthesized using a (7-diethylaminocoumarin-4-yl) methoxycarbonyl (DEACM)-cage compound. DEACM-caged aa-tRNA does not spontaneously deacylate for at least 4 h in neutral aqueous solution, and does not bind to the elongation factor Tu. On irradiation at similar to 405 nm at 125mWcm(-2), DEACM-aa-tRNA is converted into active aa-tRNA with a half-life of 19 s. Notably, this rapid uncaging induced by visible light does not impair the translation system. Translation is photoinduced when DEACM-aa-tRNA carrying a CCCG or a CUA anticodon is uncaged in the presence of mRNAs harbouring a CGGG four-base codon or a UAG amber codon, respectively. Protein synthesis is phototriggered in several model systems, including an in vitro translation system, an agarose gel, in liposomes and in mammalian cells.

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  • Photoinduced apoptosis using a peptide carrying a photosensitizer Reviewed

    Kazunori Watanabe, Hayato Fujiwara, Mizuki Kitamatsu, Takashi Ohtsuki

    Bioorganic & Medicinal Chemistry Letters   26 ( 13 )   3115 - 3118   2016.7

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    A novel molecule, TatBim-Alexa, consisting of the HIV1 Tat cell-penetrating peptide, the Bim apoptosis-inducing peptide, and Alexa Fluor 546 was synthesized for photoinducion of apoptosis. The Alexa Fluor 546 was used as a photosensitizer and covalently attached at the C-terminus of TatBim peptide by the thiol-maleimide reaction. Photo-dependent cytosolic internalization of TatBim-Alexa and photo-dependent apoptosis using TatBim-Alexa were demonstrated in several kinds of mammalian cells including human cancer cell lines. (C) 2016 Elsevier Ltd. All rights reserved.

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  • Enhanced cellular uptake of lactosomes using cell-penetrating peptides. Reviewed International journal

    Akiya Akahoshi, Eiji Matsuura, Eiichi Ozeki, Hayato Matsui, Kazunori Watanabe, Takashi Ohtsuki

    Science and technology of advanced materials   17 ( 1 )   245 - 252   2016.6

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    Polymeric micelles that are composed of synthetic polymers are generally size controllable and can be easily modified for various applications. Lactosomes (A3B-type) are biodegradable polymeric micelles composed of an amphipathic polymer, including three poly(sarcosine) blocks and a poly(l-lactic acid) block. Lactosomes accumulate in tumors in vivo through the enhanced permeability and retention (EPR) effect, even on frequently administering them. However, lactosomes cannot be efficiently internalized by cells. To improve cellular uptake of lactosomes, cell-penetrating peptide (CPP)-modified lactosomes were prepared. Seven CPPs (including EB1 and Pep1) were used, and most of them improved the cellular uptake efficiency of lactosomes. In particular, EB1- and Pep1-modified lactosomes were efficiently internalized by cells. In addition, by using CPP-modified and photosensitizer-loaded lactosomes, we demonstrated the photoinduced killing of mammalian cells, including human cancer cells. Accumulation of the EB1-modified lactosomes in NCI-N87 tumors was shown by in vivo imaging. Thus, this study demonstrated that the CPP-modified lactosome is a promising drug carrier.

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  • Photocontrolled intracellular RNA delivery by using nanoparticles or carrier-photosensitizer conjugates Invited Reviewed International journal

    Kazunori Watanabe, Takashi Ohtsuki

    Prog Mol Biol Transl Sci.   139   101 - 119   2016

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    Small interfering RNA (siRNA) and short hairpin RNA (shRNA) may potentially treat a wide variety of diseases through RNA interference-mediated silencing of specific genes. MicroRNAs (miRNAs) are endogenous regulatory RNA molecules that also modulate gene expression, and thus potential therapeutic approaches using miRNAs have attracted attention. For clinical application of these small RNAs, efficient and safe RNA delivery to target tissues and cells is necessary. Current challenges to RNA delivery are the penetration of negatively charged RNAs through the cell membrane and specific delivery of RNA into target cells. Photocontrolled intracellular RNA delivery is a promising strategy with high target specificity. This strategy includes photodependent endosomal escape of RNA or photodependent release of RNAs from carrier particles. In this chapter, photocontrolled intracellular RNA delivery methods employing gold or silver nanoparticles, upconversion nanoparticles, proteins, or polymers are discussed.

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  • The molecular mechanism of photochemical internalization of cell penetrating peptide-cargophoto-sensitizer conjugates Reviewed

    Takashi Ohtsuki, Shunya Miki, Shouhei Kobayashi, Tokuko Haraguchi, Eiji Nakata, Kazutaka Hirakawa, Kensuke Sumita, Kazunori Watanabe, Shigetoshi Okazaki

    Scientific Reports   5   18577 - 18577   2015.12

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    In many drug delivery strategies, an inefficient transfer of macromolecules such as proteins and nucleic acids to the cytosol often occurs because of their endosomal entrapment. One of the methods to overcome this problem is photochemical internalization, which is achieved using a photosensitizer and light to facilitate the endosomal escape of the macromolecule. In this study, we examined the molecular mechanism of photochemical internalization of cell penetrating peptide-cargo (macromolecule)-photosensitizer conjugates. We measured the photophysical properties of eight dyes (photosensitizer candidates) and determined the respective endosomal escape efficiencies using these dyes. Correlation plots between these factors indicated that the photogenerated O-1(2) molecules from photosensitizers were highly related to the endosomal escape efficiencies. The contribution of O-1(2) was confirmed using O-1(2) quenchers. In addition, time-lapse fluorescence imaging showed that the photoinduced endosomal escape occurred at a few seconds to a few minutes after irradiation (much longer than O-1(2) lifetime), and that the pH increased in the endosome prior to the endosomal escape of the macromolecule.

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  • A novel leucine zipper motif-based hybrid peptide delivers a functional peptide cargo inside cells Reviewed

    Hakata, Y. Tsuchiya, S, Michiue, H, Ohtsuki, T, Matsui, H, Miyazawa M, Kitamatsu, M

    Chemical Communications   51   413 - 416   2015.5

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  • A novel leucine zipper motif-based hybrid peptide delivers a functional peptide cargo inside cells Reviewed

    Y. Hakata, S. Tsuchiya, H. Michiue, T. Ohtsuki, H. Matsui, M. Miyazawa, M. Kitamatsu

    CHEMICAL COMMUNICATIONS   51 ( 2 )   413 - 416   2015

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    A hybrid comprising an autophagy-inducing peptide (AIP) and a cell-penetrating peptide (CPP) connected via heterodimeric leucine zippers was generated and delivered into cells. The hybrid successfully induced autophagy without significant cell death, while the same AIP directly connected to a CPP caused both autophagy and significant cell death.

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  • Photo-dependent protein biosynthesis using a caged aminoacyl-tRNA Reviewed

    Akiya Akahoshi, Yoshio Doi, Masahiko Sisido, Kazunori Watanabe, Takashi Ohtsuki

    Bioorganic & Medicinal Chemistry Letters   24 ( 23 )   5369 - 5372   2014.12

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    Translation systems with four-base codons provide a powerful strategy for protein engineering and protein studies because they enable site-specific incorporation of non-natural amino acids into proteins. In this study, a caged aminoacyl-tRNA with a four-base anticodon was synthesized. The caged aminoacyl-tRNA contains a photocleavable nitroveratryloxycarbonyl (NVOC) group. This study showed that the caged aminoacyl-tRNA was not deacylated, did not bind to EF-Tu, and was activated by light. Photo-dependent translation of an mRNA containing the four-base codon was demonstrated using the caged aminoacyl-tRNA. (C) 2014 Elsevier Ltd. All rights reserved.

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  • mTOR regulates the nucleoplasmic diffusion of Xrn2 under conditions of heat stress Reviewed

    Kazunori Watanabe, Kenichi Ijiri, Takashi Ohtsuki

    FEBS Letters   588 ( 18 )   3454 - 3460   2014.9

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    Stress induces various responses, including translational suppression and tRNA degradation in mammals. Previously, we showed that heat stress induces degradation of initiator tRNA(Met) (iMet) through 5'-3' exoribonuclease Xrn1 and Xrn2, respectively. In addition, we found that rapamycin inhibits the degradation of iMet under heat stress conditions. Here, we report that the mammalian target of rapamycin (mTOR) regulates the diffusion of Xrn2 from the nucleolus to the nucleoplasm, facilitating the degradation of iMet under conditions of heat stress. Our results suggest a mechanism of translational suppression through mTOR-regulated iMet degradation in mammalian cells. (C) 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

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  • Losing the stem-loop structure from metazoan mitochondrial tRNAs and co-evolution of interacting factors Reviewed

    Yoh-ichi Watanabe, Takuma Suematsu, Takashi Ohtsuki

    Frontiers in Genetics   5   109   2014.5

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    Conventional tRNAs have highly conserved sequences, four-armed cloverleaf secondary structures, and L-shaped tertiary structures. However, metazoan mitochondrial tRNAs contain several exceptional structures. Almost all tRNA(Ser) for AGY/N codons lack the D-arm. Furthermore, in some nematodes, no four-armed cloverleaf-type tRNAs are present: two tRNAs(Ser) without the D-arm and 20 tRNAs without the T-arm are found. Previously, we showed that in nematode mitochondria, an extra elongation factor Tu (EF-Tu) has evolved to support interaction with tRNAs lacking the T-arm, which interact with C-terminal domain 3 in conventional EF-Tu. Recent mitochondrial genome analyses have suggested that in metazoan lineages other than nematodes, tRNAs without the T-arm are present. Furthermore, even more simplified tRNAs are predicted in some lineages. In this review, we discuss mitochondrial tRNAs with divergent structures, as well as protein factors, including EF-Tu, that support the function of truncated metazoan mitochondrial tRNAs.

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  • Intracellular Delivery of RNA via RNA-Binding Proteins or Peptides Invited Reviewed

    Kazunori Watanabe, Takashi Ohtsuki

    Intracellular Delivery II   403 - 416   2014

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  • Near-infrared light-directed RNAi using a photosensitive carrier molecule Reviewed

    Yuka Matsushita-Ishiodori, Mika Morinaga, Kazunori Watanabe, Takashi Ohtsuki

    Bioconjugate Chemistry   24 ( 10 )   1669 - 1673   2013.10

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    Controlled activation of small RNAs, such as small interfering RNA, in cells is very useful for various biological applications. Light is an effective inducer of controlled activation
    in particular, near-infrared light is favorable because it can penetrate deeper into tissues than UV or visible light. In this study, near-infrared light control of RNA interference (RNAi) was demonstrated in mammalian cells using a photosensitive RNA carrier molecule, consisting of an RNA carrier protein and a fluorochrome. The photosensitive carrier molecule was identified from six candidates, each with a different fluorochrome. Using this carrier molecule, cytosolic RNA delivery and RNAi can be triggered by near-infrared light. Cytotoxicity was not observed after photoinduction of RNAi. © 2013 American Chemical Society.

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  • Synthesis and Properties of Peptide Dendrimers Containing Fluorescent and Branched Amino Acids Reviewed

    Mizuki Kitamatsu, Mayumi Kitabatake, Yoshiteru Noutoshi, Takashi Ohtsuki

    Biopolymers   100 ( 1 )   64 - 70   2013

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    In this report, we describe dendritic peptides possessing central fluorescent amino acids with adjacent branched amino acids. These fluorescent-peptide dendrimers were prepared using (9-fluorenyl)methoxycarbonyl (Fmoc)-based solid-phase peptide synthesis and Fmoc-derivative fluorescent and branched amino acids. The branched amino acids featured multiple carboxylic acids in their side chains, making the fluorescent-peptide dendrimers highly water-soluble compared with the analogous peptides containing the fluorescent amino acids only. The branched amino acid units also improved the fluorescence intensity of the dendrimers. Based on high-pressure liquid chromatography and fluorescence spectroscopy results, we determined that the fluorescent groups were located in the core and that the carboxylic acids were located on the surface of the dendrimers. Fluorescence resonance energy transfer was achieved among the three proximal fluorescent groups in one of the fabricated fluorescent-peptide dendrimers. (C) 2012 Wiley Periodicals, Inc.

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  • Photoinduced RNA Interference Invited Reviewed

    Yuka Matsushita-Ishiodori, Takashi Ohtsuki

    Accounts of Chemical Research   45 ( 7 )   1039 - 1047   2012.7

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    Because RNA interference (RNAi) can be applied to any gene, this technique has been widely used for studying gene functions. In addition, many researchers are attempting to use RNAi technology in RNAi-based therapies. However, several challenging and controversial issues have arisen during the widespread application of RNAi including target gene specificity, target cell specificity, and spatiotemporal control of gene silencing. To address these issues, several groups have utilized photochemistry to control the RNA release, both spatially and temporally.
    In this Account, we foam on recent studies using photocleavable protecting groups, photosensitizes, Hand gold nanoparticles for photoinduced RNAi. In 2005 the first report of photoinduced RNAi used a caged short interfering RNA (siRNA), an siRNA carrying a photocleavable protecting group. Caging groups block the bioactivities of target molecules, but allow for complete recovery of these functions via photoactivation. However, some RNAi activity can occur in these caged siRNAs, so it will be necessary to decrease this "leakage" and raise the RNAi activity restored after irradiation. This technique also uses UV light around 350 nm, which is cytotoxic, but in the near future we expect that it will be possible to use visible and near-infrared light
    We also examine the application of photochemical internalization (PCI) to RNAi technology, which involves a combination of photosensitizers and light Instead of inducing RNAi using light, the strategy behind this method was to enhance RNAi using RNA carriers. Many wellknown RNA carriers deliver siRNAs into cells by endocytosis. The siRNAs are trapped in endocytic vesicles and have to be released into the cytoplasm in order to express their activity. To achieve the endosomal escape of siRNAs, PCI technology employed photosensitizers to generate light-dependent reactive oxygen species (ROS) that disrupted the endocytic vesicles. In most studies, RNAi-mediated knockdown of the target gene was detected even without PCI. Recently, a polymer capable of trapping the siRNA in endocytic vesicles controlled RNAi almost entirely by light. CLIP-RNAi uses photosensitizing carrier proteins that can be activated over a wide range of visible light wavelengths. With this method RNA carrier/siRNA complexes are completely trapped within endosomes, and RNAi is controlled strictly by light. Such precise, light-dependent control will open up new possibilities for cellular and molecular biology and therapy.
    Most recently, gold nanoparticles (AuNPs) conjugated to siRNA have provided temporal and spatial control of RNAL The light-dependent melting of AuNPs accompanied by a shape transformation induces the release of thiolated siRNAs from AuNPs. In this method, the unique optical properties of the AuNP enable deep penetration of the excitation light into tissues at nearinfrared wavelengths.
    The development of photoinduced RNAi technology will lead to novel insights into gene functions and selective drug delivery, and many other scientific fields will continue to influence its progress.

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  • Synthesis and in situ insertion of a site-specific fluorescently labeled membrane protein into cell-sized liposomes Reviewed

    Takuma Ohtsuka, Satoshi Neki, Tamotsu Kanai, Kazunari Akiyoshi, Shin-ichiro M. Nomura, Takashi Ohtsuki

    Analytical Biochemistry   418 ( 1 )   97 - 101   2011.11

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    The integral membrane protein bacteriorhodopsin, containing a fluorescent amino acid at a specific position, was synthesized in the presence of hydrated lipid films using an in vitro translation system expanded with a four-base codon/anticodon pair. Cell-sized liposomes with the labeled protein inserted into the liposome membranes were generated after the translation reaction. This study also demonstrated that this labeling method could be used to analyze the dynamic properties of membrane proteins in situ by fluorescence correlation spectroscopy. (C) 2011 Elsevier Inc. All rights reserved.

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  • Photosensitizing Carrier Proteins for Photoinducible RNA Interference Reviewed

    Yuka Matsushita-Ishiodori, Rina Kuwabara, Hiroyuki Sakakoshi, Tamaki Endoh, Takashi Ohtsuki

    Bioconjugate Chemistry   22 ( 11 )   2222 - 2226   2011.11

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    RNA interference (RNAi) is being widely explored as a tool in functional genomics and tissue engineering, and in the therapy of intractable diseases, including cancer and neurodegenerative diseases. Recently, we developed a photoinducible RNAi method using photosensitizing carrier proteins, named CLIP-RNAi (CPP-linked REP-mediated RNA internalization and photoinduced RNAi). Novel carrier proteins were designed for this study to establish a highly efficient delivery system for small interfering RNA (siRNA) or short hairpin RNA (shRNA) and to demonstrate light-dependent gene silencing. In addition, the results suggested that the dissociation of the siRNA (or shRNA) from carrier proteins in the cytoplasm is a critical event in CLIP-RNAi-mediated gene silencing.

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  • Site-specific incorporation of arginine analogs into proteins using arginyl-tRNA synthetase Reviewed

    Akiya Akahoshi, Yoshitaka Suzue, Mizuki Kitamatsu, Masahiko Sisido, Takashi Ohtsuki

    Biochem. Biophys. Res. Commun.   414 ( 3 )   625 - 630   2011.10

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    Arginine analogs were incorporated site-specifically into proteins using an in vitro translation system. In this system, mRNAs containing a CGGG codon were translated by an aminoacyl-tRNA(CCCG), which was charged with arginine analogs using yeast arginyl-tRNA synthetase. N(G)-monomethyl-L-arginine, L-citrulline and L-homoarginine were incorporated successfully into proteins using this method. The influence of arginine monomethylation in histone H3 on the acetylation of lysine residues by histone acetyltransferase hGCN5 was investigated, and the results demonstrated that K9 acetylation was suppressed by the methylation of R8 and R17 but not by R26 methylation. K18 acetylation was not affected by the methylation of R8. R17 and R26. This site-specific modification strategy provides a way to explore the roles of post-translational modifications in the absence of heterogeneity due to other modifications. (C) 2011 Elsevier Inc. All rights reserved.

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  • Antisense effect of pyrrolidine-based oxy-peptide nucleic acids in Escherichia coli Reviewed

    Mizuki Kitamatsu, Shunsuke Kurami, Takashi Ohtsuki, Masahiko Sisido

    Bioorganic & Medicinal Chemistry Letters   21 ( 1 )   225 - 227   2011.1

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    To investigate the antisense effect of a pyrrolidine-based oxy-peptide nucleic acid (POPNA), we carried out the LacZ reporter assay using a 12-mer trans-L-POPNA conjugated with a cell-penetrating peptide (antisense reagent). The antisense effect of the conjugated POPNA (inhibition of LacZ activity) was comparable to that shown by a Nielsen-type peptide nucleic acid. Furthermore, the conjugated POPNA could switch the LacZ activity over a wide range of ambient temperatures. (C) 2010 Elsevier Ltd. All rights reserved.

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  • Synthesis of a cyclic peptide/protein using the NEXT-A reaction followed by cyclization Reviewed

    Toshimasa Hamamoto, Masahiko Sisido, Takashi Ohtsuki, Masumi Taki

    Chemical Communications   47 ( 32 )   9116 - 9118   2011

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    By using the NEXT-A reaction, we introduced a non-natural amino acid at the N-terminus of a peptide/protein that contained a cysteine unit. The side chain of the introduced amino acid spontaneously reacted with the cysteine to afford a cyclic peptide/protein.

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  • Synthesis of pyrrolidine-based oxy-peptide nucleic acids carrying four types of nucleobases and their transport into cytoplasm Reviewed

    Mizuki Kitamatsu, Akiko Takahashi, Takashi Ohtsuki, Masahiko Sisido

    Tetrahedron   66 ( 51 )   9659 - 9666   2010.12

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    We synthesized 16 pyrrolidine-based oxy-peptide nucleic acid (POPNA) monomers carrying four different nucleobases onto four different stereoisomers of pyrrolidine rings. Using these monomers, we prepared POPNA oligomers, which formed sequence-specific hybrids with DNAs. The oligomer configurations influenced the hybrid stability. The oligomers were not taken into CHO cells. However, they could enter the cell cytoplasm when mixed with the influenza virus hemagglutinin peptide-arginine heptamer conjugate. (C) 2010 Elsevier Ltd. All rights reserved.

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  • Use of EF-Tu mutants for determining and improving aminoacylation efficiency and for purifying aminoacyl tRNAs with non-natural amino acids Reviewed

    Takashi Ohtsuki, Hiromichi Yamamoto, Yoshio Doi, Masahiko Sisido

    Journal of Biochemistry   148 ( 2 )   239 - 246   2010.8

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    We present three methods relating to tRNA aminoacylation with non-natural amino acids using an Escherichia coli EF-Tu E215A/D216A mutant that can bind tightly to aa-tRNAs carrying either non-natural or natural amino acids: (i) a method for improving aminoacylation efficiency, (ii) a rapid method for analysing aminoacylation efficiency without the use of radioisotope labelling and (iii) a method for purifying aminoacyl-tRNAs. Although the EF-Tu mutant may be incompatible with some kinds of non-natural amino acids, we confirmed that the EF-Tu mutant could efficiently bind to aa-tRNAs carrying various amino acids (Arg, Ser, O-methyltyrosine, Bodipy FL-aminophenylalanine and 2-acrydonylalanine). These methods may be used for the efficient in vitro synthesis of proteins containing various non-natural amino acids.

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  • Lactobacillus-mediated RNA interference in nematode Reviewed

    Ai Kuwahara, Masashi Arita, Akira Kushiro, Yasuji Sakube, Masahiko Sisido, Takashi Ohtsuki

    J. Biosci. Bioeng.   109 ( 2 )   189 - 192   2010.2

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    We engineered Lactobacillus paracasei to produce a dsRNA that would trigger RNAi-induced silencing of an essential gene in the nematode Caenorhabditis elegans. The dsRNA-expressing L paracasei can be used in experiments conducted on culture plates and may also be used as an orally administrable dsRNA carrier for humans and other mammals. (C) 2009, The Society for Biotechnology, Japan. All rights reserved.

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  • Cellular siRNA Delivery Using TatU1A and Photo-Induced RNA Interference Invited Reviewed

    Tamaki Endoh, Takashi Ohtsuki

    RNA Interference   271 - 281   2010

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  • Detection of Bioactive Small Molecules by Fluorescent Resonance Energy Transfer (FRET) in RNA-Protein Conjugates Reviewed

    Tamaki Endoh, Ryo Shintani, Masayasu Mie, Eiry Kobatake, Takashi Ohtsuki, Masahiko Sisido

    Bioconjugate Chemistry   20 ( 12 )   2242 - 2246   2009.12

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    Bioactive small molecules such as metabolites and drugs play important roles in regulating biological functions. A technique for Visualizing such small molecules is very useful to understand their molecular mechanisms. In this study, an RNA-protein Conjugate, which consists of in RRE-RNA sensor protein (EYFP-Rev-ECFP) and an altered RRE-RNA, was constructed to detect bioactive small molecules by fluorescent resonance energy transfer (FRET). We designed a theophylline-aptamer-inserted RRE-RNA (Theo-RRE) to detect theophylline as a model target molecule. Theo-RRE formed an RNA-protein conjugate with EYFP-Rev-ECFP in the presence of theophylline. As a result, theophylline was specifically detected down to 10 mu M by the FRET increase in distinction from theophylline analogue, caffeine, in cell lysates.

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  • Spatial regulation of specific gene expression through photoactivation of RNAi Reviewed

    Tamaki Endoh, Masahiko Sisido, Takashi Ohtsuki

    Journal of Controlled Release   137 ( 3-4 )   241 - 245   2009.8

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    In this study we describe the spatial regulation of RNA interference (RNAi) using an RNA-carrier protein labeled with a fluorescent dye and a light source to trigger the RNAi. We demonstrate photo-dependent gene silencing using several dyes with different excitation wavelengths. Additionally, we use light from a halogen lamp and a photomask to produce photopatterned RNAi, and laser light to trigger single-cell RNAi on cell culture plates. (C) 2009 Elsevier B.V. All rights reserved.

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  • Cellular siRNA delivery using cell-penetrating peptides modified for endosomal escape Invited Reviewed

    Tamaki Endoh, Takashi Ohtsuki

    Advanced Drug Delivery Reviews   61 ( 9 )   704 - 709   2009.7

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    RNAi-mediated silencing of specific genes is a promising strategy for gene therapy. To utilize RNAi for therapy, an efficient and safe method for delivery of RNA into the cell cytosol is necessary. The plasma membrane is the primary, and most difficult, barrier for RNA to cross, because negatively charged RNA is strongly repulsed by the negatively charged membrane. A variety of cationic polymers can be used as RNA carriers by interacting with RNA and covering its negative charges to form a cell-penetrating complex Among the emerging candidates for RNA carriers are cationic cell-penetrating peptides (CPPs), which can cross the plasma membrane and internalize into cells together with RNA. This review focuses on CPP-based RNA delivery strategies. In using CPP-based RNA delivery, most of the RNA internalized by the cell is entrapped in endosomes. Strategies for endosomal escape of RNAs are also reviewed. (C) 2009 Elsevier B.V. All rights reserved.

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  • Carrier PNA for shRNA delivery into cells Reviewed

    Mizuki Kitamatsu, Takanori Kubo, Rino Matsuzaki, Tamaki Endoh, Takashi Ohtsuki, Masahiko Sisido

    Bioorganic & Medicinal Chemistry Letters   19 ( 13 )   3410 - 3413   2009.7

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    A peptide nucleic acid (PNA)-cell-penetrating peptide (CPP) conjugate (carrier PNA) was used as &apos;bridgebuilder&apos; to connect a CPP with an shRNA. The carrier PNA successfully formed a hybrid with an shRNA bearing complementary dangling bases and the shRNA was introduced into cells by the carrier PNA, and RNAi was induced by the shRNA. Crown Copyright (C) 2009 Published by Elsevier Ltd. All rights reserved.

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  • A protease inhibitor discovery method using fluorescence correlation spectroscopy with position-specific labeled protein substrates Reviewed

    Hidetaka Nakata, Takashi Ohtsuki, Masahiko Sisido

    Analytical Biochemistry   390 ( 2 )   121 - 125   2009.7

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    We developed novel Substrates for protease activity evaluation by fluorescence correlation spectroscopy (FCS). Substrates were labeled in a position-specific manner with a fluorophore near the N terminus and included a C-terminal, 30 kDa, highly soluble protein (elongation factor Ts [EF-Ts]). The C-terminal protein enhanced the substrate peptide Solubility and increased the molecular weight, enabling sensitive detection by FCS. Using the labeled substrates, caspase-3 and matrix metalloproteinase-9 (MMP-9) activities were confirmed by FCS. To demonstrate the Suitability of this FCS-based assay for high-throughput screening, we screened various chemical compounds for MMP-9 inhibitors. The screening results confirmed the inhibitory activity of one compound and also revealed another potential MMP-9 inhibitor. Thus, this combination of position-specific labeled protein Substrates and FCS may serve as a useful tool for evaluating activities of various proteases and for protease inhibitor screening. (C) 2009 Elsevier Inc. All rights reserved.

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  • PNA Arrays for miRNA Detection Reviewed

    Tamaki Endoh, Mizuki Kitamatsu, Masahiko Sisido, Takashi Ohtsuki

    Chemistry Letters   38 ( 5 )   438 - 439   2009.5

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    The profiling of microRNAs (miRNAs) using microarray technology has been used in many recent biological and medical studies. In this study, arrays of peptide nucleic acids (PNAs) were prepared and their miRNA capture abilities were evaluated. We found that the sensitivity of 10-mer PNA probes in targeting miRNAs was much higher than that of the corresponding DNA probes and also of longer (20-mer) DNA probes.

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  • The Central Dogma: From DNA to RNA, and to Protein Reviewed

    Takashi Ohtsuki, Masahiko Sisido

    Automation in Proteomics and Genomics   1 - 19   2009.3

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  • Chemical Synthesis and Properties of 5-Taurinomethyluridine and 5-Taurinomethyl-2-thiouridine

    Toshihiko Ogata, Tomomi Shimazaki, Tadashi Umemoto, Shinya Kurata, Takashi Ohtsuki, Tsutomu Suzuki, Takeshi Wada

    Journal of Organic Chemistry   74 ( 6 )   2585 - 2588   2009.3

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    Unique taurine-containing Uridine derivatives, 5-taurinomethyluridine (tau m(5)U) and 5-taurinomethyl-2-thiouridine (tau m(5)s(2)U), which were discovered in mammalian mitochondrial tRNAs, exist at the first position of the anticodon. In this paper, we report the first efficient synthesis of tau m(5)U and tau m(5)s(2)U and describe their physicochemical properties. These modified ribonucleosides were synthesized by the reaction of 5-substituted uridine derivatives with a tetrabutylammonium salt of taurine that is highly reactive and well-soluble in common organic solvents. UV and (1)H NMR spectrometric studies revealed the structural properties of the taurine-containing base moieties and the Sugar conformations of these modified ribonucleosides.

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  • Isolation of small RNAs using biotinylated PNAs

    Takashi Ohtsuki, Takeshi Fujimoto, Maya Kamimukai, Chisato Kumano, Mizuki Kitamatsu, Masahiko Sisido

    Journal of Biochemistry   144 ( 4 )   415 - 418   2008.10

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    In this study, an RNA isolation method was developed using a biotinylated peptide nucleic acid (PNA) that is complementary to the target RNA. Using the biotinylated PNA method, we successfully isolated several RNAs from Escherichia coli and from human total RNA in pure form. Damage to the RNA appears to be negligible by this method because the method is rapid and does not require a high temperature treatment to facilitate RNA-PNA binding.

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  • Amino acid sensing using aminoacyl-tRNA synthetase

    Akimitsu Kugimiya, Miki Morii, Takashi Ohtsuki

    Analytical Biochemistry   378 ( 1 )   90 - 92   2008.7

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    The detection of amino acids using aminoacyl-tRNA synthetases (ARSs) as the molecular recognition element was proposed, and the binding activity and specificity of ARSs were evaluated. Using this rapid and easy method, from 15 to 50 mu M tyrosine could be measured specifically. The method suggested in this article could be realized without an amino acid labeling process or a large volume of organic solvents, and the time for measurement was reasonable. (c) 2008 Elsevier Inc. All rights reserved.

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  • Modified uridines with c5-methylene substituents at the first position of the tRNA anticodon stabilize U center dot G wobble pairing during decoding

    Shinya Kurata, Albert Weixlbaumer, Takashi Ohtsuki, Tomomi Shimazaki, Takeshi Wada, Yohei Kirino, Kazuyuki Takai, Kimitsuna Watanabe, V. Ramakrishnan, Tsutomu Suzuki

    J. Biol. Chem.   283 ( 27 )   18801 - 18811   2008.7

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    Post-transcriptional modifications at the first ( wobble) position of the tRNA anticodon participate in precise decoding of the genetic code. To decode codons that end in a purine (R) (i.e. NNR), tRNAs frequently utilize 5-methyluridine derivatives (xm(5)U) at the wobble position. However, the functional properties of the C5-substituents of xm(5)U in codon recognition remain elusive. We previously found that mitochondrial tRNAs(Leu(UUR)) with pathogenic point mutations isolated from MELAS ( mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes) patients lacked the 5-taurinomethyluridine (tau m(5)U) modification and caused a decoding defect. Here, we constructed Escherichia coli tRNAs(Leu(UUR)) with or without xm(5)U modifications at the wobble position and measured their decoding activities in an in vitro translation as well as by A-site tRNA binding. In addition, the decoding properties of tRNA(Arg) lacking mnm(5)U modification in a knock-out strain of the modifying enzyme (Delta mnmE) were examined by pulse labeling using reporter constructs with consecutive AGR codons. Our results demonstrate that the xm(5)U modification plays a critical role in decoding NNG codons by stabilizing U center dot G pairing at the wobble position. Crystal structures of an anticodon stem-loop containing tau m(5)U interacting with a UUA or UUG codon at the ribosomal A-site revealed that the m(5)U center dot G base pair does not have classical U center dot G wobble geometry. These structures provide help to explain how the tau m(5)U modification enables efficient decoding of UUG codons.

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  • Cellular siRNA delivery mediated by a cell-permeant RNA-Binding protein and photoinduced RNA interference

    Tamaki Endoh, Masahiko Sisido, Takashi Ohtsuki

    Bioconjugate Chemistry   19 ( 5 )   1017 - 1024   2008.5

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    HIV-1 TAT peptide, which is a cell-penetrating peptide (CPP), was fused to the UIA RNA-binding domain (TatU1A) to generate a sequence-specific siRNA delivery system for mammalian cells. The siRNA contained a short 5'-extension that is specifically recognized by the U1A RNA-binding domain (U1AsiRNA). Specific binding of TatU1A to the U1AsiRNA was confirmed using a gel mobility shift assay. The U1AsiRNA was internalized by cells only when it was preincubated with TatU1A before addition to the cells. Although most of the internalized siRNA seemed. to be entrapped in endocytic compartments, efficient redistribution of the entrapped siRNAs was achieved by photostimulation of a fluorophore attached to TatU1A. Once in the cytoplasm, the siRNA induced RNAi-mediated gene silencing. We refer to this delivery strategy as CLIP-RNAi. CLIP-RNAi is a promising strategy for RNAi experiments and for pinpoint RNAi therapy.

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  • Elongation factor Tu mutants expand amino acid tolerance of protein biosynthesis system

    Yoshio Doi, Takashi Ohtsuki, Yoshihiro Shimizu, Takuya Ueda, Masahiko Sisido

    J. Am. Chem. Soc.   129 ( 46 )   14458 - 14462   2007.11

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    Nonnatural amino acids have been introduced into proteins using expanded protein biosynthesis systems. However, some nonnatural amino acids, especially those containing large aromatic groups, are not efficiently incorporated into proteins. Reduced binding efficiency of aminoacylated tRNAs to elongation factor Tu (EF-Tu) is likely to limit incorporation of large amino acids. Our previous studies suggested that tRNAs carrying large nonnatural amino acids are bound less tightly to EF-Tu than natural amino acids. To expand the availability of nonnatural mutagenesis, EF-Tu from the E coli translation system was improved to accept such large amino acids. We synthesized EF-Tu mutants, in which the binding pocket of the aminoacyl moiety of aminoacyl-tRNA was enlarged. L-1-Pyrenylalanine, L-2-pyrenylalanine, and DL-2anthraquinonylalanine, which are hardly or only slightly incorporated with the wild-type EF-Tu, were successfully incorporated into a protein using these EF-Tu mutants.

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  • T-armless tRNAs and elongated elongation factor Tu

    Takashi Ohtsuki, Yoh-ichi Watanabe

    IUBMB LIFE   59 ( 2 )   68 - 75   2007.2

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    Most tRNAs share a common secondary structure containing a T arm, a D arm, an anticodon arm and an acceptor stem. However, there are some exceptions. Most nematode mitochondrial tRNAs and some animal mitochondrial tRNAs lack the T arm, which is necessary for binding to canonical elongation factor Tu (EF-Tu). The mitochondria of the nematode Caenorhabditis elegans have a unique EF-Tu, named EF-Tu1, whose structure has supplied clues as to how truncated tRNAs can work in translation. EF-Tu1 has a C-terminal extension of about 60 aa that is absent in canonical EF-Tu. Recent data from our laboratory strongly suggests that EF-Tu1 recognizes the D-arm instead of the T arm by a mechanism involving this C-terminal region. Further biochemical analysis of mitochondrial tRNAs and EF-Tu from the distantly related nematode Trichinella spp. and sequence information on nuclear and mitochondrial DNA in arthropods suggest that T-armless tRNAs may have arisen as a result of duplication of the EF-Tu gene. These studies provide valuable insights into the co-evolution of RNA and RNA-binding proteins.

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  • T-armless tRNAs and elongated elongation factor Tu.

    Ohtsuki, T, Watanabe, Y

    IUBMB life   59 ( 2 )   68 - 75   2007

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  • Design of carrier tRNAs and selection of four-base codons for efficient incorporation of various nonnatural amino acids into proteins in Spodoptera frugiperda 21 (Sf21) insect cell-free translation system

    Masumi Taki, Yasunori Tokuda, Takashi Ohtsuki, Masahiko Sisido

    J. Biosci. Bioeng.   102 ( 6 )   511 - 517   2006.12

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    Spodoptera frugiperda 21 (Sf21) insect cell-free protein synthesizing system was expanded to include nonnatural amino acids. Orthogonal tRNAs that work as carriers of nonnatural amino acids in the insect system were explored. Four-base codons for assigning the positions of nonnatural amino acids were also selected. Mutated streptavidin mRNAs that contained different four-base codons were prepared and added to the insect cell-free system in the presence of various tRNAs possessing the corresponding four-base anticodons. The tRNAs were chemically amino-acylated with various types of nonnatural amino acids to examine their incorporation efficiencies. Using p-nitrophenylalanine as the nonnatural amino acid and streptavidin as the target protein, tRNA sequences and the types of four-base codons were optimized to maximize the yield of the nonnatural mutant and to minimize production of full-length proteins that do not contain the nonnatural amino acid. Among the tRNA sequences taken from a variety of tRNAs of nonstandard structures, the tRNA derived from Methanosarcina acetivorans tRNA(Pyl) was the most efficient and orthogonal tRNA. Of the CGGN-type four-base codons, CGGA and CGGG were the most efficient ones for assigning the positions of nonnatural amino acids. p-Nitrophenylalanine and 2-naphthylalanine were efficiently incorporated as in the case of Escherichia coli and rabbit reticulocyte cell-free systems. Much less efficient incorporation was observed, however, for other nonnatural amino acids, indicating that the insect system is less tolerant to the structural diversity of amino acids than the E. coli cell-free system.

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  • An evolutionary 'intermediate state' of mitochondrial translation systems found in Trichinella species of parasitic nematodes: co-evolution of tRNA and EF-Tu

    Masashi Arita, Takuma Suematsu, Arihiro Osanai, Takashi Inaba, Haruo Kamiya, Kiyoshi Kita, Masahiko Sisido, Yoh-ichi Watanabe, Takashi Ohtsuki

    NUCLEIC ACIDS RESEARCH   34 ( 18 )   5291 - 5299   2006.10

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    EF-Tu delivers aminoacyl-tRNAs to ribosomes in the translation system. However, unusual truncations found in some animal mitochondrial tRNAs seem to prevent recognition by a canonical EF-Tu. We showed previously that the chromadorean nematode has two distinct EF-Tus, one of which (EF-Tu1) binds only to T-armless aminoacyl-tRNAs and the other (EF-Tu2) binds to D-armless Ser-tRNAs. Neither of the EF-Tus can bind to canonical cloverleaf tRNAs. In this study, by analyzing the translation system of enoplean nematode Trichinella species, we address how EF-Tus and tRNAs have evolved from the canonical structures toward those of the chromadorean translation system. Trichinella mitochondria possess three types of tRNAs: cloverleaf tRNAs, which do not exist in chromadorean nematode mitochondria; T-armless tRNAs; and D-armless tRNAs. We found two mitochondrial EF-Tu species, EF-Tu1 and EF-Tu2, in Trichinella britovi. T.britovi EF-Tu2 could bind to only D-armless Ser-tRNA, as Caenorhabditis elegans EF-Tu2 does. In contrast to the case of C.elegans EF-Tu1, however, T.britovi EF-Tu1 bound to all three types of tRNA present in Trichinella mitochondria. These results suggest that Trichinella mitochondrial translation system, and particularly the tRNA-binding specificity of EF-Tu1, could be an intermediate state between the canonical system and the chromadorean nematode mitochondrial system.

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  • A protein extension to shorten RNA: elongated elongation factor-Tu recognizes the D-arm of T-armless tRNAs in nematode mitochondria Reviewed

    Masayuki Sakurai, Yoh-ichi Watanabe, Kinnitsuna Watanabe, Takashi Ohtsuki

    BIOCHEMICAL JOURNAL   399 ( 2 )   249 - 256   2006.10

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    Nematode mitochondria possess extremely truncated tRNAs. Of 22 tRNAs, 20 lack the entire T-arm. The T-arm is necessary for the binding of canonical tRNAs and EF (elongation factor)-Tu (thermo-unstable). The nematode mitochondrial translation system employs two different EF-Tu factors named EF-Tu1 and EF-Tu2. Our previous study showed that nematode Caenorhabditis elegans EF-Tu1 binds specifically to T-armless tRNA. C. elegans EF-Tu1 has a 57-amino acid C-terminal extension that is absent from canonical EF-Tu, and the T-arm-binding residues of canonical EF-Tu are not conserved. In this study, the recognition mechanism of T-armless tRNA by EF-Tu1 was investigated. Both modification interference assays and primer extension analysis of cross-linked ternary complexes revealed that EF-Tu1 interacts not only with the tRNA acceptor stem but also with the D-arm. This is the first example of an EF-Tu recogpizing the D-arm of a tRNA. The binding activity of EF-Tu1 was impaired by deletion of only 14 residues from the C-terminus, indicating that the C-terminus of EF-Tu1 is required for its binding to T-armless tRNA. These results suggest that C. elegans EF-Tu1 recognizes the D-arm instead of the T-arm by a mechanism involving its C-terminal region. This study sheds light on the co-evolution of RNA and RNA-binding proteins in nematode mitochondria.

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  • Identification of the residues involved in the unique serine specificity of Caenorhabditis elegans mitochondrial EF-Tu2

    Aya Sato, Yoh-ichi Watanabe, Tsutomu Suzuki, Makoto Komiyama, Kimitsuna Watanabe, Takashi Ohtsuki

    Biochemistry   45 ( 36 )   10920 - 10927   2006.9

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    In canonical translation systems, the single elongation factor Tu (EF-Tu) recognizes all elongator tRNAs. However, in Caenorhabditis elegans mitochondria, two distinct EF-Tu species, EF-Tu1 and EF-Tu2, recognize 20 species of T armless tRNA and two species of D armless tRNA(Ser), respectively. We previously reported that C. elegans mitochondrial EF-Tu2 specifically recognizes the serine moiety of serylated-tRNA. In this study, to identify the critical residues for the serine specificity in EF-Tu2, several residues in the amino acid binding pocket of bacterial EF-Tu were systematically replaced with corresponding EF-Tu2 residues, and the mutants were analyzed for their specificity for esterified amino acids attached to tRNAs. In this way, we obtained a bacterial EF-Tu mutant that acquired serine specificity after the introduction of 10 EF-Tu2 residues into its amino acid binding pocket. C. elegans EF-Tu2 mutants lacking serine specificity were also created by replacing seven or eight residues with bacterial residues. Further stressing the importance of these residues, we found that they are almost conserved in EF-Tu2 sequences of closely related nematodes. Thus, these three approaches reveal the critical residues essential for the unique serine specificity of C. elegans mitochondrial EF-Tu2.

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  • Binding efficiency of elongation factor Tu to tRNAs charged with nonnatural fluorescent amino acids

    H Nakata, T Ohtsuki, R Abe, T Hohsaka, M Sisido

    Analytical Biochemistry   348 ( 2 )   321 - 323   2006.1

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  • A protein extension to shorten RNA: Elongated elongation factor Tu recognizes the D-arm of T-armless tRNAs in nematode mitochondria.

    Sakurai, M, Watanabe, Y, Watanabe, K, Ohtsuki, T

    Biochemical Journal   2006

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  • Expansion of protein biosynthesis system including nonnatural amino acids

    Takashi Ohtsuki, Yoshio Doi, Taishi Manabe, Masahiko Sisido

    2006 IEEE INTERNATIONAL SYMPOSIUM ON MICRO-NANOMECHATRONICS AND HUMAN SCIENCE   323 - +   2006

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    Nonnatural amino acids, such as fluorescent amino acids, can be introduced into proteins by using an expanded protein biosynthesis system. This approach requires orthogonal tRNAs that deliver nonnatural amino acids to a ribosome and expanded codon/anticodon pairs, like four-base codon/anticodon pairs. However, in this system, some of nonnatural amino acids are not efficiently incorporated into proteins. To expand the availability of the nonnatural mugatenesis, translation factors and tRNAs must be improved. In this study, we designed and synthesized an elongation factor Tu (EF-Tu) mutant that efficiently binds to tRNAs charged with nonnatural amino acids. We also devised new tRNAs that decode four-base codons and efficiently deliver nonnatural amino acids into ribosome.

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  • Multiple incorporation of non-natural amino acids into a single protein using tRNAs with non-standard structures

    T Ohtsuki, T Manabe, M Sisido

    FEBS Letters   579 ( 30 )   6769 - 6774   2005.12

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    The ability to introduce non-natural amino acids into proteins opens up new vistas for the study of protein structure and function. This approach requires suppressor tRNAs that deliver the non-natural amino acid to a ribosome associated with an mRNA containing an expanded codon. The suppressor tRNAs must be absolutely protected from aminoacylation by any of the aminoacyl-tRNA synthetases in the protein synthesizing system, or a natural amino acid will be incorporated instead of the non-natural amino acid. Here, we found that some tRNAs with non-standard structures could work as efficient four-base suppressors fulfilling the above orthogonal conditions. Using these tRNAs, we successfully demonstrated incorporation of three different non-natural amino acids into a single protein. (c) 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

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  • Four-base codon/anticodon strategy and non-enzymatic aminoacylation for protein engineering with non-natural amino acids

    M Sisido, K Ninomiya, T Ohtsuki, T Hohsaka

    Methods   36 ( 3 )   270 - 278   2005.7

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    Techniques for position-specific incorporation of non-natural amino acids in an in vitro protein synthesizing system are described. First, a PNA-assisted non-enzymatic tRNA aminoacylation with a variety of natural and non-natural amino acids is described. With this technique, one can aminoacylate a specific tRNA simply by adding a preformed amino acid activated ester-PNA conjugate into an in vitro protein biosynthesizing system. Second, the genetic code is expanded by introducing 4-base codons that can be exclusively translated to non-natural amino acids. The most advantageous point of the 4-base codon strategy is to introduce multiple amino acids into specific positions in single proteins by using mutually orthogonal 4-base codons and orthogonal tRNAs. An easy and quick method for preparation of tRNAs possessing 4-base anticodons is also described. Combination of the non-enzymatic aminoacylation and the 4-base codon/anticodon strategy gives an easy and widely applicable technique for incorporating a variety of non-natural amino acids into proteins in vitro. (c) 2005 Elsevier Inc. All rights reserved.

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  • Isolation and physiochemical properties of protein-rich nematode mitochondrial ribosomes

    F Zhao, T Ohtsuki, K Yamada, S Yoshinari, K Kita, Y Watanabe, K Watanabe

    Biochemistry   44 ( 25 )   9232 - 9237   2005.6

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    In the present study, mitochondrial ribosomes of the nematode Ascaris suum were isolated and their physiochernical properties were compared to ribosomes of Escherichia coli. The sedimentation coefficient and buoyant density of A. suum mitochondrial ribosomes were determined. The sedimentation coefficient of the intact monosome was about 55 S. The buoyant density of formaldehyde-fixed ribosomes in cesium chloride was 1.40 g/cm(3), which suggests that the nematode mitoribosomes have a much higher protein composition than other mitoribosomes. The diffusion coefficients obtained from dynamic light scattering measurements were (1.48 +/- 0.04) x 10(-7) cm(2) s(-1) for 55 S mitoribosomes and (1.74 +/- 0.04) X 10(-7) cm(2) S-1 for the 70 S E. coli monosome. The diameter of mitoribosomes was measured by dynamic light-scattering analysis and electron microscopy. Though the nematode mitoribosome has a larger size than the bacterial ribosome, it does not differ significantly in size from mammalian mitoribosomes.

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  • Unusual usage of wobble modifications in mitochondrial tRNAs of the nematode Ascaris suum

    M Sakurai, T Ohtsuki, T Suzuki, K Watanabe

    FEBS Letters   579 ( 13 )   2767 - 2772   2005.5

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    To understand the decoding property of nematode mitochondrial tRNAs with unusual secondary structures, post-transcriptional modifications at wobble positions of Ascaris suum mitochondrial tRNAs corresponding to two-codon families ending with a purine were analyzed. 5-Carboxymethylaminomethyluridine (cmnm(5)U) was identified at the wobble positions of tRNA(Lys), tRNA(Glu) and tRNA(Gln), while 5-carboxymethylaminomethyl-2-thiouridine (cmnm(5)s(2)U) was present in tRNA(UAA)(Leu) and tRNA(Trp). In most bacterial and mitochondrial tRNAs, the 2-thiouridine derivative is present in tRNAs for Lys, Gin and Gln. These is no report that cmnm(5)s(2)U is used in tRNALeu and tRNA(Trp). The unusual usage of wobble modifications might assist decoding of nematode mitochondrial mRNAs. (c) 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

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  • A unique tRNA recognition mechanism of Caenorhabditis elegans mitochondrial EF-Tu2

    T Suematsu, A Sato, M Sakurai, K Watanabe, T Ohtsuki

    Nucleic Acids Research   33 ( 15 )   4683 - 4691   2005

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    Nematode mitochondria expresses two types of extremely truncated tRNAs that are specifically recognized by two distinct elongation factor Tu (EF-Tu) species named EF-Tu1 and EF-Tu2. This is unlike the canonical EF-Tu molecule that participates in the standard protein biosynthesis systems, which basically recognizes all elongator tRNAs. EF-Tu2 specifically recognizes Ser-tRNA(Ser) that lacks a D arm but has a short T arm. Our previous study led us to speculate the lack of the D arm may be essential for the tRNA recognition of EF-Tu2. However, here, we showed that the EF-Tu2 can bind to D arm-bearing Ser-tRNAs, in which the D-T arm interaction was weakened by the mutations. The ethylnitrosourea-modification interference assay showed that EF-Tu2 is unique, in that it interacts with the phosphate groups on the T stem on the side that is opposite to where canonical EF-Tu binds. The hydrolysis protection assay using several EF-Tu2 mutants then strongly suggests that seven C-terminal amino acid residues of EF-Tu2 are essential for its aminoacyl-tRNA-binding activity. Our results indicate that the formation of the nematode mitochondrial (mt) EF-Tu2/GTP/aminoacyl-tRNA ternary complex is probably supported by a unique interaction between the C-terminal extension of EF-Tu2 and the tRNA.

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  • Modification at position 9 with 1-methyladenosine is crucial for structure and function of nematode mitochondrial tRNAs lacking the entire T-arm

    M Sakurai, T Ohtsuki, K Watanabe

    Nucleic Acids Research   33 ( 5 )   1653 - 1661   2005

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    The mitochondria of the nematode Ascaris suum have tRNAs with unusual secondary structures that lack either the T-arm or D-arm found in most other organisms. Of the twenty-two tRNA species present in the mitochondria of A. suum, twenty lack the entire T-arm and two serine tRNAs lack the D-arm. To understand how such unusual tRNAs work in the nematode mitochondrial translation system, we analyzed post-transcriptional modifications of 11 mitochondrial tRNA species purified from A. suum, 10 of which lacked a T-arm and one of which lacked a D-arm. The most characteristic feature of nematode mitochondrial tRNAs lacking a T-arm was the presence of 1-methyladenosine at position 9 (m(1)A(9)). Synthesis of T-armless tRNAs with or without the modified nucleoside showed that T-armless tRNAs without the modification had much lower aminoacylation and EF-Tu-binding activities than native tRNAs. The addition of a single methyl group to A9 of these tRNAs was sufficient to restore nearly native levels of amino-acylation and EF-Tu-binding activity as well as tertiary structure, suggesting that m(1)A(9w) is a key residue for the activity of T-armless tRNAs. Thus, m(1)A(9) is indispensable for the structure and function of T-armless tRNAs of nematode mitochondrial origin.

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  • Glimpses of transitions from the RNA world to the RNP world in protein compensation for RNA deficit in animal mitochondrial translation systems.

    Suzuki, T, Ohtsuki, T, Watanabe, K

    Endocytobiosis Cell Research   2004

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  • Identification and characterization of an intermediate in the alkali degradation of (6-4) photoproduct-containing DNA

    M Higurashi, T Ohtsuki, A Inase, R Kusumoto, C Masutani, F Hanaoka, S Iwai

    J. Biol. Chem.   278 ( 51 )   51968 - 51973   2003.12

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    The (6-4) photoproduct formed by ultraviolet light is known as an alkali-labile DNA lesion. Strand breaks occur at (6-4) photoproducts when UV-irradiated DNA is treated with hot alkali. We have analyzed the degradation reaction of this photoproduct under alkaline conditions using synthetic oligonucleotides. A tetramer, d(GT(6-4) TC), was prepared, and its degradation in 50 mM KOH at 60 degreesC was monitored by high performance liquid chromatography. A single peak with a UV absorption spectrum similar to that of the starting material was detected after the reaction, and this compound was regarded as an intermediate before the strand break. The formation of this intermediate was compared with intermediates from the degradation of other alkali-labile lesions such as the abasic site, thymine glycol, and 5,6-dihydrothymine. The results strongly suggested that the first step of the alkali degradation of the (6- 4) photoproduct was the hydrolysis between the N3 and C4 positions of the 5'-pyrimidine component. Analyses by NMR spectroscopy and mass spectrometry supported the chemical structure of this product. Assays of the complex formation with XPC . HR23B and the translesion synthesis by DNA polymerase eta revealed that the biochemical properties are indistinguishable between the intact and hydrolyzed photoproducts.

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  • Quick two-step RNA ligation employing periodate oxidation

    S Kurata, T Ohtsuki, T Suzuki, K Watanabe

    Nucleic Acids Research   31 ( 22 )   2003.11

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    The introduction of modified or labeled nucleotides into RNA is a powerful RNA engineering tool as it enables us to investigate how native RNA modifications affect RNA function and structure. It also helps in the structural analysis of RNA. A modified nucleotide can be introduced into a specific position of RNA by the method of two-step enzymatic ligation of RNA fragments. However, this method requires a complicated purification step between the two ligation steps that results in low yields of the ligation product. Here we have developed a new ligation technique employing periodate oxide that eliminates this purification step. This increases the total yield of the ligation product and makes it a faster procedure.

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  • A unique serine-specific elongation factor Tu found in nematode mitochondria (vol 9, pg 669, 2002)

    T Ohtsuki, A Sato, Y Watanabe, K Watanabe

    NATURE STRUCTURAL BIOLOGY   10 ( 8 )   669 - 669   2003.8

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    DOI: 10.1038/nsb0803-669a

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  • Towards artificial repair of UV-damaged DNA: studies on drug binding and alkali hydrolysis. Reviewed

    S. Iwai, A. Inase, S. Jafar, M. Higurashi, T. Ohtsuki, Y. Xu, H. Sugiyama

    Nucleic acids research. Supplement (2001)   ( 3 )   181 - 182   2003

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  • Recent topics in mitochondrial translation systems Reviewed

    Watanabe, K., Ohtsuki, T., Suzuki, T.

    Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme   48 ( 4 Suppl )   365 - 374   2003

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  • Decoding property of C5 uridine modification at the wobble position of tRNA anticodon. Reviewed

    Kurata, S., Ohtsuki, T., Wada, T., Kirino, Y., Takai, K., Saigo, K., Watanabe, K., Suzuki, T.

    Nucleic acids research. Supplement (2001)   ( 3 )   245 - 246   2003

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  • Characterization of the interaction between the nucleotide exchange factor EF-Ts from nematode mitochondria and elongation factor Tu

    T Ohtsuki, M Sakurai, A Sato, K Watanabe

    Nucleic Acids Research   30 ( 24 )   5444 - 5451   2002.12

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    Caenorhabditis elegans mitochondria have two elongation factor (EF)-Tu species, denoted EF-Tu1 and EF-Tu2. Recombinant nematode EF-Ts purified from Escherichia coli bound both of these molecules and also stimulated the translational activity of EF-Tu, indicating that the nematode EF-Ts homolog is a functional EF-Ts protein of mitochondria. Complexes formed by the interaction of nematode EF-Ts with EF-Tu1 and EF-Tu2 could be detected by native gel electrophoresis and purified by gel filtration. Although the nematode mitochondrial (mt) EF-Tu molecules are extremely unstable and easily form aggregates, native gel electrophoresis and gel filtration analysis revealed that EF-Tu.EF-Ts complexes are significantly more soluble. This indicates that nematode EF-Ts can be used to stabilize homologous EF-Tu molecules for experimental purposes. The EF-Ts bound to two eubacterial EF-Tu species (E.coli and Thermus thermophilus). Although the EF-Ts did not bind to bovine mt EF-Tu, it could bind to a chimeric nematode-bovine EF-Tu molecule containing domains 1 and 2 from bovine mt EF-Tu. Thus, the nematode EF-Ts appears to have a broad specificity for EF-Tu molecules from different species.

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  • A unique serine-specific elongation factor Tu found in nematode mitochondria

    T Ohtsuki, A Sato, Y Watanabe, K Watanabe

    Nature Structural Biology   9 ( 9 )   669 - 673   2002.9

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    The translation elongation factor Tu (EF-Tu) delivers aminoacyl-tRNAs to ribosomes by recognizing the tRNA acceptor and T stems. However, the unusual truncation observed in some animal mitochondrial tRNAs seems to prevent recognition by a canonical EF-Tu. For instance, nematode mitochondria contain tRNAs lacking a T or D arm. We recently found an atypical EF-Tu (EF-Tu1) specific for nematode mitochondrial tRNAs that lack the T arm. We have now discovered a second factor, EF-Tu2, which binds only to tRNAs that lack a D arm. EF-Tu2 seems unique in its amino acid specificity because it recognizes the aminoacyl moiety of seryl-tRNAs and the tRNA structure itself. Such EF-Tu evolution might explain tRNA structural divergence in animal mitochondria.

    DOI: 10.1038/nsb826

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  • Solution structure of an RNA fragment with the P7/P9.0 region and the 3 '-terminal guanosine of the Tetrahymena group I intron

    A Kitamura, Y Muto, S Watanabe, Kim, I, T Ito, Y Nishiya, K Sakamoto, T Ohtsuki, G Kawai, K Watanabe, K Hosono, H Takaku, E Katoh, T Yamazaki, T Inoue, S Yokoyama

    RNA   8 ( 4 )   440 - 451   2002.4

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    In the second step of the two consecutive transesterifications of the self-splicing reaction of the group I intron, the conserved guanosine at the 31 terminus of the intron (omegaG) binds to the guanosine-binding site (GBS) in the intron. In the present study, we designed a 22-nt model RNA (GBS/omegaG) including the GBS and omegaG from the Tetrahymena group I intron, and determined the solution structure by NMR methods. In this structure, omegaG is recognized by the formation of a base triple with the G2649.C311 base pair, and this recognition is stabilized by the stacking interaction between omegaG and C262. The bulged structure at A263 causes a large helical twist angle (40 +/- 8degrees) between the G264.C311 and C262.G312 base pairs. We named this type of binding pocket with a bulge and a large twist, formed on the major groove, a "Bulge-and-Twist" (BT) pocket. With another twist angle between the C262.G312 and G413.C313 base pairs (45 +/- 10degrees), the axis of GBS/omegaG is kinked at the GBS region. This kinked axis superimposes well on that of the corresponding region in the structure model built on a 5.0 Angstrom resolution electron density map (Golden et al., Science, 1998, 282:345-358). This compact structure of the GBS is also consistent with previous biochemical studies on group I introns. The BT pockets are also found in the arginine-binding site of the HIV-TAR RNA, and within the 16S rRNA and the 23S rRNA.

    DOI: 10.1017/S1355838202026043

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  • The minimal tRNA: unique structure of Ascaris suum mitochondrial tRNA(UCU)(Ser) having a short T arm and lacking the entire D arm

    T Ohtsuki, G Kawai, K Watanabe

    FEBS Letters   514 ( 1 )   37 - 43   2002.3

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    The tertiary structure of Ascaris suum mitochondrial tRNA(VCU)(Ser) was examined by nuclear magnetic resonance analysis using its transcript, since tRNA(UCU)(Ser), lacking the D arm and possessing a truncated T arm, is the shortest of all the known tRNAs. Most basepairs in the proposed secondary structure of tRNA(UCU)(Ser) were shown to exist, but the connector region comprising the truncated D loop and the extra loop was flexible. This flexibility, would enable adjustment of the mutual distance between the 3'-terminus and the anticodon consistent with that of usual tRNAs. Thus, tRNA(UCU)(Ser) appears to function in a similar way to that of usual tRNAs in the ribosome. (C) 2002 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.

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  • An unnatural base pair for incorporating amino acid analogs into proteins

    Hirao, I, T Ohtsuki, T Fujiwara, T Mitsui, T Yokogawa, T Okuni, H Nakayama, K Takio, T Yabuki, T Kigawa, K Kodama, T Yokogawa, K Nishikawa, S Yokoyama

    Nature Biotechnology   20 ( 2 )   177 - 182   2002.2

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    An unnatural base pair of 2-amino-6-(2-thienyl) purine (denoted by s) and pyridin-2-one (denoted by y) was developed to expand the genetic code. The ribonucleoside triphosphate of y was site-specifically incorporated into RNA, opposite s in a template, by T7 RNA polymerase. This transcription was coupled with translation in an Escherichia coli cell-free system. The yAG codon in the transcribed ras mRNA was recognized by the CUs anticodon of a yeast tyrosine transfer RNA (tRNA) variant, which had been enzymatically aminoacylated with an unnatural amino acid, 3-chlorotyrosine. Site-specific incorporation of 3-chlorotyrosine into the Ras protein was demonstrated by liquid chromatography-mass spectrometry (LC-MS) analysis of the products. This coupled transcription-translation system will permit the efficient synthesis of proteins with a tyrosine analog at the desired position.

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  • Requirement of modified residue m1A9 for EF-Tu binding to nematode mitochondrial tRNA lacking the T arm. Reviewed

    Masayuki Sakurai, Takashi Ohtsuki, Yo-ichi Watanabe, Kimitsuna Watanabe

    NUCLEIC ACIDS RESEARCH   ( 1 )   237 - 238   2001.10

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    DOI: 10.1093/nass/1.1.237

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  • An "elongated" translation elongation factor Tu for truncated tRNAs in nematode mitochondria

    T Ohtsuki, Y Watanabe, C Takemoto, G Kawai, T Ueda, K Kita, S Kojima, Y Kaziro, J Nyborg, K Watanabe

    J. Biol. Chem.   276 ( 24 )   21571 - 21577   2001.6

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    We have found the gene for a translation elongation factor Tu (EF-Tu) homologue in the genome of the nematode Caenorhabditis elegans, Because the corresponding protein was detected immunologically in a nematode mitochondrial (mt) extract, it could be regarded as a nematode mt EF-Tu. The protein possesses an extension of about 57 amino acids (we call this domain 3') at the C terminus, which is not found in any other known EF-Tu, Because most nematode mt tRNAs lack a T stem, domain 3' may be related to this feature. The nematode EF-Tu bound to nematode T stem-lacking tRNA, but bacterial EF-Tu was unable to do so. A series of domain exchange experiments strongly suggested that domains 3 and 3' are essential for binding to T stem-lacking tRNAs, This finding may constitute a novel example of the co-evolution of a structurally simplified RNA and the cognate RNA-binding protein, the latter having apparently acquired an additional domain to compensate for the lack of a binding site(s) on the RNA.

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  • Unnatural base pairs for specific transcription

    T Ohtsuki, M Kimoto, M Ishikawa, T Mitsui, Hirao, I, S Yokoyama

    Proc. Natl. Acad. Sci. U.S.A.   98 ( 9 )   4922 - 4925   2001.4

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    An unnatural base pair of 2-amino-6-(N,N-dimethylamino)purine (designated as x) and pyridin-2-one (designated as y) has been developed for specific transcription. The ribonucleoside triphosphates of y and a modified y, 5-methylpyridin-2-one, are selectively incorporated into RNA opposite x in the templates by T7 RNA polymerase, In addition, the sequences of the DNA templates containing x can be confirmed by a dideoxynucleotide chain-terminator method supplemented with the deoxynucleoside triphosphate of y, The bulky dimethylamino group of x in the templates effectively eliminates noncognate pairing with the natural bases. These results enable RNA biosynthesis for the specific incorporation of unnatural nucleotides at the desired positions.

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  • Dual specificity of the pyrimidine analogue, 4-methylpyridin-2-one, in DNA replication

    Hirao, I, T Ohtsuki, T Mitsui, S Yokoyama

    J. Am. Chem. Soc.   122 ( 25 )   6118 - 6119   2000.6

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  • The guanosine binding mechanism of the Tetrahymena group I intron

    A. Kitamura, Y. Muto, S. Watanabe, I. Kim, T. Ito, Y. Nishiya, T. Ohtsuki, G. Kawai, K. Watanabe, K. Hosono, H. Takaku, E. Katoh, T. Yamazaki, T. Inoue, S. Yokoyama

    Nucleic Acids Symposium Series   42 ( 1 )   191 - 192   1999.11

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    Publishing type:Research paper (scientific journal)   Publisher:Oxford University Press (OUP)  

    DOI: 10.1093/nass/42.1.191

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  • Stable isotope edited NMR analysis of Ascaris suum mitochondrial tRNA(Met) having a TV-replacement loop

    T Ohtsuki, G Kawai, K Watanabe

    Journal of Biochemistry   124 ( 1 )   28 - 34   1998.7

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    Most nematode mitochondrial (mt) tRNAs have a TV-replacement loop (TV loop) which replaces the normal T arm and the variable loop in standard tRNAs with a less-structured loop. The tertiary structure of such tRNAs has been discussed theoretically with reference to the crystal structure of yeast tRNA(Phe) [Wolstenholme et al, (1994) Nucleic Acids Res. 22, 4300-4306] and examined experimentally by chemical and enzymatic probing [Watanabe et al. (1994) J, Biol. Chem. 269, 22902-22906]. The results suggest that most regions of the tRNA other than the TV loop are folded in a similar manner to yeast tRNA(Phe). To confirm this notion more clearly, the tertiary structure of Ascaris suum mt tRNA(Met) was analyzed by NMR using various synthetic tRNAs site-specifically labeled with stable isotopes, which were prepared by a combination of chemical synthesis and enzymatic ligation, Tertiary interactions involving G(L2), G(L3), U(L4), and U8 were observed in the NMR spectra of the labeled tRNAs, but those relating to G(L5) were not. On the basis of these results, a possible tertiary structural model of nematode mitochondrial tRNA(Met) was constructed.

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  • Automated chemical synthesis of biologically active tRNA having a sequence corresponding to Ascaris suum mitochondrial tRNA(Met) toward NMR measurements

    T Ohtsuki, R Vinayak, Y Watanabe, K Kita, G Kawai, K Watanabe

    Journal of Biochemistry   120 ( 6 )   1070 - 1073   1996.12

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    RNA samples corresponding to Ascaris suum mitochondrial tRNA(Met) were chemically and automatically synthesized in amounts sufficient for NMR measurement. Conventional and rapid deprotection methods gave tRNA samples with the same amino acid-accepting activity as those prepared by other method; enzymatic synthesis, and enzymatic ligation of chemically synthesized fragments. The synthetic tRNA showed the same H-1-NMR spectrum in the iminoproton region as the ligated tRNA, This rapid and reliable preparation method thus provides biologically active tRNA for NMR measurement, and further, it is applicable for synthesis of other large synthetic RNAs, by combining the site-specific isotopic labeling method.

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  • Preparation of biologically active Ascaris suum mitochondrial tRNA(Met) with a TV-replacement loop by ligation of chemically synthesized RNA fragments

    T Ohtsuki, G Kawai, Y Watanabe, K Kita, K Nishikawa, K Watanabe

    Nucleic Acids Research   24 ( 4 )   662 - 667   1996.2

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    Ascaris suum mitochondrial tRNA(Met) lacking the entire T stem was prepared by enzymatic ligation of two chemically synthesized RNA fragments. The synthetic tRNA could be charged with methionine by A.suum mitochondrial extract, although the charging activity was considerably low compared with that of the native tRNA, probably due to lack of modification, Enzymatic probing of the synthetic tRNA showed a very similar digestion pattern to that of the native tRNA(Met), which has already been concluded to take an L-shape-like structure [Watanabe et al, (1994) J. Biol. Chem., 269, 22902-22906], These results suggest that the synthetic tRNA possesses almost the same conformation as the native one, irrespective of the presence or absence of modified residues. The method of preparing the bizarre tRNA used here will provide a useful tool for elucidating the tertiary structure of such tRNAs, because they can be obtained without too much difficulty in the amounts necessary for physicochemical studies such as NMR spectroscopy.

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  • MOLLIFICATION OF CYTOTOXICITY OF SULFATED POLYSACCHARIDES BY FIBROBLAST GROWTH-FACTORS

    M KUNOU, T OHTSUKI, T AKAIKE, K HATANAKA

    Journal of Carbohydrate Chemistry   14 ( 4-5 )   659 - 665   1995

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    Sulfated polysaccharide which had a relatively high degree of sulfation showed cytotoxicity to 3T3-L1 fibroblasts. Acidic and basic fibroblast growth factors inhibited the cell damage caused by the sulfated polysaccharides, while epidermal growth factor and platelet growth factor had no effects.

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  • EFFECTS OF SYNTHETIC POLYANIONS ON 3T3-L1 FIBROBLAST PROLIFERATION STIMULATED BY FIBROBLAST GROWTH-FACTORS

    K HATANAKA, T OHTSUKI, M KUNOU

    Chemistry Letters   1407-1410 ( 8 )   1407 - 1410   1994.8

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    Effects of synthetic polyanions on the mitogenic activity of fibroblast growth factors were investigated. Poly(vinylsulfonate) contributed to potentiate the activity of the acidic fibroblast growth factor, while the carboxyl group was quite effective for basic fibroblast growth factor.

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Books

  • 化学の指針シリーズ「生物有機化学 -ケミカルバイオロジーへの展開-」

    宍戸昌彦,大槻高史

    裳華房  2008 

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  • 新規モダリティ医薬品のための新しいDDS技術と製剤化

    著者多数(含:大槻高史)( Role: Joint author)

    技術情報協会  2023.1  ( ISBN:9784861049361

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    Total pages:580p   Language:Japanese

    CiNii Books

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  • 核酸科学ハンドブック(共著)

    著者多数(含:大槻高史)( Role: Joint author)

    講談社サイエンティフク  2020.12 

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  • CSJ current review: 生体分子反応を制御する(共著)

    著者多数(含:大槻高史)( Role: Joint author)

    化学同人  2020.4 

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  • ペプチド創薬の最前線 = The frontier of peptide drug discovery

    木曽, 良明

    シーエムシー出版  2019.5  ( ISBN:9784781314174

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    Total pages:viii, 265p   Language:Japanese

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  • ペプチド医薬品のスクリーニング・安定化・製剤化技術(共著)

    技術情報協会  2017 

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  • Intracellular Delivery II

    Kazunori Watanabe, Takashi Ohtsuki( Role: Joint author ,  Intracellular delivery of RNA via RNA-binding proteins or peptides. pp 403-416)

    2014  ( ISBN:9789401788953

  • Nanotechnology Tools for the Study of RNA(共著)

    Elsevier  2014 

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  • 人工核酸によるRNAの検出と精製,In 「シングルセル解析の最前線」

    シーエムシー出版  2010 

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  • RNA Interference

    Springer  2010 

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  • The central dogma: from DNA to RNA, and to protein. In “Automation in Proteomics and Genomics”

    Wiley  2009 

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  • 蛋白質核酸酵素増刊「RNAの細胞生物学」Vol.48, No.4

    共立出版  2003 

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  • Cell-free translation system

    Springer-Verlag  2002 

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  • 日本臨床増刊「ミトコンドリアとミトコンドリア病」,Vol. 60, No.4

    日本臨床社  2002 

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  • 基礎生化学実験法 第4巻

    東京化学同人  2000 

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MISC

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Presentations

  • RNA分解を検出する蛍光寿命プローブ

    平田 陸, 平川 和貴, 島田 尚鷹, 渡邉 和則, 大槻 高史

    日本生化学会大会プログラム・講演要旨集  2021.11  (公社)日本生化学会

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  • 相分離生物学 相分離を介した核内ストレス顆粒の形成は温熱抵抗性に関与している

    渡邉 和則, 的野 恭平, 大槻 高史

    Thermal Medicine  2021.9  (一社)日本ハイパーサーミア学会

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    Event date: 2021.9

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  • Photocontrolled apoptosis induction using pre-miR-664a and PCDR method

    渡邉和則, 渡邉和則, 縄稚朋子, 奥谷瑠璃子, 大槻高史, 大槻高史

    日本分子生物学会年会プログラム・要旨集(Web)  2021 

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  • Evaluation of secondary structure and intracellular delivery by stapling of apoptosis-inducing peptide Bim

    西村風香, 北松瑞生, 藤井香帆, 大槻高史

    日本化学会春季年会講演予稿集(Web)  2021 

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  • mTOR complex1 was involved in the formation of SAFB granules under heat stress

    的野恭平, 井上歩実, 岡田真実, 山本理紗子, 大槻高史, 渡邉和則

    日本分子生物学会年会プログラム・要旨集(Web)  2021 

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  • 核内ストレス顆粒の形成は温熱抵抗性に関与している

    渡邉和則, 大槻高史

    Thermal Medicine  2020 

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  • mTOR複合体を介した核内ストレス顆粒構成因子SAFB,Satellite III RNA顆粒形成機構の解明

    的野恭平, 井上歩実, 岡田真実, 山本理紗子, 大槻高史, 大槻高史, 渡邉和則, 渡邉和則

    Thermal Medicine  2020 

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  • 温熱によるXrn1/2活性化機構の解明

    岩城 香菜子, 大槻 高史, 渡邉 和則

    Thermal Medicine  2019.9  (一社)日本ハイパーサーミア学会

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    Event date: 2019.9

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  • 温熱によるSatellite III RNAの発現上昇はmTOR複合体により制御されている

    渡邉和則, 井上歩実, 大槻高史

    日本RNA学会年会要旨集  2019 

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  • 光応答的に働く卵活性化因子の開発

    大本和正, 若井拓哉, 舟橋弘晃, 渡邉和則, 大槻高史

    日本分子生物学会年会プログラム・要旨集(Web)  2019 

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  • mTOR複合体制御による核内ストレス顆粒形成機構の解明

    渡邉和則, 井上歩実, 岡田真実, 山本理紗子, 大槻高史

    日本分子生物学会年会プログラム・要旨集(Web)  2019 

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  • mTOR複合体を介した核内ストレス顆粒形成機構の解明

    渡邉和則, 井上歩実, 岡田真実, 山本理紗子, 大槻高史

    Thermal Medicine  2019 

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  • 光応答性キャリアを用いたphotochemical internalizationの機構解明

    南條 友孝, 渡邉 和則, 大槻 高史

    日本生化学会大会プログラム・講演要旨集  2018.9  (公社)日本生化学会

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  • 光に応答し細胞質内に侵入する機能性光増感剤ペプチドの設計

    三好 祐一, 山本 怜見, 門野 真保, 北松 瑞生, 渡邉 和則, 大槻 高史

    日本生化学会大会プログラム・講演要旨集  2018.9  (公社)日本生化学会

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  • 光依存的なRNA導入法による2種類のRNAの時間差導入

    大槻 高史, 白神 かおり, 渡邉 和則

    日本生化学会大会プログラム・講演要旨集  2018.9  (公社)日本生化学会

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  • 細胞内のRNA分解に関する蛍光相関分析法による解析(Fluorescence correlation spectroscopy analysis of RNA degradation in cells)

    島田 尚鷹, 渡邉 和則, 大槻 高史

    日本生化学会大会プログラム・講演要旨集  2018.9  (公社)日本生化学会

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  • mTOR複合体を介した温熱による核内ストレス構造体形成機構の解明

    井上 歩実, 岡田 真実, 山本 理紗子, 大槻 高史, 渡邉 和則

    Thermal Medicine  2018.8  (一社)日本ハイパーサーミア学会

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  • 温熱による開始tRNA分解を介した翻訳抑制と細胞周期との関係性

    渡邉 和則, 梅本 裕介, 長房 すずか, 高橋 昭久, 井尻 憲一, 大槻 高史

    Thermal Medicine  2018.8  (一社)日本ハイパーサーミア学会

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  • 熱ストレスによる核内ストレス構造体の形成はmTOR複合体によって制御されている

    井上歩実, 岡田真実, 山本理紗子, 大槻高史, 渡邉和則

    日本生化学会大会(Web)  2018 

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  • 温熱により形成される核内ストレス顆粒形成機構の解明

    渡邉和則, 井上歩実, 岡田真実, 山本理紗子, 大槻高史

    日本分子生物学会年会プログラム・要旨集(Web)  2018 

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  • 細胞周期依存的な開始tRNA分解を介した温熱による翻訳抑制機構

    渡邉和則, 渡邉和則, 梅本裕介, 長房すずか, 高橋昭久, 井尻憲一, 大槻高史

    日本生化学会大会(Web)  2018 

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  • Pre-miR-664aを用いた光依存的な細胞死の誘導

    縄稚 朋子, 渡邉 和則, 大槻 高史

    生命科学系学会合同年次大会  2017.12  生命科学系学会合同年次大会運営事務局

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  • mTORを介した核内ストレス構造体の熱ストレス下における形成機構

    井上 歩実, 渡邉 和則, 岡田 真実, 山本 理紗子, 大槻 高史

    生命科学系学会合同年次大会  2017.12  生命科学系学会合同年次大会運営事務局

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    Language:Japanese  

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  • mTORを介した温熱による核内ストレス構造体形成機構

    渡邉 和則, 岡田 真実, 井上 歩実, 山本 理紗子, 大槻 高史

    Thermal Medicine  2017.9  (一社)日本ハイパーサーミア学会

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  • 音増感剤を用いた超音波依存的なペプチドの細胞質内導入法

    大槻 高史, 稲葉 優樹, 北松 瑞生, 中田 栄司, 原田 敦史, 渡邉 和則

    日本DDS学会学術集会プログラム予稿集  2017.6  日本DDS学会

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    Language:Japanese  

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  • ペプチドとRNA双方でアポトーシスを誘導するナノ複合体の開発

    金 亨振, 北松 瑞生, 大槻 高史

    日本DDS学会学術集会プログラム予稿集  2017.6  日本DDS学会

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  • P20 Ultrasound-dependent cytosolic delivery of bioactive molecules

    Nagahiro Shota, Inaba Yuki, Matuura Eiji, Watanabe Kazunori, Ohtsuki Takashi

    Proceedings of the Annual Meeting of the Japan Society of Sonochemistry  2017  Japan Society of Sonochemistry

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    Recently, methods for delivery of peptide and RNA to target cells are widely studied for cell research and drug delivery strategies. However, the delivery methods sometimes have problems such as cytotoxicity and ineffective delivery. In some methods, target molecules tend to be trapped in endosome and degraded by lysosome when introduced to cells. In this study, ultrasound-dependent peptide / nanoparticle delivery method was developed. We developed a molecular conjugate of a cell-penetrating peptide, a sonosensitizer and a functional peptide / nanoparticle. This molecule was trapped in endosome when cells were incubated with the molecule. However, we succeeded in cytoplasmic delivery of the molecule that escape from endosome by ultrasound irradiation.

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  • 高分子ミセル「ラクトソーム」を用いた光照射特異的なRNAi技術の開発

    大島 真, 赤星 彰也, 松浦 栄次, 小関 英一, 渡邉 和則, 大槻 高史

    日本バイオマテリアル学会大会予稿集  2016.11  日本バイオマテリアル学会

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    Event date: 2016.11

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  • 熱ストレスによる核内ストレス構造体形成機構の解明

    渡邉 和則, 岡田 真実, 山本 理紗子, 大槻 高史

    日本生化学会大会プログラム・講演要旨集  2016.9  (公社)日本生化学会

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    Event date: 2016.9

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  • 熱ストレス依存的な核内ストレス構造体形成機構の解析

    岡田真実, 山本理沙子, 渡邉和則, 大槻高史

    日本分子生物学会年会プログラム・要旨集(Web)  2016 

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  • 細胞機能を解析し創る新技術 1分子から時間空間制御まで 可視光および近赤外光による細胞内RNA導入の局所的誘導

    大槻 高史, 白神 かおり, 渡邉 和則

    日本生化学会大会・日本分子生物学会年会合同大会講演要旨集  2015.12  (公社)日本生化学会

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    Language:Japanese  

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  • RNAの分解と寿命の解明

    大西 諒, 中林 孝和, 渡邉 和則, 大槻 高史

    日本生化学会大会・日本分子生物学会年会合同大会講演要旨集  2015.12  (公社)日本生化学会

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  • 新規神経分化関連microRNAの探索

    山路 隆平, 渡邉 和則, 大槻 高史

    日本生化学会大会・日本分子生物学会年会合同大会講演要旨集  2015.12  (公社)日本生化学会

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  • 光でアポトーシスを誘導する方法の開発

    藤原隼人, 畑地祐里, 北松瑞生, 渡邉和則, 大槻高史

    日本化学会中国四国支部大会講演要旨集  2015.11.7 

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  • 熱ストレスによる開始tRNAMetは細胞周期に依存して分解促進している

    渡邉和則, 渡邉和則, 梅本裕介, 高橋昭久, 井尻憲一, 大槻高史

    日本RNA学会年会要旨集  2015 

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  • ケージドアミノアシルtRNAを用いた翻訳の光制御法の開発

    神崎重人, 渡邉和則, 大槻高史

    日本化学会中国四国支部大会講演要旨集  2015 

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  • 拡張翻訳系を用いた糖タンパク質作製法

    大槻高史, 矢形梓, 白神かおり, 渡邉和則, 畑中研一

    日本糖質学会年会要旨集  2015 

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  • 光増感剤と高分子ミセルを用いたRNAデリバリーキャリアの設計

    井野川 翔一, 渡邊 和則, 小関 英一, 松浦 栄次, 大槻 高史

    日本バイオマテリアル学会大会予稿集  2014.11  日本バイオマテリアル学会

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    Language:Japanese  

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  • 光化学的に細胞質内に侵入するペプチド分子の設計

    大槻高史, 藤原隼人, 畑地祐里, 北松瑞生, 渡邉和則

    光化学討論会要旨集(CD-ROM)  2014 

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  • 熱ストレス下では,mTORはXrn2の核小体から核質への拡散を介して開始tRNAMetの分解促進を制御している

    渡邉和則, 渡邉和則, 井尻憲一, 大槻高史

    日本分子生物学会年会プログラム・要旨集(Web)  2014 

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  • CLIP-RNAi法における多環状RNAの有効性の検討

    村上真一, 渡邉和則, 大槻高史

    日本RNA学会年会要旨集  2014 

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  • tRNA顆粒形成要因タンパク質同定の試み

    山本理紗子, 大槻高史, 渡邉和則

    日本分子生物学会年会プログラム・要旨集(Web)  2014 

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  • mTORが熱ストレスによる開始tRNAMetの分解促進を制御している

    渡邉和則, 渡邉和則, 井尻憲一, 大槻高史

    日本RNA学会年会要旨集  2014 

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  • 細胞内運搬ペプチドと光応答色素を含むアポトーシス誘導ペプチドの合成とそのエンドソーム内蓄積

    前場伊織, 北松瑞生, 大槻高史

    日本化学会講演予稿集  2013.3.8 

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  • ヘテロ二量体化ロイシンジッパーによる緑色蛍光タンパク質の細胞内輸送

    出口宝, 北松瑞生, 中島真実, 大槻高史, 道上宏之

    日本化学会講演予稿集  2013.3.8 

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  • 細胞周期とRNA関連イベントとの相関の観察手法の開発

    丸田静佳, 渡邉和則, 大槻高史

    アンチセンスシンポジウム講演要旨集  2013 

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  • ケージドアミノアシルtRNAを用いた翻訳の光制御

    赤星彰也, 土井芳朗, 木内智樹, 渡邉和則, 大槻高史

    日本RNA学会年会要旨集  2013 

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  • 多環状RNAを用いた光誘導RNAi

    村上真一, 渡邉和則, 大槻高史

    日本分子生物学会年会プログラム・要旨集(Web)  2013 

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  • 細胞内noncoding RNA局在追跡ツールの開発

    田村 佳史, 大槻 高史

    日本生化学会大会プログラム・講演要旨集  2012.12  (公社)日本生化学会

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  • 分岐状アミノ酸と蛍光性アミノ酸から成るペプチドデンドリマーの合成およびその性質

    北松瑞生, 北畠まゆみ, 能年義輝, 大槻高史

    高分子学会予稿集(CD-ROM)  2012.9.5 

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    Event date: 2012.9.5

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  • 中心に蛍光基を含んだ分岐状ペプチドの性質

    北畠まゆ美, 脇坂祐也, 石踊由佳, 大槻高史, 北松瑞生

    日本化学会西日本大会講演要旨集  2011.11.7 

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  • 細胞内侵入型PNAビーコンによる機能性RNAの検出

    西岡浩貴, 大槻高史, 北松瑞生

    日本化学会西日本大会講演要旨集  2011.11.7 

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  • 細胞内侵入型PNAによる機能性RNAの導入および検出

    西岡浩貴, 大槻高史, 北松瑞生

    アンチセンスシンポジウム講演要旨集  2011.9.1 

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  • アルギニン誘導体の部位特異的導入に基づくヒストン蛋白質の機能解析

    赤星 彰也, 鈴江 良隆, 宍戸 昌彦, 大槻 高史

    日本生化学会大会プログラム・講演要旨集  2011.9  (公社)日本生化学会

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  • リポソーム膜上における膜蛋白質への蛍光性アミノ酸導入およびその挙動解析法の開発

    大塚 巧磨, 根木 慧, 金井 保, 秋吉 一成, 野村 M. 新一郎, 大槻 高史

    日本生化学会大会プログラム・講演要旨集  2011.9  (公社)日本生化学会

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  • 近赤外光による照射領域特異的RNAi誘導法の開発

    石躍 由佳, 森長 美香, 大槻 高史

    日本生化学会大会プログラム・講演要旨集  2011.9  (公社)日本生化学会

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  • Synthesis of cyclic peptides using the NEXT-A cyclization reaction

    HAMAMOTO Toshimasa, SISIDO Masahiko, OHTSUKI Takashi, TAKI Masumi

    2011.3.1 

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  • CLIP-RNAi法によるRNAiの光誘導

    大槻高史, 米澤政樹, 松本祥, 遠藤玉樹, 石躍由佳

    日本光医学・光生物学会  2011 

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  • ケージドアミノアシルtRNAを用いた翻訳の光制御

    土井芳朗, 赤星彰也, 宍戸昌彦, 大槻高史

    日本化学会バイオテクノロジー部会シンポジウム講演要旨集  2011 

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  • 光応答性キャリア蛋白質を用いたRNAデリバリー

    大槻高史, 松本祥, 遠藤玉樹, 石躍由佳

    アンチセンスシンポジウム講演要旨集  2011 

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  • 光照射領域特異的RNAi誘導技術の開発

    石躍由佳, 桑原里奈, 米澤政樹, 遠藤玉樹, 大槻高史

    アンチセンスシンポジウム講演要旨集  2010 

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  • CLIP-RNAi法による遺伝子発現抑制の光誘導

    大槻高史, 石躍由佳, 遠藤玉樹, 宍戸昌彦

    日本化学会バイオテクノロジー部会シンポジウム講演要旨集  2010 

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  • 細胞質内RNA導入の光誘導とCLIP-RNAi

    大槻高史, 石躍由佳, 遠藤玉樹, 宍戸昌彦

    日本RNA学会年会要旨集  2010 

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  • CLIP-RNAi法による光照射領域特異的な遺伝子発現抑制

    大槻 高史, 遠藤 玉樹, 宍戸 昌彦

    日本生化学会大会プログラム・講演要旨集  2009.9  (公社)日本生化学会

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    Event date: 2009.9

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  • CLIP-RNAi法による1細胞RNAiの経時的観察

    大槻 高史, 遠藤 玉樹, 宍戸 昌彦

    バイオイメージング  2009.7.15 

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  • siRNAデリバリーのためのCPP-RBP型RNAキャリアの開発

    桑原里奈, 遠藤玉樹, 米澤政樹, 宍戸昌彦, 大槻高史

    日本分子生物学会年会講演要旨集  2009 

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  • TatEF-Tuを用いたアミノアシルtRNAの哺乳動物細胞内への輸送

    根木慧史, 大槻高史, 宍戸昌彦

    日本分子生物学会年会講演要旨集  2009 

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  • 光操作による哺乳細胞内での蛋白質発現制御

    土井芳朗, 大槻高史, 宍戸昌彦

    日本分子生物学会年会講演要旨集  2009 

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  • ペプチド核酸‐細胞内導入ペプチドコンジュゲートを用いたRNAの細胞内送達

    久保貴紀, 北松瑞生, 遠藤玉樹, 山中智史, 大庭秀樹, 大槻高史, 宍戸昌彦

    日本化学会西日本大会講演要旨集  2007.11.10 

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  • ペプチド核酸‐膜透過ペプチド オリゴマーをキャリアーとして用いたsiRNAの細胞内輸送

    北松瑞生, 久保貴紀, 大庭英樹, 遠藤玉樹, 大槻高史, 宍戸昌彦

    バイオ・高分子シンポジウム講演要旨集  2007.7.23 

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  • Antisense Effects of Pyrrolidine-Based Oxy-PNAs Conjugated with a Membrane-Permeable Peptide in Escherichia coli

    KURAMI Syunsuke, KITAMATSU Mizuki, OHTSUKI Takashi, SISIDO Masahiko

    2007.3.1 

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  • ペプチド核酸-細胞内導入ペプチドコンジュゲートを用いたRNAの細胞内送達

    久保貴紀, 北松瑞生, 遠藤玉樹, 山中智史, 大庭秀樹, 大槻高史, 宍戸昌彦

    日本化学会西日本大会講演要旨集  2007 

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  • Specific RNA binding of immobilized PNA's

    T. Ohtsuki, T. Fujimoto, Y. Sugimoto, M. Kitamatsu, M. Sisido

    JOURNAL OF PEPTIDE SCIENCE  2006  JOHN WILEY & SONS LTD

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  • interactions between nematode mitochondrial tRNAs and EF-Tus

    OHTSUKI Takashi, WATANABE Yoh-ichi, KITA Kiyoshi, TAKEMOTO Chie, KAWAI Gota, UEDA Takuya, WATANABE Kimituna

    1998.12.1 

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    Language:Japanese  

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  • NMR analysis of guanosine binding mechanism of the group I intoron

    KITAMURA Aya, MUTO Yutaka, KIM Insil, WATANABE Satoru, OOTSUKI Takashi, HOSONO Kazumi, KAWAI Gota, WATANABE Kimitsuna, TAKAKU Hiroshi, INOUE Tan, YOKOYAMA Shigeyuki

    1998.12.1 

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    Language:Japanese  

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  • 安定同位体標識NMR法による線形動物ミトコンドリアtRNAの立体構造解析

    大槻 高史, 河合 剛太, 渡辺 洋一, 西川 一八, 渡辺 公綱

    日本分子生物学会年会プログラム・講演要旨集  1996.8.1 

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  • Position specific incorporation of nonnatural amino acids into proteins in vivo

    JAACT2008  2008 

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  • tRNA構造との対応関係を越えたショウジョウバエミトコンドリアEF-TuのtRNA認識能

    第31回日本分子生物学会年会・第81回日本生化学会大会 合同大会  2008 

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  • Improvement of CPP-RBD for siRNA delivery

    JAACT2008  2008 

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  • アルギニン誘導体の蛋白質への位置特異的導入

    第31回日本分子生物学会年会・第81回日本生化学会大会 合同大会  2008 

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  • 哺乳細胞を用いた非天然アミノ酸導入蛋白質合成法の開発

    第31回日本分子生物学会年会・第81回日本生化学会大会 合同大会  2008 

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  • RNA-蛋白質ハイブリッド型FRETプローブを用いた低分子化合物の検出

    第31回日本分子生物学会年会・第81回日本生化学会大会 合同大会  2008 

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  • EF-Tu変異体によるアミノアシルtRNAの精製・定量法

    第31回日本分子生物学会年会・第81回日本生化学会大会 合同大会  2008 

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  • EF-Tu変異体を用いた非天然アミノアシルtRNAの精製と定量

    第3回バイオ関連化学合同シンポジウム  2008 

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  • 細胞内での非天然アミノ酸導入蛋白質合成法の開発」第3回バイオ関連化学合同シンポジウム

    第3回バイオ関連化学合同シンポジウム  2008 

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  • Construction of RNA-protein Hybrid for Bioimaging of Low-molecular Compounds.

    3rd International Workshop on Approaches to Single-Cell Analysis  2008 

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  • CLIP-RNAi ~photoinduced RNAi strategy~.

    3rd International Workshop on Approaches to Single-Cell Analysis  2008 

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  • 蛍光標識CPP-RBPによるshRNAの細胞内導入と遺伝子発現の光制御

    日本化学会第88春季年会  2008 

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  • RNA-蛋白質ハイブリッド型FRETプローブによる低分子化合物の検出

    日本化学会第88春季年会  2008 

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  • 蛍光標識CPP-RBPによるRNAiの光誘導

    第10回RNAミーティング  2008 

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  • 細胞内生理活性分子検出のためのRNA-タンパク質ハイブリッド型FRETプローブの構築

    第10回RNAミーティング  2008 

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  • 酵素反応を用いるアミノ酸の蛍光分析

    日本化学会第88春季年会  2008 

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  • 蛍光標識CPP-RBPによるRNAデリバリーとRNAiの光制御

    遺伝子・デリバリー研究会 第8回シンポジウム  2008 

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  • Expansion of protein biosynthesis system by EF-Tu mutants.

    12th annual meeting of RNA society  2007 

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  • 改変EF-Tuによる蛋白質への非天然アミノ酸高効率導入

    第30回日本分子生物学会年会・第80回日本生化学会大会 合同大会  2007 

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  • 生細胞内における蛋白質への位置特異的な蛍光基導入法の開発

    第30回日本分子生物学会年会・第80回日本生化学会大会 合同大会  2007 

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  • アセチル化アミノ酸やメチル化アミノ酸の蛋白質への位置特異的導入

    第30回日本分子生物学会年会・第80回日本生化学会大会 合同大会  2007 

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  • RNA interference using cell permeable protein carrier

    4th International Peptide Symposium  2007 

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  • Delivery of dsRNA with lactic acid bacteria for RNA interference

    Kuwahara, A, Arita, M, Sisido, M, Ohtsuki, T

    5th International Symposium on Nucleic Acids Chemistry  2007 

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  • Photo inducible RNA interference using cell permeable protein carrier

    Endoh, T, Sisido, M, Ohtsuki, T

    5th International Symposium on Nucleic Acids Chemistry  2007 

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  • Incorporation of large nonnatural amino acids into protein by translation with EF-Tu mutants

    4th International Peptide Symposium  2007 

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  • 蛋白質キャリアによるsiRNAの細胞内導入とRNAiの光誘導

    第212回バイオロンジル会  2007 

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  • ISFET電極を用いるアミノ酸センサーの作製と応答の評価

    日本化学会第87春季年会  2007 

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  • RNA isolation using biotinylated PNAs

    The 2nd International Workshop on Approaches to Single Cell Analysis  2007 

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  • リボソームを用いたβ-ペプチド合成に向けたアプローチ:主鎖伸長型基質導入に関与するファクター

    第22回生体機能関連化学シンポジウム  2007 

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  • 非天然アミノ酸含有タンパク質の無細胞及び細胞モデル系での発現

    特定領域『バイオ操作』若手研究者第2回ワークショップ  2007 

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  • ペプチド核酸-膜透過ペプチド オリゴマーをキャリアーとして用いたsiRNAの細胞内輸送

    第17回バイオ・高分子シンポジウム  2007 

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  • 蛍光標識Tat融合RNA結合タンパク質を用いたsiRNAの細胞内導入とRNAiの光制御

    第22回生体機能関連化学シンポジウム  2007 

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  • 改変EF-Tuを用いた翻訳系の拡張

    第22回生体機能関連化学シンポジウム  2007 

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  • Cellular delivery and photo-accelerated endosomal escape of siRNA mediated by cell permeable RNA binding protein

    12th annual meeting of RNA society  2007 

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  • RNA工学と拡張翻訳系

    第109回応用化学科セミナー  2007 

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  • 蛋白質生合成の非天然アミノ酸許容性の拡張を目的とする変異EF-Tuの作製

    日本ケミカルバイオロジー第2回年会  2007 

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  • 大腸菌に対する主鎖骨格にピロリジン環を持つオキシペプチド核酸(POPNA)のアンチセンス効果

    日本化学会第86春季年会  2006 

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  • Expansion of protein biosynthesis system including nonnatural amino acids

    Ohtsuki, T, Doi, Y, Manabe, T, Sisido, M

    IEEE International Symposium on Micromechatronics and Human Science (MHS)  2006 

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  • ペプチド核酸(PNA)-膜透過ペプチド(CPP)コンジュゲートを用いたDNAの細胞内導入

    第16回アンチセンスシンポジウム  2006 

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  • 無細胞蛋白質合成システムを用いた非天然アミノ酸導入蛋白質の合成とその応用

    第一回無細胞生命科学研究会  2006 

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  • Antisense effects of pyrrolidine-based oxy-PNAs conjugated with a membrane-permeable peptide in Escherichia coli

    43JPS-PEM4  2006 

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  • ピロリジン環型オキシPNA(POPNA)の大腸菌内アンチセンス効果

    第16回アンチセンスシンポジウム  2006 

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  • "DNA delivery into CHO cells by the use of complementary peptide nucleic acid conjugated with a cell-penetrating peptide

    NANOBIO-TOKYO2006  2006 

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  • タンパク質への部位特異的なリン酸化セリン導入法の開発

    第21回生体機能関連化学部会・第9回バイオテクノロジー部会・第9回生命化学研究会合同シンポジウム  2006 

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  • 哺乳動物細胞内における蛋白質への位置特異的蛍光基導入

    第21回生体機能関連化学部会・第9回バイオテクノロジー部会・第9回生命化学研究会合同シンポジウム  2006 

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  • 非天然アミノ酸を担持したtRNAに対して結合能の高いEF-Tuの創製

    第21回生体機能関連化学部会・第9回バイオテクノロジー部会・第9回生命化学研究会合同シンポジウム  2006 

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  • RNA isolation using biotinylated PNA.

    20th IUBMB International Congress of Biochemistry and Molecular Biology  2006 

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  • Establishment of purification method of C. elegans mitochondrial ribosome by peptide affinity tags.

    20th IUBMB International Congress of Biochemistry and Molecular Biology  2006 

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  • ISFET型電極を用いるアミノ酸センサーの開発

    日本化学会第86春季年会  2006 

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  • 寄生性線虫を用いた、異常構造をもつミトコンドリアtRNAとEF-Tuの相関関係の解明

    第75回日本寄生虫学会大会  2006 

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  • 非天然アミノ酸含有翻訳システムの拡張

    第8回日本RNA学会年会  2006 

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  • Specific RNA binding of immobilized PNAs

    29th European Peptide Symposium  2006 

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  • Creation of EF-Tu to incorporate large nonnatural amino acids into proteins

    20th IUBMB International Congress of Biochemistry and Molecular Biology  2006 

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  • 細胞膜透過性RNA結合タンパク質によるshRNAの細胞内導入

    第8回日本RNA学会年会  2006 

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  • 固定化PNAによるRNA精製法

    第8回生命化学研究会シンポジウム  2006 

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  • 人工遺伝暗号を用いた非天然アミノ酸導入系の最適化

    企画セミナー「人工遺伝暗号」プログラム  2005 

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  • RNA isolation using immobilized PNA

    Ohtsuki, T, Kumano, C, Manabe, T, Kitamatsu, M, Sisido, M

    4th International Symposium on Nucleic Acids Chemistry(第32回核酸化学シンポジウム)  2005 

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  • RNA結合蛋白質を介したshRNAの細胞内導入法

    第28回日本分子生物学会年会  2005 

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  • アミノアシルtRNA合成酵素を用いる アミノ酸センシング

    日本化学会第85春季年会  2005 

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  • 縮小化した機能性RNAからなる線形動物ミトコンドリアのタンパク質合成系

    第74回日本寄生虫学会大会  2005 

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  • ミトコンドリアtRNA-m1A9修飾酵素の探索

    第28回日本分子生物学会年会  2005 

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  • C. elegansミトコンドリアリボソームの精製を目指したペプチドタグ付リボソームタンパク質の発現

    第28回日本分子生物学会年会  2005 

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  • 非天然アミノ酸を担持したtRNAに対して結合能の高いEF-Tuの創製

    第28回日本分子生物学会年会  2005 

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  • 異常構造をもつミトコンドリアtRNAとEF-Tuの相関関係の解明

    第28回日本分子生物学会年会  2005 

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  • EF-Tu binding ability of tRNAs carrying various types of nonnatural amino acids

    4th European-Japanese Bioorganic Conference & Chemical Biology COE Program Sponsored by Okayama University  2005 

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  • セリン特異的なEF-Tuの発見とセリン特異性に関与するアミノ酸残基の解析

    第28回日本分子生物学会年会  2005 

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  • 非天然アミノ酸を担持したtRNAとEF-Tuとの結合評価

    第27回日本分子生物学会年会  2004 

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  • 線形動物ミトコンドリアEF-Tu2のtRNA認識機構の解明

    第6回RNAミーティング  2004 

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  • Orthogonal tRNAs for four-base codon translation

    International Symposium on Nucleic Acids, Membranes and Signal Transduction ---Symposium in Honor of Professor H. Gobind Khorana  2004 

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  • 線形動物ミトコンドリアにみられるtRNAとペプチド伸長因子EF-Tuの共進化

    第73回日本寄生虫学会大会  2004 

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  • 無脊椎動物ミトコンドリアのセリルtRNA合成酵素組換えタンパク質の発現

    第27回日本分子生物学会年会  2004 

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  • 4塩基コドンを用いた翻訳のための直交化tRNAの探索

    第27回日本分子生物学会年会  2004 

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  • Elongation factors Tu for truncated tRNAs

    International Symposium on Nucleic Acids, Membranes and Signal Transduction ---Symposium in Honor of Professor H. Gobind Khorana  2004 

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  • Nonnatural amino acid incorporation into protein by mitochondrial tRNAs having non-standard structure

    RNA Structure and Function: a Joint Biochemical Society/Royal Society of Chemistry Focused Meeting  2004 

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  • セリルtRNAのみを認識する伸長因子EF-Tu

    第26回日本分子生物学会年会  2003 

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  • Characteristic features of mammalian mitochondrial translation systems and functional equivalency of mitochondrial tRNA and translation factors to E. coli counterparts

    International symposium commemorating the opening of cell-free science and technology research center  2003 

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  • Decoding property of C5 uridine modification at the wobble position of tRNA anticodon.

    Kurata, S, Ohtsuki, T, Wada, T, Kirino, Y, Takai, K, Saigo, K, Watanabe, K, Suzuki, T

    30th symposium on Nucleic Acids Chemistry  2003 

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  • Unique translation systems of animal mitochondria

    International congress on protein expression & protein function  2003 

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  • Modified nucleotides in nematode mitochondrial tRNAs

    20th International tRNA Workshop  2003 

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  • Unique serine-specificity of mitochondrial EF-Tu

    20th International tRNA Workshop  2003 

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  • Towards artificial repair of UV-damaged DNA: studies on drug binding and alkali hydrolysis.

    Iwai, S, Inase, A, Jafar, S, Higurashi, M, Ohtsuki, T, Xu, Y, Sugiyama, H

    30th symposium on Nucleic Acids Chemistry  2003 

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  • Unusual elongation factors Tu for truncated tRNAs

    20th International tRNA Workshop  2003 

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  • 線形動物ミトコンドリアにみるtRNAとペプチド伸長因子EF-Tuの共進化

    第26回日本分子生物学会年会  2003 

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  • Unique characteristics of animal mitochondrial translation systems

    20th International tRNA Workshop  2003 

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  • 延長部分を持つ伸長因子TuによるTアーム欠失tRNAの新規認識機構

    第26回日本分子生物学会年会  2003 

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  • Taurine is a constituent of mammalian mitochondrial tRNAs; new insights into the functions of taurine and human mitochondrial diseases.

    19th International tRNA workshop  2002 

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  • Two distinct elongation factors Tu specific for respective T arm-lacking and D arm-lacking tRNAs in nematode mitochondria

    19th International tRNA workshop  2002 

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  • Chemical synthesis of novel taurine-containing uridine derivatives.

    Wada, T, Shimazaki, T, Nakagawa, S, Ohtsuki, T, Kurata, S, Suzuki, T, Watanabe, K, Saigo, K

    29th Symposium on Nucleic Acids Chemistry  2002 

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  • Requirement of modified residue m1A9 for EF-Tu binding to nematode mitochondrial tRNA lacking the T arm.

    Sakurai, M, Ohtsuki, T, Watanabe, Y, Watanabe, K

    28th Symposium on Nucleic Acids Chemistry  2001 

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  • 非天然型塩基対を用いた転写・翻訳系の構築 〜6塩基系による非天然型アミノ酸のタンパク質中への取り込み

    第3回 RNA ミーティング  2001 

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  • Structural and functional compensation for deficit in RNA with proteins in animal mitochondrial translation systems

    International conference in honor of Alexander Spirin:PROEIN SYNTHESIS  2001 

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  • Unnatural base pairs between 2-amino-6-(2-thienyl)purine and the complementary bases.

    Hirao, I, Fujiwara, T, Kimoto, M, Mitsui, T, Okuni, T, Ohtsuki, T, Yokoyama, S

    27th Symposium on Nucleic Acids Chemistry  2000 

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Industrial property rights

  • ホウ素含有化合物およびそれを含む薬剤

    北松瑞生, 副島哲朗, 山形尚紀, 道上宏之, 大槻高史

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    Application no:特願2019- 008817  Date applied:2019.1.22

    Announcement no:特開2020-117479  Date announced:2020.8.6

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  • 修飾RNAを含有する分子集合体及びそれを用いたRNA送達システム

    大槻高史, 松浦栄次, 小渕浩嗣, 赤星彰也, 小関 英一

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    Applicant:株式会社島津製作所・岡山大学

    Application no:特願2016-557827  Date applied:2014.11.8

    Patent/Registration no:特許6586103  Date registered:2019.9.13 

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  • 近赤外光によりRNAを細胞質内に送達するための新規なキャリア分子ならびに方法

    大槻高史, 石躍由佳

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    Application no:特願2011-063953  Date applied:2011.11.24

    Patent/Registration no:特許5900895 

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  • 新規ペプチド複合体、そのハイブリッド複合体およびその用途

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    Application no:特願2011-233812  Date applied:2011.10.25

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  • 乳酸菌により二本鎖RNAを生成するキット及びその利用

    大槻高史, 宍戸昌彦, 公文裕巳, 柏倉祐司, 落合和彦

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    Application no:特願2008-279228  Date applied:2008.10.30

    Patent/Registration no:特許5660537 

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  • 乳酸菌において二本鎖RNAを生成するキット及びその利用

    大槻高史, 宍戸昌彦

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    Application no:特願2008-1174884  Date applied:2008.4.28

    Announcement no:特開2008-301812 

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  • 修飾型PNA/RNA複合体

    北松瑞生, 大槻高史, 宍戸昌彦, 久保貴紀, 大庭英樹

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    Application no:特願2007-37041  Date applied:2007.2.16

    Announcement no:特開2008-301812 

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  • 部位特異的にリン酸化した蛋白質の合成方法、当該方法に用いるホスホセリルtRNA及び当該方法を実施するための試薬キット

    大槻高史, 宍戸昌彦, 横川隆志, 大野敏, 西川一八

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    Application no:特願2006-240828  Date applied:2006.9.5

    Announcement no:特開2008-061538 

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  • 部位特異的アミノ酸導入法のための新規な直交化tRNA

    川井淳, 川上文清, 大槻高史, 宍戸昌彦

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    Application no:特願2005-102868  Date applied:2005.3.31

    Patent/Registration no:特許4406731 

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  • 細胞内へ核酸を導入する為の新規な分子並びに細胞内へ導入する核酸および細胞内へ核酸を導入する為の新規な方法

    川井淳, 川上文清, 大槻高史, 宍戸昌彦

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    Application no:特願2005-103703  Date applied:2005.3.31

    Patent/Registration no:特許4709971 

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  • アミノ酸分析用バイオセンシング装置およびアミノ酸分析用バイオセンサーおよびアミノ酸分析用アミノアシルtRNAシンセターゼ

    釘宮章光, 大槻高史, 菅野憲一

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    Application no:特願2004-359328  Date applied:2004.12.13

    Announcement no:特開2006-166709 

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Awards

  • 研究功績賞

    2017   岡山大学工学部  

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  • 内山勇三科学技術賞

    2016  

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    Country:Japan

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  • The Journal of Biochemistry/OUP Poster Prize

    2009  

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    Country:Japan

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  • 岡山大学若手トップリサーチャー研究奨励賞

    2008  

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    Country:Japan

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  • 生体機能関連化学シンポジウム講演賞

    2007  

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    Country:Japan

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Research Projects

  • mRNAのPhotochemical Internalization

    2022.04 - 2024.03

    科研費 挑戦的研究(萌芽) 

    大槻 高史

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    Authorship:Principal investigator  Grant type:Competitive

    Grant amount:\5000000

  • 生命機能の光制御のための機能性光増感ペプチドの開発

    2018.04 - 2022.03

    科研費 基盤研究(B) 

    大槻 高史

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    Authorship:Principal investigator  Grant type:Competitive

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  • Creation of mammalian oocytes with high fertility by spatiotemporal regulation of the function of mitochondria

    Grant number:18H02328  2018.04 - 2022.03

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Scientific Research (B)

    FUNAHASHI Hiroaki

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    Grant amount:\17290000 ( Direct expense: \13300000 、 Indirect expense:\3990000 )

    The aim of this study was to generate oocytes with high developmental competence by regulating the mitochondrial (mt) DNA copy number in the ooplasm. Porcine ooplasmic mtDNA copy number did not change during in vitro maturation (IVM) and was lower in oocytes from small than medium follicles. Drp1 (mt mitogen) was reduced during early development, and its inhibition impaired the mt distribution in oocytes and early developmental competence. Overexpression of PGC1alpha (mt synthesis-related genes) resulted in increased reactive oxygen species and mtDNA copy number reduction in oocytes, and reduced IVM rate and early developmental competence. The autophagic potential of oocytes varied greatly during IVM and early development, and differed significantly by follicle diameter of origin. Attempts to introduce photosensitizers and peptide/protein molecular tools into oocytes have not resulted in significant improvement.

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  • ケージドアミノアシルtRNAによる初期胚における翻訳の時空間的制御

    2017.04 - 2019.03

    科研費 挑戦的研究(萌芽) 

    大槻 高史

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  • Live imaging of microRNA by intracellular-invasion peptide nucleic acid beacon

    Grant number:16KT0195  2016.07 - 2019.03

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Scientific Research (C)

    KITAMATSU Mizuki

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    Grant amount:\4680000 ( Direct expense: \3600000 、 Indirect expense:\1080000 )

    We aim to develop methods to detect microRNAs that are indicators of disease in cells. In particular, dell-penetrating peptide and peptide nucleic acid (PNA) beacon were conjugated for direct detection of microRNA inside cells. We also used the PNA beacon to enable detection of the target microRNA based on the color of the fluorescence emission by modified with two fluorescent groups that generate fluorescence resonance energy transfer at N- and C- terminal on the PNA. We successfully detected the RNA by using the PNA beacon in vitro.

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  • 細胞内におけるRNA分解の観測法の開発とストレス応答研究への応用

    2015.04 - 2017.03

    科研費 挑戦的萌芽研究 

    大槻 高史

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    Authorship:Principal investigator  Grant type:Competitive

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  • 光増感剤修飾分子を用いたPCI の分子科学

    2014.04 - 2016.03

    科研費 新学術領域研究 

    大槻 高史

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    Authorship:Principal investigator  Grant type:Competitive

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  • PCDR技術による細胞機能の光制御

    2013.04 - 2016.03

    科研費 基盤研究(B) 

    大槻 高史

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    Authorship:Principal investigator  Grant type:Competitive

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  • Sonochemical internalization法の確立

    2013.04 - 2015.03

    科研費 挑戦的萌芽研究 

    大槻 高史

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    Authorship:Principal investigator  Grant type:Competitive

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  • 光増感によるエンドソーム脱出の分子科学

    2012.04 - 2014.03

    科研費 新学術領域研究 

    大槻 高史

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    Authorship:Principal investigator  Grant type:Competitive

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  • RNAが引き起こす細胞内イベントの細胞周期依存性の解析

    2011.04 - 2013.03

    科研費 挑戦的萌芽研究 

    大槻 高史

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    Authorship:Principal investigator  Grant type:Competitive

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  • CLIP-RNAi法の開発と応用

    2009.04 - 2012.03

    科研費 若手研究(A) 

    大槻 高史

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    Authorship:Principal investigator  Grant type:Competitive

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  • RNAi創薬を目指したRNAデリバリー技術の開発

    2008.04 - 2010.03

    科研費 特定領域研究 

    大槻 高史

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    Authorship:Principal investigator  Grant type:Competitive

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  • 拡張型蛋白質合成系を含む細胞システムの構築

    2008.04 - 2010.03

    科研費 特定領域研究 

    大槻 高史

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    Authorship:Principal investigator  Grant type:Competitive

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  • 人工核酸によるRNAの検出と精製

    2007.04 - 2009.03

    科研費 特定領域研究 

    大槻 高史

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    Authorship:Principal investigator  Grant type:Competitive

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  • New development in study of mitochondrial translation system of theparasitic nematode

    Grant number:19590425  2007 - 2008

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Scientific Research (C)

    YOSHINARI Shigeo, WATANABE Yoh-ichi, OHTSUKI Takashi, YOKOBORI Shin-ichi

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    Grant amount:\4550000 ( Direct expense: \3500000 、 Indirect expense:\1050000 )

    動物ミトコンドリア(mt)のタンパク質合成系は、より短い機能性RNAを用いる。我々は、短縮化したmt tRNA を持つ種と持たない種が混在する節足動物で、短縮化したtRNAを持たない昆虫の2つのEF-Tu のうちの1つが、短縮化したtRNA と結合できることを示した。また、植物寄生性線虫ミトコンドリアで遺伝子が未同定だったtRNA のうち1種の発現を同定した。このtRNA は発現が確認された中で最も短い。

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  • 非天然アミノ酸含有タンパク質の無細胞及び細胞モデル系での発現

    2006.04 - 2008.03

    科研費 特定領域研究 

    大槻 高史

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    Authorship:Principal investigator  Grant type:Competitive

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  • RNAの新規細胞内導入法の開発と応用

    2006.01 - 2008.12

    NEDO  産業技術研究助成事業 

    大槻 高史

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    Authorship:Principal investigator  Grant type:Competitive

    Grant amount:\48100000

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  • 生細胞中での蛋白質の部位特異的蛍光標識法の開発

    2005.04 - 2007.03

    科研費 若手研究(B) 

    大槻 高史

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  • Study of parasite mitochondrial translation system which have truncated functional RNAs

    Grant number:17590368  2005 - 2006

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Scientific Research (C)

    WATANABE Yoh-ichi, OHTSUKI Takashi

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

    Translation system in metazoan mitochondria is quite different from those in eukaryotic cytoplasm and prokaryote in many points. Among those, based on mitochondrial DNA sequences, translation system in nematode mitochondria has been predicted to have shortest tRNAs and shortest rRNA. From the results of analysis of mitochondrial translation system of parasitic and free-living nematodes, we are hoping to reveal that what will happen when functional RNAs in translation system is truncated. In this study, we analyzed these issues and pursued to reveal why truncation of functional RNAs in translation system had happened. We obtained the following results during the last two years:
    1.To purify a larger amount of mitochondrial ribosome more efficiently, we prepared recombinant nematode expressing a mitochondrial ribosomal protein with affinity tags. However, we could not obtained mitochondrial ribosome from the transformant, suggesting that optimization of combination of ribosomal protein and tag sequence will be necessary.
    2.Using mutant thermophilic bacterial EF-Tu proteins possessing residues found in Caenorhabditis elegans mitochondrial EF-Tu2, we revealed which residues in EF-Tu2 contribute the specificity against seryl-moiety in seryl-tRNA binding.
    3.We revealed specificities of aminoacyl-tRNA binding of mitochondrial EF-Tu proteins in parasitic nematode Trichinella spp.
    4.We revealed specificities of aminoacyl-tRNA binding of mitochondrial EF-Tu proteins in the arthropod.
    5.We found a candidate tRNA methyltransferase.

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  • Chemical Expansion of the Central Dogma towards Synthetic Microorganisms

    Grant number:15101008  2003 - 2007

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Scientific Research (S)

    SISIDO Masahiko, OHTSUKI Takashi, TAKI Masumi

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    Grant amount:\103610000 ( Direct expense: \79700000 、 Indirect expense:\23910000 )

    This research project aims to expand molecules like amino acids, nucleic acids and proteins constitute the protein biosynthesizing system to synthesize novel type of proteins with artificial functions. The final goal is to create synthetic microorganisms that live with 21 type of amino acids. During the 5 year we obtained the following results. (1) Some nonnatural amino acids were successfully charged onto a specific tRNA by the use of a peptide nucleic acid (PNA) that is complementary to the tRNA. The PNA-assisted aminoacylation mold be carried out in an E.coli in vitro protein biosynthesizing system to create a protein incorporated with the nonnatural amino add. (2) tRNAs and an elongation faint Tu. (EF-Tu) were expanded to bring large-sized nonnatural amino adds into E.col ribosome and subsequently to a protein. (3) An orthogonal set of aminoacyl tRNAsynthetase (ARS) and tRNA was screened to bring some nonnatural amino acids into proteins in living mammalian cells. (4) Finally these expanded molecular systems were introduced into mammalian cells and the synthesis of proteins containing nonnatural amino acids was detected, although its quantity was very small.
    In the last year, an attempt was made to apply these techniques to attach fluorescent amino acids to peptides and proteins. The fluorescently labeled peptides were screened to dimmer those that bind to cancer cells or to cancer-cell specific proteins.

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  • Study of nematode mitochondrial translation system using parasitic nematodes

    Grant number:15590365  2003 - 2004

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Scientific Research (C)

    WATANABE Yoh-ichi, OHTSUKI Takashi, KITA Kiyoshi, KAMIYA Haruo

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

    Most nematode mitochondria have D-armless tRNAs, which also exist in most metazoan mitochondria, and T-armless tRNAs. On the other hand, bacterial translation elongation factor Tu (EF-Tu) needs T-arm of aminoacyl-tRNA for the complex formation. In order to investigate how nematode mitochondrial EF-Tu recognizes the T-armless tRNA, we cloned EF-Tus from various nemaotdes. Nematode have two distinct EF-Tus. Two EF-Tus, namely EF-Tu1 and EF-Tu2, have novel C-terminal extensions (about 40-60 residues and about 15 residues, respectively) when compared with bacterial EF-Tu. Caernarhabditis elegans and Trichinella britovi EF-Tu1 bound to the T-armless tRNAs, while EF-Tu2 bound to D-armless tRNAs. C.elegans EF-Tus and T.britovi EF-Tu2 could not bind to conventional tRNAs, while T.britovi EF-Tu1 could bind to conventional tRNAs. These results consist well with the fact that C.elegans mitochondria do not have any conventional tRNA but Trichinella mitochondria have. Deletion of 10 residues of C-terminus of both of C.elegans EF-Tus lead to loss of binding to the D-armless or the T-armless tRNAs, suggesting the novel C-Terminal extensions may involve in interaction between EF-Tus and the truncated nematode tRNAs. In summary, the nematode mitochondrial EF-Tu adapted to the deletion of D-arm or T-arm of tRNAs by the gene duplication and aquirment of novel C-terminal extension to make a new interaction between the truncated tRNA and EF-Tu.

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  • 異常tRNAを用いた翻訳系

    2002.04 - 2003.10

    科学技術振興調整費 

    大槻 高史

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    Authorship:Principal investigator  Grant type:Competitive

    Grant amount:\34000000 ( Direct expense: \29130000 )

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  • Elucidation of basic principle in genetic information translation system by using the specialty of animal mitochondria

    Grant number:14208077  2002 - 2003

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Scientific Research (A)

    WATANABE Kimitsuna, SUZUKI Tsutomu, OHTSUKI Takashi

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    Grant amount:\48880000 ( Direct expense: \37600000 、 Indirect expense:\11280000 )

    The aim of this project is to elucidate the basic principle in genetic information translation system by using the specialty of animal mitochondria. This can be done by combining the well-studied E.coli translation system with the special translation system of animal mitochondria. (1)The minimal prerequisite structure of tRNA : The higher-order structure of nematode mitochondrial (mt) tRNA^<Ser>_<UCU>, which is the smallest tRNA in the extant organisms (54 nucleotides, having no D arm and small T arm consisting of 3 base pairs and a loop with 4 bases), was examined by using NMR. It turned out that almost all the base-pairs predicted from its clover-leaf structure existed in solution and the connector region consisting of the truncated D loop and extra loop had a rather flexible structure. This mobility is indispensable for the tRNA^<Ser>_<UCU> to maintain the relative arrangement of the 3'-terminus and the anticodon equal to that of the usual tRNAs. (2)The minimal prerequisite structure of ribosome : In the animal mt ribosome, the content of rRNA is about a half, but that of proteins is doubled, as compared with the counterparts of E.coli ribosome. The functional regions of mt rRNA are strictly conserved in the central parts of two ribosomal subunits and the peripheral regions of rRNA lacking in the mt ribosome are embedded by proteins so as to maintain the whole shapes of ribosomes almost equal between mt and E.coli. It turned out that some of the stem/loop structures in the peripheral regions of mt ribosome are dispensable. (3)What is the trigger in the hydrolysis reaction of GTP which is an energy source in the translation system : It was suggested that the trigger in the hydrolysis reaction of GTP is L7/L12 protein in 50S ribosomal subunit, on the basis of the fact that GTPase activity of EF-G is activated by L7/L12. We have confirmed this proposal by elucidating that E.coli EF-G can function on the mt ribosome in which only the L7/L12 protein is replaced by the counterpart of E.coli ribosome, although E.coli EF-G cannot work on the native mt ribosome.

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  • 伸長因子EF-TuによるTアームを欠くtRNAの認識機構

    2001.04 - 2002.03

    科研費 若手研究(B) 

    大槻 高史

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    Authorship:Principal investigator  Grant type:Competitive

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  • Elucidation of the construction principle of the mitochondria translation system by means of structural biology

    Grant number:11308024  1999 - 2001

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Scientific Research (A)

    WATANABE Kimitsuna, OHTSUKI Takashi, SUZUKI Tsutomu

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    Grant amount:\34720000 ( Direct expense: \32800000 、 Indirect expense:\1920000 )

    The aim of this research is to elucidate the construction principle of the mitochondria! translation system, by analyzing the functional structures of each component of the animal mitochondrial translation system using X-ray analysis and NMR spectroscopy, focusing on the functional characteristics and structural abnormalities of the components. The following results have been obtained for these three years.
    (1) Bovine mitochondrial in vitro translation system has been established by optimizing concentration of each component and reaction conditions, which has similar efficiency to that of 」. co// system in terms of poly(U)-dependent poly(Phe) synthesis.
    (2) Ribosomes are exchangeable between E. coli and mitochondrial systems, as far as tRNA and translation factors of the same origins are used, and EF-Tu of E. coli origin is used. Thus, the ribosomes are functionally equivalent between E. coli and mitochondrial systems.
    (3) It was verified that 2 species of serine tRNAs with unusual secondary structures can be functional. The low efficiency of the D arm-lacking serine tRNA is derived from a defect in its binding to the ribosomal A site.
    (4) These two serine tRNAs were recognized by a single seryl-tRNA synthetase (serRS) and its recognition sites on the two tRNAs were identified.
    (5) A unique EF-Tu was found which recognized almost all the T arm-lacking tRNAs in nematode mitochondria. It had 57 amino acid extension at the C terminus which was considered to complement the T arm-lacking part of tRNAs. Another EF-Tu recognizing two D arm-lacking serine tRNAs was specific for serine.
    (6) Crystallization of these translation factors are being under way. We have obtained a needle-like srystal for SerRS. The X-ray analysis will be made in future.

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