Analysis of molecular mechanism of transcription reaction by zero-mode waveguides

• Project/Area Number: 17K19351
• Research Category: Grant-in-Aid for Challenging Research (Exploratory)
• Allocation Type: Multi-year Fund
• Research Field: Molecular and Genome biology and related fields
• Research Institution: Osaka University
• Principal Investigator: Harada Yoshie 大阪大学, 蛋白質研究所, 教授 (10202269)
• Research Collaborator: Tadakuma Hisashi ; Nakao Kimiko ; Han Yong-Woon ; Fukumoto Kodai ; Masubuchi Takeya
• Project Period (FY): 2017-06-30 – 2019-03-31
• Project Status: Completed (Fiscal Year 2018)
• Budget Amount: ¥6,500,000 (Direct Cost: ¥5,000,000、Indirect Cost: ¥1,500,000); Fiscal Year 2018: ¥3,250,000 (Direct Cost: ¥2,500,000、Indirect Cost: ¥750,000); Fiscal Year 2017: ¥3,250,000 (Direct Cost: ¥2,500,000、Indirect Cost: ¥750,000)
• Keywords: ナノバイオロジー / 生物物理 / ナノバイオ / 蛋白質

Outline of Final Research Achievements

Transcription is the first step of gene expression. To visualize the real-time process of transcription, we established the method using Zero-mode waveguides (ZMWs). We optimized the fabrication process and glass surface modification, which allowed us to minimize the fluctuation of ZMW-chip quality. In addition, it is important to control the precise three-dimensional (3D) layout of transcription factors, e.g. RNA polymerase (RNAP), as the 3D layout on the flexible DNA affects the interaction of RNAP and regulatory factors. Therefore, using DNA origami technology, we established new approach to control the molecular layout at nano-meter resolution. These methods should contribute to future elucidation of transcription mechanism.

Academic Significance and Societal Importance of the Research Achievements

タンパク質や核酸など生体分子間の相互作用は、生命現象の基本であり、本研究で確立した方法は、これらの分子機構を理解する上で有用である。本研究では、精製した因子を用いているが、作製したナノ開口チップには、細胞抽出液や細胞そのものを載せる事も可能であり、将来的には、1分子レベルの高感度検出が可能な利点を生かし、臨床検査などへの展開も期待される。

Research Products

• [Journal Article] Enrichment of ODMR-active nitrogen-vacancy centres in five-nanometre-sized detonation- synthesized nanodiamonds: Nanoprobes for temperature, angle and position (2018)
  • Author(s): Sotoma S, Terada D, Segawa TF, Igarashi R, Harada Y, Shirakawa M.
  • Journal Title: Sci.Rep. Volume: 8 Issue: 1 Pages: 5463-5463
  • DOI: 10.1038/s41598-018-23635-5
  • NAID: 120006517896
  • Peer Reviewed / Open Access
• [Journal Article] Construction of integrated gene logic-chip (2018)
  • Author(s): Takeya Masubuchi, Masayuki Endo, Ryo Iizuka, Ayaka Iguchi, Dong Hyun Yoon, Tetsushi Sekiguchi, Hao Qi, Ryosuke Iinuma, Yuya Miyazono, Shuichi Shoji, Takashi Funatsu, Hiroshi Sugiyama, Yoshie Harada, Takuya Ueda, Hisashi Tadakuma
  • Journal Title: Nat. Nanotechnol. Volume: 13 Issue: 10 Pages: 933-940
  • DOI: 10.1038/s41565-018-0202-3
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] One-Pot Synthesis of Highly Dispersible Fluorescent Nanodiamonds for Bioconjugation (2018)
  • Author(s): Terada Daiki、Sotoma Shingo、Harada Yoshie、Igarashi Ryuji、Shirakawa Masahiro
  • Journal Title: Bioconjugate Chemistry Volume: 29 Issue: 8 Pages: 2786-2792
  • DOI: 10.1021/acs.bioconjchem.8b00412
  • Peer Reviewed
• [Journal Article] Commonly stabilized cytochromes c from deep-sea Shewanella and Pseudomonas (2018)
  • Author(s): Fujii Sotaro、Masanari-Fujii Misa、Kobayashi Shinya、Kato Chiaki、Nishiyama Masayoshi、Harada Yoshie、Wakai Satoshi、Sambongi Yoshihiro
  • Journal Title: Bioscience, Biotechnology, and Biochemistry Volume: 82 Issue: 5 Pages: 792-799
  • DOI: 10.1080/09168451.2018.1448255
  • Peer Reviewed
• [Journal Article] Live Cell Imaging Using Photoswitchable Diarylethene-Doped Fluorescent Polymer Dots (2017)
  • Author(s): Osakada Yasuko、Fukaminato Tuyoshi、Ichinose Yuma、Fujitsuka Mamoru、Harada Yoshie、Majima Tetsuro
  • Journal Title: Chemistry - An Asian Journal Volume: 12 Issue: 20 Pages: 2660-2665
  • DOI: 10.1002/asia.201701038
  • Peer Reviewed / Open Access
• [Presentation] Studies on Biomolecules Using Single-Molecule Imaging Technique (2019)
  • Author(s): Yoshie Harada
  • Organizer: Deutsch-Japan Kolloquien 2019
  • Int'l Joint Research / Invited
• [Presentation] Quantitative analysis of biomolecular complexes using Zero-Mode Waveguides (ZMW) (2018)
  • Author(s): Kimiko Nakao, Hisashi Tadakuma, Yong-Woon Han, Yoshie Harada
  • Organizer: 第56回日本生物物理学会年会
• [Presentation] Poly-lysine tag increase the binding rate of SNAPf-fused protein to DNA origami (2018)
  • Author(s): Kodai Fukumoto, Yuya Miyazono, Hisashi Tadakuma, Yoshie Harada
  • Organizer: 第56回日本生物物理学会年会
• [Presentation] ポリリジン残基の付加は、DNAオリガミへのSNAPf融合蛋白質の結合速度を向上させる (2018)
  • Author(s): 福本紘大、宮園侑也、多田隈尚史、原田慶恵
  • Organizer: 2019年生体運動研究合同班会議
• [Presentation] 超解像顕微鏡で探る哺乳動物細胞の収縮環形成機構 (2018)
  • Author(s): 上条桂樹， 加藤薫， 高橋正行， 馬渕一誠， 細谷浩史 ， 藤原敬宏， 原田慶恵
  • Organizer: 生体運動合同班会議
• [Presentation] Rational design of orthogonal gene transcription nano device on DNA origami (2017)
  • Author(s): Takeya Masubuchi, Hisashi Tadakuma, Masayuki Endo, Ryo Iizuka, Hiroshi Sugiyama, Yoshie Harada, Takashi Funatsu, Takuya Ueda
  • Organizer: 第19回日本RNA学会
• [Presentation] in vivo RNA labelling reveals dynamic regulation of ribonucleic foci in living neurons (2017)
  • Author(s): Ikumi Oomoto, Hiroki Umeshima, Yoshie Harada, Yong-Woon Han, Akimitsu Okamoto, Ohtan Wang
  • Organizer: 第55回日本生物物理学会
• [Presentation] High-throughput fabrication of Zero-Mode Waveguide (ZMW) and its application to observation of bio-molecules (2017)
  • Author(s): Kimiko Nakao, HisashiTadakuma, Yong-Woon Han, Kodai Fukumoto, Yoshie Harada
  • Organizer: 第55回日本生物物理学会
• [Presentation] Rational design of orthogonal gene transcription nano device on DNA origami (2017)
  • Author(s): Takeya Masubuchi, Masayuki Endo, Ryo Iizuka, Ayaka Iguchi, Dong Hyun Yoon, Tetsushi Sekiguchi, Hao Qi, Ryosuke Iinuma, Yuya Miyazono, Shuichi Shoji, Takashi Funatsu, Hiroshi Sugiyama, Yoshie Harada, Takuya Ueda and Hisashi Tadakuma
  • Organizer: CBI(情報計算化学生物)学会
• [Presentation] DNAオリガミを用いた直行性を有する転写デバイスの合理的設計 (2017)
  • Author(s): 増渕岳也, 遠藤政幸, 飯塚怜, 井口彩香, Dong Hyun Yoon, 関口哲志, Hao Qi, 飯沼良介, 宮薗侑也, 庄子習一, 船津高志, 杉山弘, 原田慶恵, 上田卓也, 多田隈尚史
  • Organizer: 生命科学系学会合同年次大会
• [Remarks] 大阪大学 蛋白質研究所 原田慶恵研 研究テーマ
  • URL: https://www.ccc.osaka-u.ac.jp/protein/nanobiology/research/
• [Remarks] 大阪大学 蛋白質研究所 原田慶恵研 研究テーマ
  • URL: https://www.ccc.osaka-u.ac.jp/protein/nanobiology/research/