DNA－タンパク質相互作用のデジタルカウンティング

• Project Area: Spying minority in biological phenomena -Toward bridging dynamics between individual and ensemble processes-
• Project/Area Number: 26115708
• Research Category: Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)
• Allocation Type: Single-year Grants
• Review Section: Biological Sciences
• Research Institution: Kyoto University
• Principal Investigator: 原田 慶恵 京都大学, 物質－細胞統合システム拠点, 教授 (10202269)
• Project Period (FY): 2014-04-01 – 2016-03-31
• Project Status: Discontinued (Fiscal Year 2015)
• Budget Amount: ¥12,090,000 (Direct Cost: ¥9,300,000、Indirect Cost: ¥2,790,000); Fiscal Year 2015: ¥6,240,000 (Direct Cost: ¥4,800,000、Indirect Cost: ¥1,440,000); Fiscal Year 2014: ¥5,850,000 (Direct Cost: ¥4,500,000、Indirect Cost: ¥1,350,000)
• Keywords: ナノバイオ

Outline of Annual Research Achievements

「少数性生物学」では数を知ることが重要である。分子の数を数える方法として、蛍光1分子イメージング技術がある。現在用いられている全反射照明による蛍光1分子イメージング法では、蛍光色素分子の濃度がおよそ50 nMを越えると、常に近接した領域に複数の蛍光標識生体分子が存在するようになり、個々の分子の観察が非常に困難になる。そのため、全反射照明蛍光顕微鏡法による蛍光1分子イメージングで解析できるのは、基質との親和性が十分高い酵素に限られていた。高濃度条件下で1分子蛍光イメージングを行うことができる新しい蛍光1分子イメージング法としてナノ開口が開発された。ナノ開口はガラス基板上の厚さ100 nm程度のアルミフィルムに作製した直径80～100 nmの穴である。穴の直径に比べて、励起光の波長が大きいため、ガラス基板の下から励起光を照射すると、励起光は穴の外には出ることができず、穴の底面ごく近傍に近接場光が生じる。全反射照明法より、さらに微小領域を励起するこの方法を用いることで、数μMでも1分子観察が可能になる。ガラス基板上にナノメートルサイズの加工を施す技術は確立されておらず、ナノ開口の作製は容易ではない。これまでにいくつかのナノ開口基板作製手法が報告されているが、我々はMetal Lift-off法でナノ開口基板を作製している。昨年度は条件検討を繰り返し、30×20 mm角の石英ガラス基板に安定してナノ開口を作製することができるようになった。今年度はナノ開口を大量に作製するため、直径4インチの石英ウェハを使って、1回の作業で60枚のナノ開口を作製する方法を確立した。その結果、安定して大量のナノ開口が作製できるようになった。このナノ開口を使って、エピジェネティクス制御の１つであるDNAメチル化パターン維持に関与するタンパク質の機能解析を行っている。

Research Progress Status

27年度が最終年度であるため、記入しない。

Strategy for Future Research Activity

27年度が最終年度であるため、記入しない。

Research Products

• [Journal Article] ECHO-liveFISH: in vivo RNA labeling reveals dynamic regulation of nuclear RNA foci in living tissues. (2015)
  • Author(s): Oomoto, I., Suzuki-Hirano, A., Umeshima, H., Han, YW., Yanagisawa, H., Carlton, P., Harada, Y., Kengaku, M., Okamoto, A., Shimogori, T. and Wang, DO.
  • Journal Title: Nucleic Acids Res. Volume: 43(19) Issue: 19 Pages: e126-e126
  • DOI: 10.1093/nar/gkv614
  • NAID: 120005619785
  • Peer Reviewed / Open Access
• [Journal Article] Suppression of Non-Specific Protein-Nanodiamond Adsorption Enabling Specific Targeting of Nanodiamonds to Bio-molecules of Interest (2015)
  • Author(s): Shingo Sotoma, Ryuji Igarashi, Jun Iimura, Yuta Kumiya, Hedehito Tochio, Yoshie Harada and Masahiro Shirakawa,
  • Journal Title: Chemistry letters Volume: 44 Issue: 3 Pages: 354-356
  • DOI: 10.1246/cl.141036
  • NAID: 130004868380
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Synergistic effect of ATP for RuvA-RuvB-Holliday junction DNA complex formation (2015)
  • Author(s): Takuma Iwasa, Yong-Woon Han, Ryo Hiramatsu, Hiroaki Yokota, Kimiko Nakao, Ryuji Yokokawa, Teruo Ono and Yoshie Harada
  • Journal Title: Scientific Reports Volume: 印刷中
  • NAID: 120005712940
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] The application of Fluorescence-Conjugated Pyrrole/Imidazole Polyamides in the Characterization of Protein-DNA Complex Formation (2015)
  • Author(s): Yong-Woon Han, Hiroshi Sugiyama and Yoshie Harada
  • Journal Title: Biomaterials Science Volume: 印刷中
  • Peer Reviewed / Open Access
• [Journal Article] Optically Detected Magnetic Resonance of Nanodiamonds In Vivo; Implementation of Selective Imaging and Fast Sampling. (2015)
  • Author(s): Yoshinari, Y., Mori, S., Igarashi, R., Sugi, T., Yokota, H., Ikeda, K., Sumiya, H., Mori, I, Tochio, H., *Harada, Y. and Shirakawa, M.
  • Journal Title: J Nanosci Nanotechnol. Volume: 15 Issue: 2 Pages: 1014-1021
  • DOI: 10.1166/jnn.2015.9739
  • Peer Reviewed / Open Access
• [Journal Article] Comprehensive and quantitative analysis for controlling the physical/chemical states and particle properties of nanodiamonds for biological applications (2015)
  • Author(s): Sotoma, S., Akagi, K., Hosokawa, S., Igarashi, R., Tochio, H., Harada, Y., and Shirakawa
  • Journal Title: RSC Advances Volume: 5 Issue: 18 Pages: 13818-13827
  • DOI: 10.1039/c4ra16482b
  • Peer Reviewed / Open Access
• [Journal Article] Hard X-ray-induced Optical Luminescence via Biomolecule-directed Metal Clusters (2014)
  • Author(s): Y. Osakada, T. Teranishi, B. Cui et al.
  • Journal Title: Chem. Commun. Volume: 50 Issue: 27 Pages: 3549-3551
  • DOI: 10.1039/c3cc48661c
  • Peer Reviewed
• [Journal Article] Real-time single-cell imaging of protein secretion (2014)
  • Author(s): Yamagishi, M. Suzuki, N. Izawa, K. Nakahara, A. Mizuno, J. Shoji, S. Heike, T. Harada, Y. Nishikomori, R. Ohara, O.
  • Journal Title: Sci Rep Volume: 4 Issue: 1 Pages: 4736-4736
  • DOI: 10.1038/srep04736
  • Peer Reviewed / Open Access
• [Journal Article] Quantitative in vivo fluorescence cross-correlation analyses highlight the importance of competitive effects in the regulation of protein-protein interactions (2014)
  • Author(s): Sadaie W, Harada Y, Matsuda M, Aoki K
  • Journal Title: Molecular and Cellular Biology Volume: 34 Issue: 17 Pages: 3272-3290
  • DOI: 10.1128/mcb.00087-14
  • NAID: 120005625632
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Effective Production of Fluorescent Nanodiamond Containing Negatively-Charged Nitrogen-Vacancy Center by Ion Irradiation (2014)
  • Author(s): Shingo Sotoma, Yosuke Yoshinari, Ryuji Igarashi, Akiyoshi Yamazaki, Shige H. Yoshimura, Hidesato Tochio, Masahiro Shirakawa and Yoshie Harada
  • Journal Title: Diamond and Related Materials Volume: 49 Pages: 33-38
  • DOI: 10.1016/j.diamond.2014.07.011
  • NAID: 120005474529
  • Peer Reviewed / Open Access
• [Journal Article] ナノ開口基板を用いたDNAメチル化パターン維持機構に関与する生体分子の機能解析 (2014)
  • Author(s): 韓龍雲、原田慶恵
  • Journal Title: 生体の科学 Volume: 65 Pages: 553-558
• [Presentation] Application of fluorescent diamond nanoparticles to bio-imaging (2015)
  • Author(s): Ryuji Igarashi, Shingo Sotoma, Masahiro Shirakawa and Yoshie Harada
  • Organizer: JSAP-OSA Joint Symposia 2015
  • Place of Presentation: 名古屋国際会議場
  • Year and Date: 2015-09-16
  • Invited
• [Presentation] Force generation mechanism of the RNA polymerase (2015)
  • Author(s): Yoshie Harada
  • Organizer: The 53th Annual Meeting of the Biophysical Society of Japan 2015
  • Place of Presentation: 金沢大学
  • Year and Date: 2015-09-14
  • Invited
• [Presentation] Characterization of RuvA-RuvB-Holliday junction DNA complex formation using Zero Mode Waveguides (2015)
  • Author(s): Yong-Woon Han, Takuma Iwasa, Ryo Hiramatsu, Hiroaki Yokota, Kimiko Nakao, Ryuji Yokokawa, Teruo Ono and Yoshie Harada
  • Organizer: The 53th Annual Meeting of the Biophysical Society of Japan 2015
  • Place of Presentation: 金沢大学
  • Year and Date: 2015-09-14
• [Presentation] Rational desigh of orthogonal gene transcription nano device on DNA origami (2015)
  • Author(s): Takeya Masubuchi, Hisashi Tadakuma, Ryo Iizuka, Masayuki Endo, Takashi Funatsu, Hiroshi Sugiyama and Yoshie Harada
  • Organizer: The 53th Annual Meeting of the Biophysical Society of Japan 2015
  • Place of Presentation: 金沢大学
  • Year and Date: 2015-09-14
• [Presentation] Direct observation of the swimming motility of deep-sea bacterium at high-pressure conditions (2015)
  • Author(s): Masayoshi Nishiyama, Tiaki Kato and Yoshie Harada
  • Organizer: The 53th Annual Meeting of the Biophysical Society of Japan 2015
  • Place of Presentation: 金沢大学
  • Year and Date: 2015-09-13
• [Presentation] Application of fluorescent diamond nanoparticles to bio-imaging (2015)
  • Author(s): Yoshie Harada
  • Organizer: Diamond Quantum Sensing Workshop 2015
  • Place of Presentation: 香川国際会議場
  • Year and Date: 2015-08-07
  • Invited
• [Presentation] Characterization of RuvA-RuvB-Holliday junction DNA complex formation process using Single molecule fluorescence imaging technique (2015)
  • Author(s): Yong-Woon Han, Takuma Iwasa, Ryo Hiramatsu, Hiroaki Yokota, Ryuji Yokokawa, Teruo Ono, and Yoshie Harada
  • Organizer: The 18th iCeMS International Symposium & The 15th International Membrane Research Forum
  • Place of Presentation: Kyoto University Kyoto
  • Year and Date: 2015-03-03
• [Presentation] ナノ開口基板を用いたDNAメチル化パターン維持機構に関与する生体分子の機能解析 (2015)
  • Author(s): 韓龍雲、原田慶恵
  • Organizer: 光・量子デバイス研究会
  • Place of Presentation: ゆふいん七色の風 大分
  • Year and Date: 2015-01-26
• [Presentation] 1分子イメージング技術を使った生体分子機能解析 (2014)
  • Author(s): 原田慶恵
  • Organizer: 大阪大学蛋白研究所未来コロキウム
  • Place of Presentation: 大阪大学蛋白質研究所
  • Year and Date: 2014-12-08
  • Invited
• [Presentation] ナノ開口を用いたDNAとDNA結合タンパク質の相互作用解析 (2014)
  • Author(s): 原田慶恵
  • Organizer: 日本細胞生物学会大会
  • Place of Presentation: 奈良県新公会堂 奈良
  • Year and Date: 2014-06-12
• [Presentation] Studies on biomolecules using single-molecule imaging techniques (2014)
  • Author(s): Yoshie Harada
  • Organizer: The 19th Annual Conference of the Biophysical Society of ROC
  • Place of Presentation: Cheng Kung University, Tainan, Taiwan
  • Year and Date: 2014-05-08
  • Invited
• [Remarks] 原田研究室 研究テーマ
  • URL: http://www.harada.icems.kyoto-u.ac.jp/research.html