Development of functional nucleic acid whose activity is altered between inside and outside the cell by utilizing in-cell NMR

• Project/Area Number: 17K14515
• Research Category: Grant-in-Aid for Young Scientists (B)
• Allocation Type: Multi-year Fund
• Research Field: Bio-related chemistry
• Research Institution: Kyoto University
• Principal Investigator: YAMAOKI Yudai 京都大学, エネルギー理工学研究所, 研究員 (00778095)
• Project Period (FY): 2017-04-01 – 2020-03-31
• Project Status: Completed (Fiscal Year 2019)
• Budget Amount: ¥4,550,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥1,050,000); Fiscal Year 2018: ¥2,080,000 (Direct Cost: ¥1,600,000、Indirect Cost: ¥480,000); Fiscal Year 2017: ¥2,470,000 (Direct Cost: ¥1,900,000、Indirect Cost: ¥570,000)
• Keywords: in-cell NMR / 四重鎖核酸 / リボザイム / アプタマー / カリウムイオン

Outline of Final Research Achievements

The potassium ion concentration inside cells (100-150 mM) is much higher than that outside the cells (3.5-5.5 mM), therefore functional RNAs that sense the potassium ion concentration can switch their activities ON/OFF between the intra- and extracellular environments. Here we developed an RNA aptamer whose binding activity is altered in response to potassium ion via quadruplex structure formation. Furthermore, in order to monitoring the functional RNA inside the living cells, we tried to measure in-cell NMR spectra of RNAs in living human cells. Here NMR signals of DNA and RNA were successfully observed in living human cells. The observed signals indicated the formation of DNA and RNA hairpin structures in living human cells.

Academic Significance and Societal Importance of the Research Achievements

カリウムイオン濃度を感知して細胞内に取り込まれた時のみ活性がオンになる新規機能性RNA分子は細胞内分子を標的とする核酸医薬品および同分野で課題となっているドラッグデリバリーシステム開発の一助と成り得る。また、細胞内は試験管内環境とは異なり、様々な生体分子が密に詰まった分子混雑環境である。このような混雑環境中では核酸の挙動が試験管内環境とは異なる。本研究で確立したヒト細胞を用いた核酸のin-cell NMR法はこのような分子混雑環境下で核酸の挙動を詳細に調べる手法と成り得る。これは核酸の制御する様々な生命現象を調べるツールと成ることが期待される。

Research Products

• [Journal Article] RNA sequence and length contribute to RNA-induced conformational change of TLS/FUS (2020)
  • Author(s): Hamad Nesreen、Mashima Tsukasa、Yamaoki Yudai、Kondo Keiko、Yoneda Ryoma、Oyoshi Takanori、Kurokawa Riki、Nagata Takashi、Katahira Masato
  • Journal Title: Scientific Reports Volume: 10 Issue: 1 Pages: 2629-2629
  • DOI: 10.1038/s41598-020-59496-0
  • Peer Reviewed / Open Access
• [Journal Article] Recent progress of in-cell NMR of nucleic acids in living human cells (2020)
  • Author(s): Yamaoki, Y., Nagata, T., Sakamoto, T., and Katahira, M.
  • Journal Title: Biophysical Reviews Volume: - Issue: 2 Pages: 411-417
  • DOI: 10.1007/s12551-020-00664-x
  • Peer Reviewed / Open Access
• [Journal Article] Versatility of RNA-Binding Proteins in Living Cells (2019)
  • Author(s): R. Kurokawa et al.
  • Journal Title: Biomedical Sciences Volume: 5 Issue: 1 Pages: 7-13
  • DOI: 10.11648/j.bs.20190501.12
  • Peer Reviewed / Open Access
• [Journal Article] The anti‐prion RNA aptamer R12 disrupts the Alzheimer's disease‐related complex between prion and amyloid β (2019)
  • Author(s): Iida Mamiko、Mashima Tsukasa、Yamaoki Yudai、So Masatomo、Nagata Takashi、Katahira Masato
  • Journal Title: The FEBS Journal Volume: 印刷中 Issue: 12 Pages: 2355-2365
  • DOI: 10.1111/febs.14819
  • Peer Reviewed / Open Access
• [Journal Article] 2023年に向けて (2019)
  • Author(s): 武藤梨沙、河合(久保田)寿子、池上貴久
  • Journal Title: Seibutsu Butsuri Volume: 59 Issue: 1 Pages: 001-001
  • DOI: 10.2142/biophys.59.001
  • NAID: 130007557811
  • ISSN: 0582-4052, 1347-4219
  • Peer Reviewed / Open Access
• [Journal Article] Analysis of Interactions between Telomeric i-Motif DNA and a Cyclic Tetraoxazole Compound (2018)
  • Author(s): Sedghi Masoud Shadi、Yamaoki Yudai、Ma Yue、Marchand Adrien、Winnerdy Fernaldo Richtia、Gabelica Valerie、Phan Anh Tuan、Katahira Masato、Nagasawa Kazuo
  • Journal Title: ChemBioChem Volume: 19 Issue: 21 Pages: 2268-2272
  • DOI: 10.1002/cbic.201800425
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Multiplicity in Long Noncoding RNA Biomedical Sciences (2018)
  • Author(s): 3.R. Kurokawa, R. Komiya, T. Oyoshi, Y. Matsuno, H. Tani, M. Katahira, K. Hitachi, Y. Iwashita, T. Yamashita, K. Kondo, R. Yoneda, Y. Yamaoki, N. Ueda, T. Mashima, N. Kobayashi, T. Nagata, A. Kiyoishi, M. Miyake, F. Kano, M. Murata1, N. Hamad, K. Sasaki, N. Shoji
  • Journal Title: Biomedical Sciences Volume: 4 Issue: 2 Pages: 18-23
  • DOI: 10.11648/j.bs.20180402.11
  • Peer Reviewed / Open Access
• [Journal Article] Selective alkylation of T-T mismatched DNA using vinyldiaminotriazine-acridine conjugate (2018)
  • Author(s): Onizuka Kazumitsu、Usami Akira、Yamaoki Yudai、Kobayashi Tomohito、Hazemi Madoka E、Chikuni Tomoko、Sato Norihiro、Sasaki Kaname、Katahira Masato、Nagatsugi Fumi
  • Journal Title: Nucleic Acids Res. Volume: 46 Issue: 3 Pages: 1059-1068
  • DOI: 10.1093/nar/gkx1278
  • NAID: 120006466620
  • Peer Reviewed / Open Access
• [Journal Article] The first successful observation of in-cell NMR signals of DNA and RNA in living human cells (2018)
  • Author(s): Yamaoki Yudai、Kiyoishi Ayaka、Miyake Masayuki、Kano Fumi、Murata Masayuki、Nagata Takashi、Katahira Masato
  • Journal Title: Phys Chem Chem Phys Volume: 20 Issue: 5 Pages: 2982-2985
  • DOI: 10.1039/c7cp05188c
  • NAID: 130007557810
  • Peer Reviewed / Open Access
• [Journal Article] Development of an RNA aptamer that acquires binding capacity against HIV-1 Tat protein via G-quadruplex formation in response to potassium ions (2017)
  • Author(s): Yamaoki, Y., Nagata, T., Mashima, T., Katahira, M.
  • Journal Title: Chemical communications Volume: 53 Issue: 52 Pages: 7056-7059
  • DOI: 10.1039/c7cc03312e
  • Peer Reviewed / Open Access
• [Presentation] In-cell NMR法を用いた非標準構造を形成する核酸の構造安定性の解析 (2020)
  • Author(s): 阪本知樹, 山置佑大, 加納ふみ，村田昌之，永田崇, 片平正人
  • Organizer: 第100回日本化学会年会
• [Presentation] Disruption of the Alzheimer's disease-related complex betweeen prion and amyloid b by an anti-prion aptamer (2019)
  • Author(s): T. Mashima, M. Iida, Y. Yamaoki, M. So, T. Nagata, M. Katahira
  • Organizer: Commemorative International Symposium of the Japan Society of Nucleic Acids Chemistry (CISNAC)
  • Int'l Joint Research
• [Presentation] In-cell NMR法を用いたヒト生細胞内の核酸の構造と安定性の評価 (2019)
  • Author(s): 山置佑大, 永田崇，清石彩華，三宅雅之，Lin Kuan-Heng, 高見昇平, 加納ふみ，村田昌之，片平正人
  • Organizer: 第21回日本RNA学会年会
• [Presentation] The observation of in-cell NMR signals of nucleic acids in living human cells (2019)
  • Author(s): Y. Yamaoki, A. Kiyoishi, M. Miyake, K. H. Lin, F. Kano, M. Murata, T. Nagata, M. Katahira
  • Organizer: 7th International Meeting on Quadruplex Nucleic Acids
  • Int'l Joint Research
• [Presentation] Evaluation of the structure and dynamics of nucleic acids inside the living human cells by in-cell NMR spectroscopy (2019)
  • Author(s): Y. Yamaoki, T. Nagata, A. Kiyoishi, M. Miyake, F. Kano, M. Murata, M. Katahira
  • Organizer: 第57回日本生物物理学会年会
  • Invited
• [Presentation] Development of anti-prion RNA aptamers and destruction of the Alzheimer's disease-related complex (2019)
  • Author(s): T. Mashima, M. Iida, J. H. Lee, Y. Yamaoki, M. So, Y. O. Kamatari, T. Hayashi, M. Kinoshita, K. Kuwata, T. Nagata, M. Katahira
  • Organizer: International Symposium on Nucleic Acids Chemistry 2019(ISNAC2019)
  • Int'l Joint Research
• [Presentation] In-cell NMR法を用いたヒト生細胞内のDNAおよびRNAの構造安定性評価 (2019)
  • Author(s): 山置佑大，永田崇，高見昇平，阪本知樹，片平正人
  • Organizer: 第58回NMR討論会
• [Presentation] Promotor associated non-coding RNA, pncRNA, can diminish the aggregation of TLS caused by shearing stress (2019)
  • Author(s): N. Hamad, T. Mashima, Y. Yamaoki, K. Kondo, R. Yoneda, R. Kurokawa, T. Nagata, M. Katahira
  • Organizer: 第42回分子生物学会
• [Presentation] In-cell NMR法によるヒト生細胞内の核酸の構造およびダイナミクスの解析 (2019)
  • Author(s): 山置佑大, 永田崇, 阪本知樹, 高見昇平, 加納ふみ, 村田昌之, 片平正人
  • Organizer: 第42回分子生物学会
• [Presentation] In-cell NMR法によるヒト生細胞内における核酸‐リガンド相互作用の解析 (2019)
  • Author(s): 高見昇平, 山置佑大, 加納ふみ，村田昌之，坂本秦一，原田和雄，永田崇, 片平正人
  • Organizer: 第42回分子生物学会
• [Presentation] ヒト生細胞内における核酸の非標準構造および構造安定性のin-cell NMR法を用いた解析 (2019)
  • Author(s): 阪本知樹, 山置佑大, 加納ふみ，村田昌之，永田崇, 片平正人
  • Organizer: 第42回分子生物学会
• [Presentation] In-cell NMR法によるヒト細胞内DNAおよびRNAの観測 (2018)
  • Author(s): 山置佑大
  • Organizer: 第2回長鎖非コードRNA勉強会
• [Presentation] In-cell NMR法によるヒト細胞内DNAおよびRNAの観測 (2018)
  • Author(s): 山置佑大
  • Organizer: 第19回若手NMR研究会
  • Invited
• [Presentation] Conformational change detection of translocated in liposarcoma, TLS, upon binding to promoter-associated non-coding RNA, pncRNA (2018)
  • Author(s): Nesreen Hamad, Tsukasa Mashima, Yudai Yamaoki, Keiko Kondo, Hiroki Watanabe, Takayuki Uchihashi, Riki Kurokawa, Takashi Nagata, Masato Katahira
  • Organizer: International Conference on Biochemistry, Molecular Biology and Biotechnology 2018 (ICBMBB2018)
  • Int'l Joint Research
• [Presentation] Observation of imino proton signals of DNA and RNA introduced inside the living human cells by using in-cell NMR spectroscopy (2018)
  • Author(s): Yudai Yamaoki, Takashi Nagata, Ayaka Kiyoishi, Masayuki Miyake, Fumi Kano, Masayuki Murata, Masato Katahira
  • Organizer: XXVIII International Conference on Magnetic Resonance in Biological Systems (ICMRBS2018)
  • Int'l Joint Research
• [Presentation] In-cell NMR studies on structure and dynamics of DNA and RNA introduced inside the living human cells (2018)
  • Author(s): Yudai Yamaoki, Takashi Nagata, Ayaka Kiyoishi, Masayuki Miyake, Kuan-Heng Lin, Shohei Takami, Fumi Kano, Masayuki Murata, Masato Katahira
  • Organizer: The 45th International Symposium on Nucleic Acids Chemistry (ISNAC2018)
  • Int'l Joint Research
• [Presentation] In-cell NMR法を用いたヒト生細胞内のDNAおよびRNAの構造とダイナミクスの解析 (2018)
  • Author(s): 山置佑大, 永田崇, 清石彩華, 三宅雅之, 加納ふみ, 村田昌之, 片平正人
  • Organizer: 第57回NMR討論会
• [Presentation] In-cell NMR法によるヒト生細胞内環境下の核酸の解析 (2018)
  • Author(s): 山置佑大, 永田崇, 清石彩華, 三宅雅之, Lin Kuan-Heng, 高見昇平, 加納ふみ, 村田昌之, 片平正人
  • Organizer: 核酸化学若手フォーラム2018
• [Presentation] In-cell NMR analysis for structure and dynamics of nucleic acids introduced inside the living human cells (2018)
  • Author(s): Yudai Yamaoki, Takashi Nagata, Ayaka Kiyoishi, Masayuki Miyake, Kuan-Heng Lin, Shohei Takami, Fumi Kano, Masayuki Murata, Masato Katahira
  • Organizer: The 41st Annual Meeting of the Molecular Biology Society of Japan
• [Presentation] Evaluation of the structural stability and dynamics of nucleic acids inside the living cells by using in-cell NMR spectroscopy (2018)
  • Author(s): Takashi Nagata, Yudai Yamaoki, Masato Katahira
  • Organizer: The 18th Annual Meeting of the Protein Science Society of Japan
  • Invited
• [Presentation] Evaluation of the structural stability and dynamics of nucleic acids inside the living cells by using in-cell NMR spectroscopy (2018)
  • Author(s): Nagata Takashi、Yamaoki Yudai、Katahira Masato
  • Organizer: The 18th Annual Meeting of the Protein Science Society of Japan
• [Presentation] In-cell NMR法によるヒト細胞内DNAおよびRNAの観測 (2017)
  • Author(s): 山置佑大、清石彩華、三宅雅之、加納ふみ、村田昌之、永田崇、片平正人
  • Organizer: 第56回 NMR討論会
• [Presentation] Interaction of a cyclic tetraoxazole with i-motif DNA and its effect on this structure (2017)
  • Author(s): Masoud Shadi Sedghi、Yamaoki Yudai、Ma Yue、Katahira Masato、Nagasawa Kazuo
  • Organizer: The 44rd International Symposium on Nucleic Acids Chemistry (ISNAC)
  • Int'l Joint Research
• [Presentation] An anti-prion aptamer inhibits the formation of prion protein-amyloid β oligomer complex that is related to Alzheimer's disease (2017)
  • Author(s): Iida Mamiko、Mashima Tsukasa、Yamaoki Yudia、Nagata Takashi、Katahira Masato
  • Organizer: 第55回 日本生物物理学会年会
• [Presentation] DNA中のT-Tミスマッチ構造を選択的にアルキル化する小分子の開発 (2017)
  • Author(s): 鬼塚和光、宇佐美彬、山置佑大、小林倫仁、Madoka Eurika Hazemi、千国友子、片平正人、永次史
  • Organizer: 第11回バイオ関連化学シンポジウム
• [Presentation] Analysis of conformational changes of translocated in liposarcoma, TLS, upon binding to promoter-associated non-coding RNA, pncRNA (2017)
  • Author(s): Hamad Nesreen、Mashima Tsukasa、Yamaoki Yudai、Kondo Keiko、Nagata Takashi、Katahira Masato
  • Organizer: 3rd International Conference on New Horizons in Basic and Applied Science
  • Int'l Joint Research
• [Presentation] Invention of RNA aptamer and ribozyme whose activities switch on in response to potassium ion via G-quadruplex formation (2017)
  • Author(s): Yamaoki Yudai、Mashima Tsukasa、Nagata Takashi、Katahira Masato
  • Organizer: 6th International Meeting on Quadruplex Nucleic Acids
  • Int'l Joint Research
• [Presentation] Synthesis of cyclic polyoxazole compound as i-motif binder (2017)
  • Author(s): Masoud Shadi Sedghi、Ma Yue、Marchand Adrien、Gabelica Valerie、Yamaoki Yudai、Katahira Masato、Nagasawa Kazuo
  • Organizer: 6th International Meeting on Quadruplex Nucleic Acids
  • Int'l Joint Research
• [Presentation] Development of K+-responsive RNA aptamer and ribozyme, and in-cell NMR of nucleic acids in human cells (2017)
  • Author(s): Yamaoki Yudai、Kiyoishi Ayaka、Mashima Tsukasa、Kano Fumi、Murata Masayuki、Nagata Takashi、Katahira Masato
  • Organizer: International Symposium for NMR of Nucleic Acids
  • Int'l Joint Research / Invited
• [Remarks] 京都大学エネルギー理工学研究所 エネルギー構造生命科学研究分野 片平研究室
  • URL: http://www.iae.kyoto-u.ac.jp/bio/