Structural probing of RNA molecules with in-cell NMR

• Project/Area Number: 24310163
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Partial Multi-year Fund
• Section: 一般
• Research Field: Chemical biology
• Research Institution: Tokushima Bunri University (2015); Tohoku University (2012-2014)
• Principal Investigator: TANAKA Yoshiyuki 徳島文理大学, 薬学部, 教授 (70333797)
• Co-Investigator(Kenkyū-buntansha): KOJIMA Chojiro 横浜国立大学, 大学院工学研究科, 教授 (50333563)
• Co-Investigator(Renkei-kenkyūsha): TOCHIO Hidehito 京都大学, 大学院理学研究科, 教授 (70336593) ; ONO Akira 神奈川大学, 工学部, 教授 (10183253)
• Project Period (FY): 2012-04-01 – 2016-03-31
• Project Status: Completed (Fiscal Year 2015)
• Budget Amount: ¥19,500,000 (Direct Cost: ¥15,000,000、Indirect Cost: ¥4,500,000); Fiscal Year 2014: ¥6,370,000 (Direct Cost: ¥4,900,000、Indirect Cost: ¥1,470,000); Fiscal Year 2013: ¥6,240,000 (Direct Cost: ¥4,800,000、Indirect Cost: ¥1,440,000); Fiscal Year 2012: ¥6,890,000 (Direct Cost: ¥5,300,000、Indirect Cost: ¥1,590,000)
• Keywords: 生体分子計測 / NMR / 安定同位体標識 / バイオプローブ / 細胞内化学反応

Outline of Final Research Achievements

In-cell NMR has been rarely applied to nucleic acids. In order to apply in-cell NMR into nucleic acids, there are several issues. 1) There has been no appropriate site-specific labeling technique of long RNA molecules. 2) There was no established method for introducing nucleic acids into cells. Therefore, at first, Tanaka (principal investigator: PI) developed a site-specific labeling technique of RNA molecules by using a recently discovered primer-extension ability of RNA polymerases. Interestingly, by using the derived labeled RNA molecule (HAC1 mRNA), PI identified that conserved C883 and G888 residues were base-paired within a loop. Then, in collaboration with grant team members, a method for introducing RNA molecules into cells was established by using an electroporation technique. It should be also mentioned that chemical reactions occurred to the introduced molecule was detected by using appropriate 19F-labeling tag. Thus, the team accomplished main purposes of this project.

Research Products

• [Int'l Joint Research] チェコ科学アカデミー(チェコ)
• [Int'l Joint Research] VU University Amsterdam(ドイツ)
• [Journal Article] Benchmark Theoretical and Experimental Study on 15N NMR Shifts of Oxidatively Damaged Guanine (2016)
  • Author(s): Martin Dracinsky, Michal Sala, Blanka Klepetarova, Jakub Sebera, Jiri Fukal, Veronika Holeckova, Yoshiyuki Tanaka, Radim Nencka, and Vladimir Sychrovsky
  • Journal Title: Journal of Physical Chemistry B Volume: 120 Pages: 915-925
  • DOI: 10.1021/acs.jpcb.5b11428
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Direct Detection of the Mercury-Nitrogen Bond in the Thymine-HgII-Thymine Base-pair with 199Hg NMR Spectroscopy (2015)
  • Author(s): Takenori Dairaku, Kyoko Furuita, Hajime Sato, Jakub Sebera, Daichi Yamanaka, Hiroyuki Otaki, Shoko Kikkawa, Yoshinori Kondo, Ritsuko Katahira, F. Matthias Bickelhaupt, Celia Fonseca Guerra, Akira Ono, Vladimir Sychrovsky, Chojiro Kojima and Yoshiyuki Tanaka
  • Journal Title: Chem. Commun. Volume: 51 Pages: 8488-8491
  • DOI: 10.1039/c5cc02423d
  • Peer Reviewed / Open Access / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] Exploring a DNA sequence for the three-dimensional structure determination of a silver(I)-mediated C-C base pair in a DNA duplex by 1H NMR spectroscopy (2015)
  • Author(s): Takenori Dairaku, Kyoko Furuita, Hajime Sato, Yoshinori Kondo, Chojiro Kojima, Akira Ono and Yoshiyuki Tanaka
  • Journal Title: Nucleosides Nucleotides Nucleic Acids Volume: 34 Pages: 877-890
  • DOI: 10.1080/15257770.2015.1088160
  • Peer Reviewed / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] High-resolution crystal structure of AgI-RNA hybrid duplex containing Watson-Crick-like C-AgI-C metallo-base pairs (2015)
  • Author(s): Jiro Kondo, Yoshinari Tada, Takenori Dairaku, Hisao Saneyoshi, Itaru Okamoto, Yoshiyuki Tanaka, Akira Ono
  • Journal Title: Angew. Chem. Int. Ed Volume: 54 Pages: 13323-13326
  • DOI: 10.1002/anie.201507894
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Structures, physicochemical properties, and applications of T-HgII-T, C-AgI-C, and other metallo-base-pairs. (2015)
  • Author(s): Yoshiyuki Tanaka, Jiro Kondo, Vladimir Sychrovsky, Jakub Sebera, Takenori Dairaku, Hisao Saneyoshi, Hidehito Urata, Hidetaka Torigoe and Akira Ono
  • Journal Title: Chem. Comm. Volume: 51 Pages: 17313-17360
  • DOI: 10.1039/c5cc02693h
  • Peer Reviewed / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] The activation of N-glycosidic bond cleavage operated by base-excision repair enzyme hOGG1; theoretical study of the role of Lys 249 residue in activation of G, OxoG and FapyG. (2014)
  • Author(s): Jakub Sebera, Lukas Trantirek, Yoshiyuki Tanaka, Radim Nencka, Jiri Fukal, Vladimir Sychrovsky
  • Journal Title: RSC Advances Volume: 4 Pages: 44043-44051
  • DOI: 10.1039/c4ra08278h
  • Peer Reviewed
• [Journal Article] Pseudoknot interaction-mediated activation of hammerhead ribozyme which is applicable to gene-therapeutic agents (2014)
  • Author(s): Mituhiro Kuriyama, Yoshinori Kondo, and Yoshiyuki Tanaka
  • Journal Title: Nucleosides, Nucleotides & Nucleic Acids Volume: (印刷中)(掲載確定) Pages: 466-480
  • DOI: 10.1080/15257770.2014.887098
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Hg²⁺-Trapping Beads: Hg²⁺-Specific Recognition through Thymine–Hg(II)–Thymine Base Pairing (2014)
  • Author(s): 栗山光博、他12名
  • Journal Title: CHEMICAL & PHARMACEUTICAL BULLETIN Volume: 62 Issue: 7 Pages: 709-12
  • DOI: 10.1248/cpb.c13-00918
  • NAID: 130004053812
  • ISSN: 0009-2363, 1347-5223
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Mechanism of base excision with the hOGG1 DNA repair enzyme. (2014)
  • Author(s): Jakub Sebera, Lukas Trantirek, Jiri Fukal, Yoshiyuki Tanaka and Vladimir Sychrovsky
  • Journal Title: Chemicke Listy Volume: 108 Pages: 364-367
  • Peer Reviewed
• [Journal Article] Theoretical and experimental study of charge transfer through DNA: impact of mercury mediated T-Hg-T base pair. (2014)
  • Author(s): Irena Kratochvilova, Martin Golan, Martin Vala, Miroslava Sperova, Martin Weiter, Ondrej Pav, Jakub Sebera, Ivan Rosenberg, Vladimir Sychrovsky, Yoshiyuki Tanaka and F. Matthias Bickelhaupt
  • Journal Title: The Journal of Physical Chemistry B Volume: 118 Pages: 5374-5381
  • DOI: 10.1021/jp501986a
  • Peer Reviewed
• [Journal Article] NMR spectroscopic studies on metallo-base-pair in DNA duplex. (2014)
  • Author(s): Yoshiyuki Tanaka, Itaru Okamoto, Kyoko Furuita, Jakub Sebera, Jiro Kondo, Hidetaka Torigoe, Hidehito Urata, Takenori Dairaku, Akira Ono, Chojiro Kojima, Vladimir Sychrovsky
  • Journal Title: Journal of Biological Inorganic Chemistry Volume: 19
  • DOI: 10.1007/s00775-014-1157-y
• [Journal Article] Syntheses of DNA duplexes containing metal ion mediated base pairs. (2014)
  • Author(s): Akira Ono, Itaru Okamoto, Hidehito Urata, Hidetaka Torigoe, Hisao Saneyoshi, Jiro Kondo, Yoshiyuki Tanaka
  • Journal Title: Journal of Biological Inorganic Chemistry Volume: 19
  • DOI: 10.1007/s00775-014-1156-z
• [Journal Article] Proton and nitrogen-15 NMR spectroscopic studies of AgI-mediated C-C base-pairs. (2014)
  • Author(s): Takenori Dairaku, Itaru Okamoto, Kyoko Furuita, Shuji Oda, Daichi Yamanaka, Hidetaka Torigoe, Tetsuo Kozasa, Yoshinori Kondo, Akira Ono, Chojiro Kojima, Vladimir Sychrovsky, Yoshiyuki Tanaka
  • Journal Title: Journal of Biological Inorganic Chemistry Volume: 19
  • DOI: 10.1007/s00775-014-1163-0
• [Journal Article] Crystal structure of metallo-DNA duplex containing consecutive Watson-Crick-like T-Hg(II)-T base pairs (2014)
  • Author(s): Jiro Kondo, Tom Yamada, Chika Hirose, Itaru Okamoto, Yoshiyuki Tanaka, Akira Ono
  • Journal Title: Angewandte Chemie International Edition Volume: 53 Pages: 2385-2388
  • DOI: 10.1002/anie.201309066
  • Peer Reviewed
• [Journal Article] The structure of metallo-DNA with consecutive T-HgII-T base-pairs explains positive entropy for the metallo-base-pair formation (2014)
  • Author(s): Yamaguchi H
  • Journal Title: Nucleic Acids Research Volume: 42 Pages: 4094-4099
  • DOI: 10.1093/nar/gkt1344
  • Peer Reviewed
• [Journal Article] Formation of the T-HgII-T Metal-Mediated DNA Base Pair; Proposal and Theoretical Calculation of the Reaction Pathway (2013)
  • Author(s): Sebera J
  • Journal Title: Chemistry - A European Journal Volume: 19 Pages: 9884-9894
  • DOI: 10.1002/chem.201300460
  • Peer Reviewed
• [Journal Article] Organocatalytic functionalization of heteroaromatic N-oxides with C-nucleophiles using in situ generated onium amide bases (2013)
  • Author(s): Kiyofumi Inamoto, Yuta Araki, Shoko Kikkawa, Misato Yonemoto, Yoshiyuki Tanaka, Yoshinori Kondo
  • Journal Title: Organic & Biomolecular Chemistry Volume: 11 Pages: 3773-3775
  • DOI: 10.1039/c3ob40782a
  • Peer Reviewed
• [Journal Article] Carboxylation of alkynylsilanes with carbon dioxide mediated by cesium fluoride in DMSO (2013)
  • Author(s): Misato Yonemoto-Kobayashi, Kiyofumi Inamoto, Yoshiyuki Tanaka and Yoshinori Kondo
  • Journal Title: Organic & Biomolecula Chemistry Volume: 11 Pages: 3773-3775
  • DOI: 10.1039/c3ob40760h
  • Peer Reviewed
• [Journal Article] Structure Formation and Catalytic Activity of DNA in Organic Solvents (2013)
  • Author(s): Hiroshi Abe, Naoko Abe, Aya Shibata, Keiji Ito, Yoshiyuki Tanaka, Mika Ito, Hisao Saneyoshi, Satoshi Shuto, and Yoshihiro Ito
  • Journal Title: Chem. Int. Ed Volume: 51 Pages: 6475-6479
  • DOI: 10.1002/anie.201201111
  • Peer Reviewed
• [Journal Article] Raman spectroscopic detection of the T-HgII-T base pair and the ionic characteristics of mercury (2013)
  • Author(s): Tomomi Uchiyama, Takashi Miura, Hideo Takeuchi, Takenori Dairaku, Tomoyuki Komuro, Takuya Kawamura, Yoshinori Kondo, Ladislav Benda, Vladimir Sychrovsky, Petr Bour, Itaru Okamoto, Akira Ono and Yoshiyuki Tanaka
  • Journal Title: Nucleic Acids Res. Volume: 40 Pages: 5766-5774
  • DOI: 10.1093/nar/gks208
  • Peer Reviewed
• [Journal Article] Detection of Mercury-TpT Dinucleotide Binding by the Raman Spectra. A Computational Study (2013)
  • Author(s): Ladislav Benda, 他4名(最終著者)
  • Journal Title: The Journal of Physical Chemisty A Volume: 116 Pages: 8313-8320
  • DOI: 10.1021/jp3045077
  • Peer Reviewed
• [Journal Article] Thermodynamic and structural properties of the specific binding between Ag+ ion and C:C mismatched base pair in duplex DNA to form C-Ag-C metal-mediated base pair (2013)
  • Author(s): Hidetaka Torigoe
  • Journal Title: Biochimie, Volume: 94 Pages: 2431-2440
  • DOI: 10.1016/j.biochi.2012.06.024
  • Peer Reviewed
• [Journal Article] Pyramidalization of the Glycosidic Nitrogen Provides the Way for Efficient Cleavage of the N-glycosidic Bond of 8-OxoG with the hOGGl DNA Repair Protein (2013)
  • Author(s): Jakub Sebera, Jakub Sebera, Lukas Tranti'rek, 田中 好幸, Vladimir Sychrovsky
  • Journal Title: The Journal of Physical Chemistry B Volume: 116 Pages: 12535-12544
  • DOI: 10.1021/jp309098d
  • Peer Reviewed
• [Presentation] C-Ag(I)-C塩基対を含むメタロDNAの立体構造解析 (2015)
  • Author(s): 古板 恭子, 大樂 武範, 児嶋 長次郎, 田中 好幸
  • Organizer: 第54回NMR討論会
  • Place of Presentation: 習志野市
  • Year and Date: 2015-11-06
• [Presentation] Structure of C-Ag(I)-C metallo base pair in RNA duplex (2015)
  • Author(s): Yoshinari Tada, Takenori Dairaku, Itaru Okamoto, Hisao Saneyoshi, Yoshiyuki Tanaka, Akira Ono, Jiro Kondo
  • Organizer: 第42回国際核酸化学シンポジウム
  • Place of Presentation: 姫路
  • Year and Date: 2015-09-23
  • Int'l Joint Research
• [Presentation] NMR structure of a DNA duplex containing a silver-mediated C-C base pair (2015)
  • Author(s): Kyoko Furuita, Takenori Dairaku, Chojiro Kojima, Yoshiyuki Tanaka
  • Organizer: 第42回国際核酸化学シンポジウム
  • Place of Presentation: 姫路
  • Year and Date: 2015-09-23
  • Int'l Joint Research
• [Presentation] NMR spectroscopic studies on metallo-base-pair in DNA duplex. (2014)
  • Author(s): ◯Yoshiyuki Tanaka, Itaru Okamoto, Kyoko Furuita, Jakub Sebera, Jiro Kondo, Hidetaka Torigoe, Hidehito Urata, Takenori Dairaku, Akira Ono, Chojiro Kojima, Vladimir Sychrovsky
  • Organizer: 12th European Biological Inorganic Chemistry Conference (EuroBIC-12)
  • Place of Presentation: チューリッヒ、スイス
  • Year and Date: 2014-08-24 – 2014-08-28
  • Invited
• [Presentation] Syntheses of DNA duplexes containing metal ion mediated base pairs (2014)
  • Author(s): ◯Akira Ono, Itaru Okamoto, Hidehito Urata, Hidetaka Torigoe, Hisao Saneyoshi, Jiro Kondo, Yoshiyuki Tanaka
  • Organizer: 12th European Biological Inorganic Chemistry Conference (EuroBIC-12)
  • Place of Presentation: チューリッヒ、スイス
  • Year and Date: 2014-08-24 – 2014-08-28
  • Invited
• [Presentation] Proton and nitrogen-15 NMR spectroscopic studies of AgI-mediated C-C base-pairs. (2014)
  • Author(s): ◯Takenori Dairaku, Itaru Okamoto, Kyoko Furuita, Shuji Oda, Daichi Yamanaka, Hidetaka Torigoe, Tetsuo Kozasa, Yoshinori Kondo, Akira Ono, Chojiro Kojima, Vladimir Sychrovsky, Yoshiyuki Tanaka
  • Organizer: 12th European Biological Inorganic Chemistry Conference (EuroBIC-12)
  • Place of Presentation: チューリッヒ、スイス
  • Year and Date: 2014-08-24 – 2014-08-28
• [Presentation] 多核NMR分光法による機能性核酸分子の局所観測およびJ値・化学シフト値による状態解析 (2014)
  • Author(s): 田中好幸
  • Organizer: 第15回若手NMR研究会
  • Place of Presentation: 千葉県長生郡長柄町
  • Year and Date: 2014-07-12
  • Invited
• [Presentation] 化学シフト値・結合定数J（金属錯体の配位化学・構造化学） (2013)
  • Author(s): 田中好幸
  • Organizer: 日本分光学会NMR部会2013年NMR講習会
  • Place of Presentation: 名古屋
  • Invited
• [Presentation] 水銀イオンのT-Tミスマッチ塩基対に対する親和性の構造化学的要因（口頭発表） (2013)
  • Author(s): ◯田中好幸, 大樂武範, 根東義則, 内山朋美, 三浦隆史, 竹内英夫, 古板恭子, 児嶋長次郎, Ladislav Benda, Jakub Sebera, Vladimir Sychrovsky, 岡本到, 小野晶
  • Organizer: 第回 生体分子科学討論会
  • Place of Presentation: 大阪
• [Presentation] DNAを用いた水銀除去剤の研究（口頭発表） (2013)
  • Author(s): ◯栗山 光博、春田 佳一郎、小野 晶、森田 勇人、根東 義則、田中 好幸
  • Organizer: 第7回 バイオ関連シンポジウム
  • Place of Presentation: 名古屋
• [Presentation] 金属内包DNAナノワイヤー創成に向けたメタロ塩基対の構造解析（口頭発表） (2013)
  • Author(s): ◯大樂武範、岡本到、古板恭子、Vladimir Sychrovsky、山中大地、織田修司、児嶋長次郎、小野晶、根東義則、田中好幸
  • Organizer: 第51回 日本薬学会東北支部大会
  • Place of Presentation: 仙台
• [Presentation] 部位特異的RNA切断酵素Ire1pによるHAC1 mRNA 切断機構の解析 (2013)
  • Author(s): ◯河原郁美、芦原悠太、春田佳一郎、児嶋長次郎、田中好幸
  • Organizer: 第51回 日本生物物理学会年会
  • Place of Presentation: 京都
• [Presentation] Structure of Ag(I)-mediated C-C base pair determined by hetero-nuclear NMR spectroscopy (2013)
  • Author(s): Takenori Dairaku, Itaru Okamoto, Kyoko Furuita, Jukub Sebera, Mathias Bickelhaupt, Shuji Oda, Daichi Yamanaka, Yoshinori Kondo, Vladimir Sychrovsky, Chojiro Kojima, Akira Ono, ◯Yoshiyuki Tanaka
  • Organizer: 40th International Symposium on Nucleic Acids Chemistry
  • Place of Presentation: 横浜
• [Presentation] Construction of expression system of human 8-oxoguanine glycosylase 1 for its mechanistic analysis (2013)
  • Author(s): ◯Daichi Sato, Momoko Yoneyama, Ikumi Kawahara, Kyoko Fruita, Yoshinori Kondo, Chojiro Kojima, Yoshiyuki Tanaka
  • Organizer: 40th International Symposium on Nucleic Acids Chemistry
  • Place of Presentation: 横浜
• [Presentation] Hammerhead ribozyme which is responsible for a point mutation in a substrate RNA (2013)
  • Author(s): ◯Mituhiro Kuriyama, Hisaaki Tateoka, Yoshinori Kondo, Yoshio Kato, and Yoshiyuki Tanaka
  • Organizer: 40th International Symposium on Nucleic Acids Chemistry
  • Place of Presentation: 横浜
• [Presentation] NMR analysis of HAC1 mRNA using site-specific isotope labeling (2013)
  • Author(s): ◯Ikumi Kawahara, Yuta Ashihara, Kaichiro Haruta, Yoshiyuki Tanaka and Chojiro Kojima
  • Organizer: 40th International Symposium on Nucleic Acids Chemistry
  • Place of Presentation: 横浜
• [Presentation] Site-specific labeling techniques of RNA molecules for structural and mechanistic studies with NMR spectroscopy (2012)
  • Author(s): 田中好幸, 他6名
  • Organizer: 39th International Symposium on Nucleic Acids Chemistry
  • Place of Presentation: 名古屋, 日本(国際会議)(口頭発表)
  • Year and Date: 2012-11-17
• [Presentation] 高分子量機能性RNAの局所構造・状態解析のため部位特異的標識法 (2012)
  • Author(s): 田中好幸, 他4名
  • Organizer: 第51回NMR討論会
  • Place of Presentation: 名古屋(口頭発表)
  • Year and Date: 2012-11-10
• [Presentation] NMR分光法を利用したHammerhead Ribozymeの活性残基の物性解析 (2012)
  • Author(s): 山中大地, 他3名
  • Organizer: 第51回日本薬学会東北支部大会
  • Place of Presentation: 青森(口頭発表)
  • Year and Date: 2012-10-07
• [Presentation] シュードノット構造を持つハンマーヘッド型リボザイムを利用した遺伝子治療薬創製研究 (2012)
  • Author(s): 栗山光博, 他4名
  • Organizer: アンチセンス・遺伝子・デリバリーシンポジウム
  • Place of Presentation: 仙台(ポスター)
  • Year and Date: 2012-09-17
• [Presentation] NMR分光法によるRNA分子の局所構造プローブ法の開発 (2012)
  • Author(s): 芦原悠太, 他7名
  • Organizer: 14th RNA meeting in Tohoku
  • Place of Presentation: 仙台(ポスター)
  • Year and Date: 2012-07-19
• [Presentation] スプライソソーム非依存的スプライシングを可能にするHACL mRNAの構造特性 (2012)
  • Author(s): 河原郁美, 他4名
  • Organizer: 14th RNA meeting in Tohoku
  • Place of Presentation: 仙台(ポスター)
  • Year and Date: 2012-07-18
• [Presentation] 小胞体ストレスセンサーIrelpにより細胞質スプライシングを受けるHACL mRNAの構造解析 (2012)
  • Author(s): 田中好幸, 他4名
  • Organizer: 第39回生体分子科学討論会
  • Place of Presentation: 仙台(口頭発表)
  • Year and Date: 2012-06-09