Modular RNA engineering of RNA molecular switches (riboswitches)

• Project/Area Number: 23310161
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Chemical biology
• Research Institution: University of Toyama (2013); Kyushu University (2011-2012)
• Principal Investigator: IKAWA YOSHIYA 富山大学, 理工学研究部(理学), 教授 (70281087)
• Co-Investigator(Renkei-kenkyūsha): FURUTA Hiroyuki 九州大学, 大学院・工学研究院, 教授 (40244157)
• Project Period (FY): 2011-04-01 – 2014-03-31
• Project Status: Completed (Fiscal Year 2013)
• Budget Amount: ¥14,040,000 (Direct Cost: ¥10,800,000、Indirect Cost: ¥3,240,000); Fiscal Year 2013: ¥2,730,000 (Direct Cost: ¥2,100,000、Indirect Cost: ¥630,000); Fiscal Year 2012: ¥2,730,000 (Direct Cost: ¥2,100,000、Indirect Cost: ¥630,000); Fiscal Year 2011: ¥8,580,000 (Direct Cost: ¥6,600,000、Indirect Cost: ¥1,980,000)
• Keywords: リボスイッチ / RNA / モジュール工学 / ケミカルバイオロジー / リボザイム / シンセティック・バイオロジー / 合成生物学 / シンセフィック・バイオロジー

Research Abstract

Riboswitches are attractive not only as drug targets for novel antibiotics but also as modular tools for controlling gene expression. Riboswitches frequently have GNRA loop/receptor interaction modules in thier aptamer modules. In this study, the functional importance of the GAAA loop-receptor interaction in the c-diGMP riboswitch was examined. A series of variant c-diGMP riboswitches with mutations in the GAAA loop/receptor interaction were assayed for their switching abilities. These analyses strongly suggested that the GAAA loop/receptor interaction does not simply establish the RNA 3D structure but docking of P2 GAAA loop rigidifies the P3 element. This mechanism was applied to design an artificial riboswitch bearing a theophylline aptamer module in P3 the structural rigidity of which could be modulated by the small molecule theophylline. We also generated a new class of aptamer module that specifically recognizes a GAAC RNA tetraloop by employing in vitro directed evolution.

Research Products

• [Journal Article] Installation of orthogonality to the interface that assembles two modular domains in the Tetrahymena group I ribozyme. (2014)
  • Author(s): T. Tanaka, H. Furuta, and Y. Ikawa
  • Journal Title: J. Biosci. Bioeng. Volume: 117 Issue: 4 Pages: 407-411
  • DOI: 10.1016/j.jbiosc.2013.10.008
  • NAID: 110009817054
  • Peer Reviewed
• [Journal Article] Fixation and accumulation of thermotolerant catalytic competence of a pair of ligase ribozymes through complex formation and cross ligation (2013)
  • Author(s): N. Isomoto, Y. Maeda, T. Tanaka, H. Furuta & Y. Ikawa
  • Journal Title: J. Mol. Evol Volume: 76 Pages: 48-58
  • Peer Reviewed
• [Journal Article] An in vitro selected RNA receptor for the GAAC loop : modular receptor for non-GNRA-type tetraloop (2013)
  • Author(s): J. Ishikawa, H. Furuta, & Y. Ikawa
  • Journal Title: Nucleic Acids Res Volume: 41 Pages: 3748-3759
  • Peer Reviewed
• [Journal Article] Colorimetric/fluorogenic detection of thiols by N-fused porphyrin in water (2013)
  • Author(s): Y. Ikawa, S. Touden, S. Katsumata, & H. Furuta
  • Journal Title: Bioorg. Med. Chem Volume: 21 Pages: 6501-6505
  • Peer Reviewed
• [Journal Article] RNA Tectonics (tectoRNA) for RNA nanostructure design and its application in synthetic biology (2013)
  • Author(s): J. Ishikawa, H. Furuta, & Y. Ikawa
  • Journal Title: WIREs RNA Volume: 4 Pages: 651-664
  • Peer Reviewed
• [Journal Article] Colorimetric/fluorogenic detection of thiols by N-fused porphyrin in water. (2013)
  • Author(s): Y. Ikawa, S. Touden, S. Katsumata, Y. Ikawa and H. Furuta
  • Journal Title: Bioorg. Med. Chem. Volume: 21 Issue: 21 Pages: 6501-6505
  • DOI: 10.1016/j.bmc.2013.08.037
  • Peer Reviewed
• [Journal Article] RNA Tectonics (tectoRNA) for RNA nanostructure design and its application in synthetic biology. (2013)
  • Author(s): J. Ishikawa, H. Furuta, and Y. Ikawa
  • Journal Title: WIREs RNA Volume: 4 Issue: 6 Pages: 651-664
  • DOI: 10.1002/wrna.1185
  • Peer Reviewed
• [Journal Article] Fixation and accumulation of thermotolerant catalytic competence of a pair of ligase ribozymes through complex formation and cross ligation. (2013)
  • Author(s): N. Isomoto, Y. Maeda, T. Tanaka, H. Furuta and Y. Ikawa
  • Journal Title: J. Mol. Evol. Volume: 76 Issue: 1-2 Pages: 48-58
  • DOI: 10.1007/s00239-012-9536-x
  • Peer Reviewed
• [Journal Article] An in vitro-Selected RNA Receptor for the GAAC loop : Modular Receptor for Non-GNRA-Type Tetraloop (2013)
  • Author(s): Junya Ishikawa
  • Journal Title: Nucleic Acids Research Volume: (in press) Issue: 6 Pages: 3748-3759
  • DOI: 10.1093/nar/gkt040
  • Peer Reviewed
• [Journal Article] Functional roles of a tetraloop/receptor interacting module in a cyclic-di-GMP riboswitch (2012)
  • Author(s): Y. Fujita, T. Tanaka, H. Furuta, & Y. Ikawa
  • Journal Title: J. Biosci. Bioeng Volume: 113 Pages: 141-145
  • NAID: 110009418659
  • Peer Reviewed
• [Journal Article] TectoRNP : self-assembling RNAs with peptide-recognition motifs as templates for chemical peptide ligation. TectoRNP: self-assembling RNAs with peptide-recognition motifs as templates for chemical peptide ligation (2012)
  • Author(s): K. Yamashita, T. Tanaka, H. Furuta & Y. Ikawa
  • Journal Title: J. Pep. Sci Volume: 18 Pages: 635-642
  • Peer Reviewed
• [Journal Article] A two-piece derivative of a group I intron RNA as a platform for designing self-assembling RNA templates to promote peptide ligation (2012)
  • Author(s): T. Tanaka, H. Furuta & Y. Ikawa
  • Journal Title: J. Nucleic Acids Volume: vol. 2012 Pages: 10-10
  • DOI: 10.1155/2012/305867
  • Peer Reviewed
• [Journal Article] RNA立体構造のボトムアップ構築とリボザイム・リボスイッチの人工改変 (2012)
  • Author(s): T. Tanaka, H. Furuta, & Y. Ikawa
  • Journal Title: Antisense Volume: 16 Pages: 13-23
• [Journal Article] TectoRNP: self-assembling RNAs with peptide-recognition motifs as templates for chemical peptide ligation. (2012)
  • Author(s): K. Yamashita, T. Tanaka, H. Furuta and Y. Ikawa
  • Journal Title: J. Pep. Sci. Volume: 18 Issue: 10 Pages: 635-642
  • DOI: 10.1002/psc.2444
  • Peer Reviewed
• [Journal Article] Functional roles of a tetraloop/receptor interacting module in a cyclic-di-GMP riboswitch (2012)
  • Author(s): Y.Fujita, T.Tanaka, H.Furuta, Y.Ikawa
  • Journal Title: J.Biosci.Bioeng. Volume: 113 Issue: 2 Pages: 141-145
  • DOI: 10.1016/j.jbiosc.2011.10.004
  • NAID: 110009418659
  • Peer Reviewed
• [Journal Article] Turnover ability of a designed RNA acting as a template for chemical peptide ligation (2011)
  • Author(s): K. Yamashita, N. Kashiwagi, H. Furuta & Y. Ikawa
  • Journal Title: Biosci. Biotech. Biochem Volume: 75 Pages: 2021-2024
  • NAID: 10029873198
  • Peer Reviewed
• [Journal Article] N-fused porphyrin with pyridinium side-arms : A new class of aromatic ligand with DNA-binding ability (2011)
  • Author(s): Y. Ikawa, S. Touden & H. Furuta
  • Journal Title: Org. Biomol. Chem Volume: 9 Pages: 8068-8078
  • NAID: 120005227444
  • Peer Reviewed
• [Presentation] Formation of a catalytic supramolecular RNA 1D-array through self-assembly of an engineered group I intron RNA enzyme (2013)
  • Author(s): Narumi Uehara, Hiroyuki Furuta, Yoshiya Ikawa
  • Organizer: RNA 2013 The 18th Annual Meeting of RNA society
  • Place of Presentation: スイス
• [Presentation] Self-dimerizing group I ribozymes as a new class of modular units for RNA synthetic biology (2013)
  • Author(s): Takahiro Tanaka, Hiroyuki Furuta, Yoshiya Ikawa
  • Organizer: RNA 2013 The 18th Annual Meeting of RNA society
  • Place of Presentation: スイス
• [Presentation] c-di-GMP応答型リボスイッチの発現調節機構の解明 (2013)
  • Author(s): 西村圭一郎, 古田弘幸, 井川善也
  • Organizer: 第50回化学関連支部合同九州大会
  • Place of Presentation: 北九州
• [Presentation] An in vitro-selected RNA receptor for the GAAC loop: The first modular receptor for non-GNRA-type tetraloop (2013)
  • Author(s): Yoshiya Ikawa
  • Organizer: 2nd Meeting on RNA and Biofunctions-Asia Study
  • Place of Presentation: Fukuoka
  • Invited
• [Presentation] UCG ループを認識する人工RNA レセプターの進化工学的創製 (2012)
  • Author(s): 冨永雄人・古田弘幸・井川善也
  • Organizer: 第35回日本分子生物学会年会
  • Place of Presentation: 福岡
• [Presentation] 二分子型グループI イントロンを用いた自己組織型ペプチド連結の鋳型RNA (2012)
  • Author(s): 田中貴大, 古田弘幸, 井川善也
  • Organizer: 第35回日本分子生物学会年会
  • Place of Presentation: 福岡
• [Presentation] AUCGループを認識する人工RNAレセプターの進化工学的創製 (2012)
  • Author(s): 冨永雄人、古田弘幸、井川善也
  • Organizer: RNAフロンティアミーティング2012
  • Place of Presentation: 熊本
• [Presentation] AUCGループを認識する人工RNAレセプターの進化工学的創製 (2012)
  • Author(s): 冨永雄人、古田弘幸、井川善也
  • Organizer: 第35回日本分子生物学会年会
  • Place of Presentation: 福岡
• [Presentation] Modular Engineering of a RNA molecular switch controlled by c-diGMP (2011)
  • Author(s): Yoshiya Ikawa
  • Organizer: RNAアジア会議(RM Asia Studies)
  • Place of Presentation: 台北(台湾)(招待講演)
  • Year and Date: 2011-11-12
• [Presentation] 人工RNA創製における二刀流のススメ:分子デザインと進化工学 (2011)
  • Author(s): 井川善也
  • Organizer: 生体機能関連化学部会若手の会第23回サマースクール
  • Place of Presentation: 広島(招待講演)
  • Year and Date: 2011-07-22
• [Presentation] Functional roles of a conserved GAAA/receptor interaction in a cyclic di-GMP riboswitch (2011)
  • Author(s): Yoshiya Ikawa, et al
  • Organizer: RNA 2011
  • Place of Presentation: Kyoto
  • Year and Date: 2011-06-15
• [Presentation] Construction of a peptide ligation system based on a bimolecular derivative of the Tetrahymena group I intron (2011)
  • Author(s): 田中貴大・古田弘幸・井川善也
  • Organizer: 第16回日本RNA学会
  • Place of Presentation: 京都
• [Presentation] In vitro selection and characterization of a receptor for GAAC loop (2011)
  • Author(s): 石川隼也・古田弘幸・井川善也
  • Organizer: 第16回日本RNA学会
  • Place of Presentation: 京都
• [Presentation] 二分子型グループI イントロンを基盤としたペプチド連結システム (2011)
  • Author(s): 田中貴大・古田弘幸・井川善也
  • Organizer: 第48回化学関連支部合同九州大会
  • Place of Presentation: 北九州
• [Presentation] An RNA-assisted peptide ligation system based on a bimolecular derivative of the Tetrahymena group I intron (2011)
  • Author(s): 田中貴大・古田弘幸・井川善也
  • Organizer: The 38th International Symposium on Nucleic Acid Chemistry (ISNAC 2011)
  • Place of Presentation: 北海道
• [Presentation] c-di-GMP応答型リボスイッチの発現プラットフォームの機能解析
  • Author(s): 西村圭一郎, 古田弘幸, 井川善也
• [Presentation] Formation of a catalytic supramolecular RNA 1D-array through self-assembly of an engineered group I intron RNA enzyme
  • Author(s): Narumi Uehara, Hiroyuki Furuta, Yoshiya Ikawa
  • Organizer: The 18th Annual meeting of the RNA society
  • Place of Presentation: ダボス・スイス
• [Presentation] c-di-GMP応答型リボスイッチの発現調節機構の解明
  • Author(s): 西村圭一郎、古田弘幸、井川善也
  • Organizer: 第50回化学関連支部合同九州大会
  • Place of Presentation: 北九州
• [Presentation] c-di-GMP応答型リボスイッチの発現プラットフォームの機能解析
  • Author(s): 西村圭一郎、古田弘幸、井川善也
  • Organizer: 第15回日本RNA学会年会
  • Place of Presentation: 松山（愛媛）
• [Book] グループI イントロン研究の新たな潮流 : 機能構造と分子進化 (実験医学増刊) (2013)
  • Author(s): 井川善也
  • Publisher: 羊土社
• [Book] Natural selection and structural polymorphism of RNA 3D structures involving GNRA loops and their receptor motifs in RNA Nanotechnology and Therapeutics (ed. Guo, P.) (2013)
  • Author(s): T. Tanaka, H. Furuta & Y. Ikawa
  • Publisher: CRC press
• [Book] in vitro 進化分子工学による人工RNA 触媒の創製およびその機能解析と応用利用 (2013)
  • Author(s): 井川善也
  • Publisher: NTS出版
• [Book] RNA Nanotechnology and Therapeutics (Ed. Guo, P.) (2013)
  • Author(s): Tanaka, T. Furuta, H. and Ikawa, Y. (分担著者)
  • Total Pages: 611
  • Publisher: CRC Press (Florida, USA)
• [Book] モジュール工学と進化工学によるRNA 触媒の人工創製 (2011)
  • Author(s): 井川善也
  • Publisher: 化学同人
• [Remarks] HP
  • URL: http://www3.u-toyama.ac.jp/orgsyn3/