Design of L-DNA/SNA circuit for detection of RNA in cell with high sensitivity and accuracy

• Project/Area Number: 17K14514
• Research Category: Grant-in-Aid for Young Scientists (B)
• Allocation Type: Multi-year Fund
• Research Field: Bio-related chemistry
• Research Institution: Nagoya University
• Principal Investigator: Murayama Keiji 名古屋大学, 工学研究科, 助教 (70779595)
• Project Period (FY): 2017-04-01 – 2020-03-31
• Project Status: Completed (Fiscal Year 2019)
• Budget Amount: ¥4,420,000 (Direct Cost: ¥3,400,000、Indirect Cost: ¥1,020,000); Fiscal Year 2019: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000); Fiscal Year 2018: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000); Fiscal Year 2017: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
• Keywords: 人工核酸 / シグナル増幅 / RNA検出 / SNA / L-DNA / DNA / 直交性 / 増幅回路 / キラリティ伝播 / 修飾核酸塩基 / 核酸 / バイオテクノロジー / 蛍光性プローブ

Outline of Final Research Achievements

We attempted to develop the RNA detection system achieved by transfer of RNA information into L-DNA, mirror of natural DNA, and amplified the fluorescent signal. As a result, we successfully establish RNA detection without inhibition by contaminating and nucleases. We also found that L-DNA-bound SNA induced left-handed helicity that significantly reduced hybridization of SNA with right-handed natural nucleic acids.

Academic Significance and Societal Importance of the Research Achievements

従来のRNAの蛍光検出法では、細胞内に夾雑する非標的核酸や核酸分解酵素によって正確な検出が困難であった。本研究で開発した手法では、非標的核酸や核酸分解酵素による影響を受けず細胞内のRNAを正確かつ高感度で検出する手法として有力である。
また、核酸の巻き方向が一本鎖部分に伝播することで結合力が変化する現象はこれまでに報告例がなく、学術的に全く新しい知見であるため、今後様々な研究へと展開されることが期待できる。

Research Products

• [Journal Article] A triplex-forming linear probe for sequence-specific detection of duplex DNA with high sensitivity and affinity (2020)
  • Author(s): Chen Yanglingzhi、Murayama Keiji、Kashida Hiromu、Kamiya Yukiko、Asanuma Hiroyuki
  • Journal Title: Chemical Communications Volume: 印刷中 Issue: 40 Pages: 5358-5361
  • DOI: 10.1039/d0cc01865a
  • Peer Reviewed
• [Journal Article] Incorporation of Pseudo‐complementary Bases 2,6‐Diaminopurine and 2‐Thiouracil into Serinol Nucleic Acid (SNA) to Promote SNA/RNA Hybridization (2020)
  • Author(s): Kamiya Yukiko、Sato Fuminori、Murayama Keiji、Kodama Atsuji、Uchiyama Susumu、Asanuma Hiroyuki
  • Journal Title: Chemistry ? An Asian Journal Volume: 15 Issue: 8 Pages: 1266-1271
  • DOI: 10.1002/asia.201901728
  • Peer Reviewed
• [Journal Article] A Quencher‐Free Linear Probe from Serinol Nucleic Acid with a Fluorescent Uracil Analogue (2019)
  • Author(s): Murayama Keiji、Asanuma Hiroyuki
  • Journal Title: ChemBioChem Volume: 21 Issue: 1-2 Pages: 120-128
  • DOI: 10.1002/cbic.201900498
  • Peer Reviewed
• [Journal Article] 8-Pyrenylvinyl Adenine Controls Reversible Duplex Formation between Serinol Nucleic Acid and RNA by [2 + 2] Photocycloaddition (2019)
  • Author(s): Murayama Keiji、Yamano Yuuhei、Asanuma Hiroyuki
  • Journal Title: Journal of the American Chemical Society Volume: 141 Issue: 24 Pages: 9485-9489
  • DOI: 10.1021/jacs.9b03267
  • Peer Reviewed
• [Journal Article] cis‐on/trans‐off of DNA hybridization with alkylthio‐azobenzene on L‐threoninol responding to visible light (2019)
  • Author(s): Asanuma Hiroyuki、Ishikawa Teruchika、Murayama Keiji、Liang Xingguo、Yamano Yuuhei
  • Journal Title: ChemPhotoChem Volume: 印刷中 Issue: 6 Pages: 418-424
  • DOI: 10.1002/cptc.201900060
  • Peer Reviewed
• [Journal Article] Orthogonally Photocontrolled Non-Autonomous DNA Walker (2019)
  • Author(s): ?kugor Marko、Valero Juli?n、Murayama Keiji、Centola Mathias、Asanuma Hiroyuki、Famulok Michael
  • Journal Title: Angewandte Chemie International Edition Volume: 印刷中 Issue: 21 Pages: 6948-6951
  • DOI: 10.1002/anie.201901272
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] The Use of Serinol Nucleic Acids as Ultrasensitive Molecular Beacons (2019)
  • Author(s): Murayama, K.; Kashida H.; Asanuma, H.
  • Journal Title: Method in Molecular Biology Volume: 1973 Pages: 261-279
  • Peer Reviewed
• [Journal Article] Introduction of 2,6-Diaminopurines into Serinol Nucleic Acid Improves Anti-miRNA Performance (2017)
  • Author(s): Kamiya Yukiko、Donoshita Yuka、Kamimoto Hiroshi、Murayama Keiji、Ariyoshi Jumpei、Asanuma Hiroyuki
  • Journal Title: ChemBioChem Volume: 18 Issue: 19 Pages: 1917-1922
  • DOI: 10.1002/cbic.201700272
  • Peer Reviewed
• [Journal Article] D-aTNA circuit orthogonal to DNA can be operated by RNA input via SNA (2017)
  • Author(s): Murayama Keiji、Nagao Ryuya、Asanuma Hiroyuki
  • Journal Title: ChemistrySelect Volume: 2 Issue: 20 Pages: 5624-5627
  • DOI: 10.1002/slct.201701126
  • Peer Reviewed
• [Presentation] 人工核酸L-aTNAを基にした新たな非環状型人工核酸の合成 (2020)
  • Author(s): 村山恵司, 飯塚洋平, 浅沼浩之
  • Organizer: 日本化学会第100春季年会
• [Presentation] Photo-control of reversible duplex formation between SNA and RNA by [2+2] photocycloaddition of 8-pyrenylvinyl adenine (2019)
  • Author(s): Keiji Murayama, Yuuhei Yamano, Hiroyuki Asanuma
  • Organizer: ISNAC 2019
• [Presentation] Artificial Nucleic Acids Composed of Acyclic Backbone for Fluorescent Probe and Antisense Reagent (2019)
  • Author(s): Keiji Murayama, Hiromu Kashida, Hiroyuki Asanuma
  • Organizer: 15th Annual Meeting Of The OTS
  • Int'l Joint Research
• [Presentation] 8-ピレニルビニルアデニンの可逆的[2+2]光環化反応を利用した人工核酸SNAの光制御 (2019)
  • Author(s): 山野雄平, 村山恵司, 浅沼浩之
  • Organizer: 第68回高分子討論会
• [Presentation] 人工核酸SNAを利用したRNA/L-DNAシグナル変換と直交性シグナル増幅回路の構築 (2019)
  • Author(s): 曽根務史, 村山恵司, 浅沼浩之
  • Organizer: 第13回バイオ関連化学シンポジウム
• [Presentation] 人工核酸SNAを用いたRNA/L-DNAシグナル変換と直交性シグナル増幅回路の構築 (2019)
  • Author(s): 曽根務史, 村山恵司, 浅沼浩之
  • Organizer: 第169回東海高分子研究会講演会(夏期合宿)
• [Presentation] 光応答性修飾核酸塩基の導入による機能性SNAの開発 (2019)
  • Author(s): 山野雄平, 村山恵司, 浅沼浩之
  • Organizer: 第29回バイオ・高分子シンポジウム
• [Presentation] Development of novel stimuli-responsive supramolecular hydrogel containing artificial nucleic acids (2019)
  • Author(s): MURAYAMA, Keiji; SHIGEMATSU, Yuuki; ASANUMA, Hiroyuki
  • Organizer: 日本化学会第99春季年会
• [Presentation] Design of novel fluorescent probe with fluorescent nucleobase on artificial acyclic nucleic acid (2018)
  • Author(s): Keiji Murayama, Hiroyuki Asanuma
  • Organizer: International Symposium on Photochemistry (27th PhotoIUPAC)
  • Int'l Joint Research
• [Presentation] 修飾核酸塩基導入による人工核酸SNAの高機能化 (2018)
  • Author(s): 村山恵司, 山野雄平, 浅沼浩之
  • Organizer: 第6回バイオ関連化学シンポジウム若手フォーラム
• [Presentation] 蛍光性核酸塩基導入による非環状型人工核酸プローブの開発 (2018)
  • Author(s): 村山恵司, 浅沼浩之
  • Organizer: 第67回高分子討論会
• [Presentation] Development of signal amplification circuit composed of acyclic artificial nucleic acids for detection of natural nucleic acids (2017)
  • Author(s): Keiji Murayama, Ryuya Nagao, Hiroyuki Asanuma
  • Organizer: 第66回高分子学会年次大会
• [Presentation] Acyclic artificial nucleic acid circuit for sensitive detection of RNA (2017)
  • Author(s): Keiji Murayama, Ryuya Nagao, Hiroyuki Asanuma
  • Organizer: The Second International Symposium on Biofunctional Chemistry (ISBC2017)
  • Int'l Joint Research
• [Presentation] 非環状型人工核酸で構成されるシグナル増幅回路の開発とRNA可視化への応用 (2017)
  • Author(s): 村山恵司, 長尾竜弥, 浅沼浩之
  • Organizer: 第66回高分子討論会