Development of DNA modification enzymes based on sequence-specificity of DNA binding domains and their application for genome editing.

• Project/Area Number: 25410171
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Bio-related chemistry
• Research Institution: Tokyo Medical and Dental University
• Principal Investigator: Nomura Wataru 東京医科歯科大学, 生体材料工学研究所, 准教授 (80463909)
• Project Period (FY): 2013-04-01 – 2016-03-31
• Project Status: Completed (Fiscal Year 2015)
• Budget Amount: ¥5,200,000 (Direct Cost: ¥4,000,000、Indirect Cost: ¥1,200,000); Fiscal Year 2015: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000); Fiscal Year 2014: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000); Fiscal Year 2013: ¥2,080,000 (Direct Cost: ¥1,600,000、Indirect Cost: ¥480,000)
• Keywords: タンパク質 / メチル化 / 人工酵素 / エピゲノム編集 / 核酸 / ケミカルバイオロジー / 合成生物学

Outline of Final Research Achievements

Improvement of genome editing tools such as CRISPR-Cas9 system leads to the increased demands for other gene modification tools such as DNA recombination and methylation. The split DNA methylase bearing split M.HhaI domains and zinc finger domains show the potential for the highly specific DNA methylation. Structural modelling study suggested that the DNA-split methylase complex could contain steric hindrance in some parts. Thus altered DNA assembly of split methylase on the target gene and several linker mutations were tested to obtain the increased activity in DNA methylation without losing sequence specificity. As the results, the assembly pattern in which ZFPs bind to the different strand on the target DNA showed increased activity in DNA methylation. In addition, the extension of linker length between the domains also affected on the increased activity. Above all, the results give new insights into the assembly of split protein domains especially on the target DNA.

Research Products

• [Journal Article] Application and Potential of Genome Engineering by Artificial Enzymes (2015)
  • Author(s): 野村 渉
  • Journal Title: YAKUGAKU ZASSHI Volume: 135 Issue: 3 Pages: 405-414
  • DOI: 10.1248/yakushi.14-00240-5
  • NAID: 130004756508
  • ISSN: 0031-6903, 1347-5231
  • Year and Date: 2015-03-01
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] ターゲットタンパク質を特異的に認識するプローブ (2014)
  • Author(s): 野村 渉、玉村 啓和
  • Journal Title: 化学工業 Volume: 65 Pages: 8-14
• [Journal Article] Screening for Protein Kinase C Ligands Using Fluorescence Resonance Energy Transfer (2014)
  • Author(s): Ohashi N, Nomura W, Minato N, Tamamura H.
  • Journal Title: Chemical and Pharmaceutical Bulletin Volume: 62 Issue: 10 Pages: 1019-1025
  • DOI: 10.1248/cpb.c14-00419
  • NAID: 130004677396
  • ISSN: 0009-2363, 1347-5223
  • Peer Reviewed / Open Access
• [Journal Article] Development of a fluoride-responsive amide bond cleavage device that is potentially applicable to a traceable linker (2014)
  • Author(s): J. Yamamoto, N. Maeda, C. Komiya, T. Tanaka, M. Denda, K. Ebisuno, W. Nomura, H. Tamamura, Y. Sato, A. Yamauchi, A. Shigenaga, and A. Otaka
  • Journal Title: Tetrahedron Volume: 70 Issue: 34 Pages: 5122-5127
  • DOI: 10.1016/j.tet.2014.05.110
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Development of the 8-aza-3-bromo-7-hydroxycoumarin-4-ylmethyl group as a new entry of photolabile protecting groups (2014)
  • Author(s): Takano, H., Narumi, T., Ohashi, N., Suzuki, A., Furuta, T., Nomura, W., Tamamura, H.
  • Journal Title: Tetrahedron Volume: XX Issue: 29 Pages: 4400-4404
  • DOI: 10.1016/j.tet.2014.04.063
  • Peer Reviewed
• [Journal Article] Development of a traceable linker containing a thiol-responsive amino acid for the enrichment and selective labelling of target proteins (2014)
  • Author(s): J. Yamamoto, M. Denda, N. Maeda, M. Kita, C. Komiya, T. Tanaka, W. Nomura, H. Tamamura, Y. Sato, A. Yamauchi, A. Shigenaga, and A. Otaka
  • Journal Title: Org. Biomol. Chem. Volume: 12 Issue: 23 Pages: 3821-3826
  • DOI: 10.1039/c4ob00622d
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] A CD4 Mimic as an HIV Entry Inhibitors: Pharmacokinetics (2013)
  • Author(s): Hashimoto, C., Narumi, T., Otsuki, H., Hirota, Y., Arai, H., Yoshimura, K., Harada, S., Ohashi, N., Nomura, W., Miura, T., Igarashi, T., Matsuhita, S., Tamamura, H.
  • Journal Title: Bioorg. Med. Chem. Volume: 21 Issue: 24 Pages: 7884-7889
  • DOI: 10.1016/j.bmc.2013.10.005
  • Peer Reviewed
• [Journal Article] CXCR4-derived synthetic peptides inducing anti-HIV-1 antibodies. (2013)
  • Author(s): Hashimoto C, Nomura W, Narumi T, Fujino M, Nakahara T, Yamamoto N, Murakami T, Tamamura H.
  • Journal Title: Bioorg. Med. Chem. Volume: 21 Issue: 22 Pages: 6878-6885
  • DOI: 10.1016/j.bmc.2013.09.037
  • Peer Reviewed
• [Journal Article] Anti-HIV-1 Peptide Derivatives Based on the HIV-1 Co-receptor CXCR4. (2013)
  • Author(s): Hashimoto C, Nomura W, Narumi T, Fujino M, Tsutsumi H, Haseyama M, Yamamoto N, Murakami T, Tamamura H.
  • Journal Title: ChemMedChem Volume: 8 Issue: 10 Pages: 1668-1672
  • DOI: 10.1002/cmdc.201300289
  • Peer Reviewed
• [Journal Article] Cell-Permeable Stapled Peptides Based on HIV-1 Integrase Inhibitors Derived from HIV-1 Gene Products. (2013)
  • Author(s): Nomura W, Aikawa H, Ohashi N, Urano E, Metifiot M, Fujino M, Maddali K, Ozaki T, Nozue A, Narumi T, Hashimoto C, Tanaka T, Pommier Y, Yamamoto N, Komano JA, Murakami T, Tamamura H.
  • Journal Title: ACS Chem. Biol. Volume: 8 Issue: 10 Pages: 2235-2244
  • DOI: 10.1021/cb400495h
  • Peer Reviewed
• [Journal Article] Peptide-based Ligand Screening and Functional Analysis of Protein Kinase C (2013)
  • Author(s): Ohashi, N., Nomura, W., Narumi, T., Tamamura, H.
  • Journal Title: Biopolymers: Peptide Science Volume: 100 Issue: 6 Pages: 613-620
  • DOI: 10.1002/bip.22324
  • Peer Reviewed
• [Journal Article] Multimerized CHR-Derived Peptides as HIV-1 Fusion Inhibitors. (2013)
  • Author(s): Nomura W, Hashimoto C, Suzuki T, Ohashi N, Fujino M, Murakami T, Yamomoto N, Tamamura H.
  • Journal Title: Bioorg. Med. Chem. Volume: 21 Issue: 15 Pages: 4452-4458
  • DOI: 10.1016/j.bmc.2013.05.060
  • Peer Reviewed
• [Presentation] Efficient conversion of genomic promoter region by genome engineering systems (2015)
  • Author(s): Wataru Nomura, Akemi Masuda, Hirokazu Tamamura
  • Organizer: Pacifichem 2015
  • Place of Presentation: ホノルル（アメリカ合衆国）
  • Year and Date: 2015-12-15
  • Int'l Joint Research
• [Presentation] Simultaneous digestion by site-specific nucleases for efficient gene deletion: Study of hTERT promoter function (2015)
  • Author(s): Wataru Nomura, Akemi Masuda, Hirokazu Tamamura
  • Organizer: Conference on Transposition and Genome Engineering 2015
  • Place of Presentation: 奈良春日野国際フォーラム（奈良県・奈良市）
  • Year and Date: 2015-11-17
  • Int'l Joint Research
• [Presentation] Genome deletion by ZFN and CRISPR-Cas targeting promoter region for analysis of hTERT function (2015)
  • Author(s): Wataru Nomura, Akemi Masuda, Hirokazu Tamamura
  • Organizer: 細胞を創る 研究会8.0
  • Place of Presentation: 大阪大学吹田キャンパス銀杏会館（大阪府・吹田市）
  • Year and Date: 2015-11-12
• [Presentation] 人工転写因子の協奏的な働きによる遺伝子発現調節機構の構築 (2015)
  • Author(s): 杉井太亮、野村 渉、玉村啓和
  • Organizer: 第59回日本薬学会関東支部大会
  • Place of Presentation: 日本大学薬学部（千葉県・船橋市）
  • Year and Date: 2015-09-12
• [Presentation] 配列特異的分割型DNAメチル化酵素の作用機構解析 (2015)
  • Author(s): 野村 渉、大浦伊代、玉村啓和
  • Organizer: 第59回日本薬学会関東支部大会
  • Place of Presentation: 日本大学薬学部（千葉県・船橋市）
  • Year and Date: 2015-09-12
• [Presentation] ペプチド性機能分子のデザインと応用 (2015)
  • Author(s): 野村 渉
  • Organizer: 第47回若手ペプチド夏の勉強会
  • Place of Presentation: アスティかたおか（長野県・塩尻市）
  • Year and Date: 2015-08-11
  • Invited
• [Presentation] 遺伝子を標的とした創薬ケミカルバイオロジー研究 (2015)
  • Author(s): 野村 渉
  • Organizer: 長浜バイオ大学大学院講義
  • Place of Presentation: 長浜バイオ大学（滋賀県・長浜市）
  • Year and Date: 2015-08-10
  • Invited
• [Presentation] ゲノム編集技術を用いた複数箇所切断によるプロモーター領域の配列変換 (2015)
  • Author(s): 野村 渉、増田朱美、玉村啓和
  • Organizer: 日本ケミカルバイオロジー学会第10回年会
  • Place of Presentation: 東北大学川内キャンパス萩ホール（宮城県・仙台市）
  • Year and Date: 2015-06-10
• [Presentation] 人工DNA修飾酵素の構築戦略とゲノム・エピゲノム編集への展開 (2015)
  • Author(s): 野村 渉
  • Organizer: 日本薬学会第135年会
  • Place of Presentation: 神戸学院大学
  • Year and Date: 2015-03-26 – 2015-03-28
  • Invited
• [Presentation] ゲノム編集法による効率的な配列欠損反応 (2014)
  • Author(s): 野村 渉、増田 朱美、玉村 啓和
  • Organizer: 細胞を創る会7.0
  • Place of Presentation: 東京大学弥生講堂
  • Year and Date: 2014-11-13 – 2014-11-14
• [Presentation] プロモーター領域を標的としたDNA二重鎖切断による配列欠損反応の解析 (2014)
  • Author(s): 野村 渉、増田 朱美、玉村 啓和
  • Organizer: 第4回ゲノム編集研究会
  • Place of Presentation: 広島国際会議場
  • Year and Date: 2014-10-06 – 2014-10-07
• [Presentation] ゲノム編集法による複数箇所同時切断が示す配列欠損反応効率の向上 (2014)
  • Author(s): 野村 渉、増田 朱美、玉村 啓和
  • Organizer: 第8回バイオ関連化学シンポジウム
  • Place of Presentation: 岡山大学津島キャンパス
  • Year and Date: 2014-09-11 – 2014-09-13
• [Presentation] Efficient Gene Disruption at hTERT Promoter Region by Simultaneous Digestion by Pairs of ZFNs or Guide RNAs of CRISPR/Cas System. (2014)
  • Author(s): Wataru Nomura, Akemi Masuda, Hirokazu Tamamura
  • Organizer: The 28th annual symposium of protein society
  • Place of Presentation: San Diego, USA
  • Year and Date: 2014-07-27 – 2014-07-30
  • Invited
• [Presentation] Enhanced Gene Disruption at Specific Promoter Region By Simultaneous Digestion of ZFN or CRISPR/Cas System. (2014)
  • Author(s): Wataru Nomura, Akemi Masuda, Hirokazu Tamamura
  • Organizer: The Synthetic Biology: Engineering, Evolution & Design (SEED) conference 2014
  • Place of Presentation: Manhattan Beach, USA
  • Year and Date: 2014-07-14 – 2014-07-17
• [Presentation] Enhanced Gene Disruption by Simultaneous Digestion of ZFN or CRISPR/Cas System at hTERT Promoter Region. (2014)
  • Author(s): Wataru Nomura, Akemi Masuda, Hirokazu Tamamura
  • Organizer: FASEB SRC "Genome Engineering Cutting-Edge Research and Applications
  • Place of Presentation: Nassau, Bahamas
  • Year and Date: 2014-06-22 – 2014-06-27
• [Presentation] 人工酵素によるゲノム編集工学とその応用、可能性 (2014)
  • Author(s): 野村渉
  • Organizer: 日本薬学会第134年会
  • Place of Presentation: ホテル日航熊本
  • Invited
• [Presentation] ジンクフィンガーヌクレアーゼによるテロメア活性の制御 (2013)
  • Author(s): 野村渉、増田朱美、玉村啓和
  • Organizer: 第7回バイオ関連化学シンポジウム
  • Place of Presentation: 名古屋大学東山キャンパス
• [Presentation] Effects of Zinc Finger Nucleases Targeting Telomerase Promoter Region (2013)
  • Author(s): Wataru Nomura, Akemi Masuda, Hirokazu Tamamura
  • Organizer: The 2nd Annual Conference of the International Chemical Biology Society
  • Place of Presentation: 京都大学紫蘭会館
• [Presentation] DNA Modification Enzymes Utilizing Sequence-Specificity of Zinc Finger Domains (2013)
  • Author(s): Wataru Nomura, Hirokazu Tamamura
  • Organizer: CBI学会2013年大会
  • Place of Presentation: 船堀タワーホール
• [Presentation] Creation of Zinc Finger Nucleases Targeting Telomerase Promoter Region (2013)
  • Author(s): Wataru Nomura, Akemi Masuda, Hirokazu Tamamura
  • Organizer: 4th Asia-Pacific International Peptide Symposium
  • Place of Presentation: ホテル阪急エキスポパーク
• [Presentation] Development of Peptide-derived Molecular Probes and Inhibitors Based on the Interactions with Biomacromolecules (2013)
  • Author(s): Wataru Nomura
  • Organizer: 4th Asia-Pacific International Peptide Symposium
  • Place of Presentation: ホテル阪急エキスポパーク
  • Invited
• [Presentation] テロメラーゼプロモーター領域を標的としたゲノム編集 (2013)
  • Author(s): 野村渉、増田朱美、玉村啓和
  • Organizer: 細胞を創る会6.0
  • Place of Presentation: 慶應義塾大学鶴岡タウンキャンパス
• [Presentation] ジンクフィンガーからゲノム編集ツールへの展開 (2013)
  • Author(s): 野村渉
  • Organizer: 2013年東日本スクリプス会
  • Place of Presentation: ゆうぽうと五反田
  • Invited
• [Book] pp19-20, Peptide Science 2014, Akira Otaka (Ed.) (2015)
  • Author(s): Takano H, Narumi T, Ohashi N, Nomura W, Furuta T, Tamamura H.
  • Total Pages: 369
  • Publisher: The Japanese Peptide Society, Osaka
• [Book] pp75-76, Peptide Science 2014, Akira Otaka (Ed.) (2015)
  • Author(s): Nomura W, Koseki T, Mizuguchi T, Tamamura H.
  • Total Pages: 369
  • Publisher: The Japanese Peptide Society, Osaka
• [Book] pp103-104, Peptide Science 2014, Akira Otaka (Ed.) (2015)
  • Author(s): Honda Y, Mizuguchi T, Nomura W, Tamamura H.
  • Total Pages: 369
  • Publisher: The Japanese Peptide Society, Osaka
• [Book] Peptide Science 2012 (2013)
  • Author(s): Masuda A, Nomura W, Tamamura H.
  • Total Pages: 419
  • Publisher: Kazuhisa Sugimura (Ed.), The Japanese Peptide Society, Osaka
• [Book] Peptide Science 2012 (2013)
  • Author(s): Hashimoto C, Nomura W, Komano JA, Tamamura H.
  • Total Pages: 419
  • Publisher: Kazuhisa Sugimura (Ed.), The Japanese Peptide Society, Osaka
• [Book] Peptide Science 2012 (2013)
  • Author(s): Nomura W, Narumi T, Aikawa H, Tamamura H.
  • Total Pages: 419
  • Publisher: Kazuhisa Sugimura (Ed.), The Japanese Peptide Society, Osaka
• [Remarks] 東京医科歯科大学 玉村研究室 ホームページ
  • URL: http://tamamura-tmd.sakura.ne.jp/
• [Remarks] 東京医科歯科大学生体材料工学研究所メディシナルケミストリー分野 研究業績
  • URL: http://www.tmd.ac.jp/i-mde/www/molb/publication/index.html