Development of single molecule observation technique for the interaction analysis of DNA and protein

2011 Fiscal Year Final Research Report

• Project/Area Number: 21770183
• Research Category: Grant-in-Aid for Young Scientists (B)
• Allocation Type: Single-year Grants
• Research Field: Molecular biology
• Research Institution: Gunma University
• Principal Investigator: OSHIGE Masahiko 群馬大学, 大学院・工学研究科, 准教授 (00451716)
• Project Period (FY): 2009 – 2011
• Keywords: 1分子解析 / 形態制御 / 可視化技術 / 1本鎖DNA結合タンパク質 / 1本鎖DNA結合ペプチド

Research Abstract

Intercalation of fluorescent dyes such as YOYO-1 and SYTOX Orange has been the standard method for observing single molecules of double-stranded DNA (dsDNA), but effective fluorescent dyes for observing single molecules of single-stranded DNA (ssDNA) have not been found. To facilitate direct single-molecule observations of DNA metabolism reactions, it is necessary to establish methods for discriminating ssDNA and dsDNA. To observe ssDNA directly, we prepared a fusion protein consisting of the 70 kDa DNA-binding domain of replication protein A and enhanced yellow fluorescent protein (RPA-YFP). This fusion protein had ssDNA-binding activity. In this experiment, dsDNA was stained by SYTOX Orange and ssDNA by RPA-YFP, and we succeeded in staining ssDNA and dsDNA by using RPA-YFP and SYTOX Orange simultaneously and in observing of DNA synthesis reaction by Klenow fragment (3'-5'exo-).

Research Products

• [Journal Article] Direct Observation Method of Individual Single-Stranded DNA Molecules Using Fluorescent Replication Protein A. (2011)
  • Author(s): Oshige, M., Kawasaki, S., Takano, H., Yamaguchi, K., Kurita, H., Mizuno, T., Matsuura, S. I., Mizuno, A., Katsura, S.
  • Journal Title: Journal of Fluoresce Volume: 21(3) Pages: 1189-1194
  • Peer Reviewed
• [Journal Article] A new DNA combing method for biochemical analysis. (2010)
  • Author(s): Oshige, M., Yamaguchi, K., Matsuura, S. I., Kurita, H., Mizuno, A., Katsura, S.
  • Journal Title: Analytical Biochemistry Volume: 400(1) Pages: 145-147
  • Peer Reviewed
• [Presentation] 開発した2種類の1本鎖DNA可視化方法の比較検討 (2011)
  • Author(s): 村上太滝, 川崎祥平, 高橋俊介, 大重真彦, 栗田弘史, 松浦俊一, 水野武, 水野彰, 桂進司
  • Organizer: 第34回日本分子生物学会年会
  • Place of Presentation: パシフィコ横浜(神奈川)
  • Year and Date: 2011-12-13
• [Presentation] Real-Time観測を目指した1本鎖DNA可視化技術の開発 (2010)
  • Author(s): 川崎祥平, 大重真彦, 水野武, 栗田弘史, 松浦俊一, 水野彰, 桂進司
  • Organizer: BMB2010(第33回日本分子生物学会年会・第83回日本生化学会大会合同大会)
  • Place of Presentation: 神戸ポートアイランド(兵庫)
  • Year and Date: 2010-12-09
• [Presentation] 1本鎖DNA結合ペプチドを用いた1本鎖DNAの可視化 (2010)
  • Author(s): 村上太滝, 野中潤, 川崎祥平, 大重真彦, 栗田弘史, 松浦俊一, 水野彰, 桂進司
  • Organizer: BMB2010(第33回日本分子生物学会年会・第83回日本生化学会大会合同大会)
  • Place of Presentation: 神戸ポートアイランド(兵庫)
  • Year and Date: 2010-12-09
• [Presentation] 1分子解析のためのDNA形態制御 (2009)
  • Author(s): 山口晃史, 川崎祥平, 大重真彦, 松浦俊一, 栗田弘史, 水野彰, 桂進司
  • Organizer: 第32回日本分子生物学会年会
  • Place of Presentation: パシフィコ横浜(神奈川)
  • Year and Date: 2009-12-09
• [Presentation] 1分子観察技術を用いたDNA-タンパク質相互作用解析の試み (2009)
  • Author(s): 山口晃史, 川崎祥平, 大重真彦, 松浦俊一, 水野武, 栗田弘史, 水野彰, 坂口謙吾, 桂進司
  • Organizer: 第32回日本分子生物学会年会
  • Place of Presentation: パシフィコ横浜(神奈川)
  • Year and Date: 2009-12-09
• [Presentation] 1本鎖DNA結合因子を用いた1本鎖DNAの可視化技術の開発 (2009)
  • Author(s): 川崎祥平, 山口晃司, 大重真彦, 水野武, 栗田弘史, 坂口謙吾, 水野彰, 桂進司
  • Organizer: 第32回日本分子生物学会年会
  • Place of Presentation: パシフィコ横浜(神奈川)
  • Year and Date: 2009-12-09
• [Book] 【執筆し章のタイトル】Manipulatio and Direct Observation Techniques for investigation of DNA Metabolic Reactions (2011)
  • Author(s): Oshige, M., Katsura, S.
  • Total Pages: 384
  • Publisher: Nova Science Publishers
• [Book] 【執筆した章のタイトル】第7章DNA代謝反応の1分子観察とDNA分子の形態制御技術 (2011)
  • Author(s): 桂進司, 大重真彦
  • Total Pages: 153
  • Publisher: シーエムシー出版