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Direct Observation Method of Individual Single-Stranded DNA Molecules Using Fluorescent Replication Protein A

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Abstract

Direct observation studies of single molecules have revealed molecular behaviors usually hidden in the ensemble and time-averaging of bulk experiments. Direct single DNA molecule analysis of DNA metabolism reactions such as DNA replication, repair, and recombination is necessary to fully understand these essential processes. 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 our experiments, 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.

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Acknowledgments

We thank Prof. Hiroshi Ueda (Department of Chemistry and Biotechnology, University of Tokyo) for the kind gift of the eYFP fusion protein expression system of the pET32 modified vector (pET32-eYFP).

This work was partially supported by the Nakatani Foundation of Electronic Measuring Technology Advancement (to S.K.). M.O. was supported by a Grant-in-Aid for Young Scientists (Start-up: 19870003 and B: 21770183).

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Authors and Affiliations

  1. Department of Chemical and Environmental Engineering, Graduate School of Engineering, Gunma University, 1-5-1 Tenjin-cho, Kiryu, Gunma, 376-8515, Japan

    Masahiko Oshige, Shohei Kawasaki, Hiroki Takano, Kouji Yamaguchi & Shinji Katsura

  2. Department of Environmental and Life Sciences, Graduate School of Engineering, Toyohashi University of Technology, Aichi, 441-8580, Japan

    Hirofumi Kurita & Akira Mizuno

  3. Cellular Dynamics Laboratory, Advanced Science Institute, RIKEN, Wako, Saitama, 351-0198, Japan

    Takeshi Mizuno

  4. Research Center for Compact Chemical System, National Institute of Advanced Industrial Science and Technology (AIST), Miyagi, 983-8551, Japan

    Shun-ichi Matsuura

Authors
  1. Masahiko Oshige
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  2. Shohei Kawasaki
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  3. Hiroki Takano
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  4. Kouji Yamaguchi
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  5. Hirofumi Kurita
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  6. Takeshi Mizuno
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  7. Shun-ichi Matsuura
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  8. Akira Mizuno
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  9. Shinji Katsura
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Correspondence to Shinji Katsura.

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Oshige, M., Kawasaki, S., Takano, H. et al. Direct Observation Method of Individual Single-Stranded DNA Molecules Using Fluorescent Replication Protein A. J Fluoresc 21, 1189–1194 (2011). https://doi.org/10.1007/s10895-010-0797-8

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  • DOI: https://doi.org/10.1007/s10895-010-0797-8

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