Abstract
We developed two labeling methods for the direct observation of single-stranded DNA (ssDNA), using a ssDNA binding protein and a ssDNA recognition peptide. The first approach involved protein fusion between the 70-kDa ssDNA-binding domain of replication protein A and enhanced yellow fluorescent protein (RPA-YFP). The second method used the ssDNA binding peptide of Escherichia coli RecA labeled with Atto488 (ssBP-488; Atto488-IRMKIGVMFGNPETTTGGNALKFY). The labeled ssλDNA molecules were visualized over time in micro-flow channels. We report substantially different dynamics between these two labeling methods. When ssλDNA molecules were labeled with RPA-YFP, terminally bound fusion proteins were sheared from the free ends of the ssλDNA molecules unless 25-mer oligonucleotides were annealed to the free ends. RPA-YFP-ssλDNA complexes were dissociated by the addition of 0.2 M NaCl, although complex reassembly was possible with injection of additional RPA-YFP. In contrast to the flexible dynamics of RPA-YFP-ssλDNA complexes, the ssBP-488-ssλDNA complexes behaved as rigid rods and were not dissociated even in 2 M NaCl.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Kabata H, Kurosawa O, Arai I, Washizu M, Margarson SA, Glass RE, Shimamoto N (1993) Visualization of single molecules of RNA polymerase sliding along DNA. Science 262:1561–1563
Harada Y, Funatsu T, Murakami K, Nonoyama Y, Ishihama A, Yanagida T (1999) Single-molecule imaging of RNA polymerase-DNA interactions in real time. Biophys J 76:709–715
Reuter M, Parry F, Dryden DT, Blakely GW (2010) Single-molecule imaging of Bacteroides fragilis AddAB reveals the highly processive translocation of a single motor helicase. Nucleic Acids Res 38:3721–3731
Matsuura S, Kurita H, Nakano M, Komatsu J, Takashima K, Katsura S, Mizuno A (2002) One-end immobilization of individual DNA molecules on a functional hydrophobic glass surface. J Biomol Struct Dyn 20:429–436
Kurita H, Inaishi K, Torii K, Urisu M, Nakano M, Katsura S, Mizuno A (2008) Real-time direct observation of single-molecule DNA hydrolysis by exonuclease III. J Biomol Struct Dyn 25:473–480
Kurita H, Torii K, Yasuda H, Takashima K, Katsura S, Mizuno A (2009) The effect of physical form of DNA on ExonucleaseIII activity revealed by single-molecule observations. J Fluoresc 19:33–40
Hilario J, Kowalczykowski SC (2010) Visualizing protein-DNA interactions at the single-molecule level. Curr Opin Chem Biol 14:15–22
Fili N, Mashanov GI, Toseland CP, Batters C, Wallace MI, Yeeles JTP, Dillingham MS, Webb MR, Molloy JE (2010) Visualizing helicases unwinding DNA at the single molecule level. Nucleic Acids Res 38:4448–4457
Mameren J, Modesti M, Kanaar R, Wyman C, Wuite GJ, Peterman EJ (2006) Dissecting elastic heterogeneity along DNA molecules coated partly with Rad51 using concurrent fluorescence microscopy and optical tweezers. Nucleic Acids Res 91:L78–L80
Mameren JV, Modesti M, Kanaar R, Wyman C, Wuite GJL, Peterman EJG (2006) Dissecting elastic heterogeneity along DNA molecules coated partly with Rad51 using concurrent fluorescence microscopy and optical tweezers. Biophys J 91:L78–L80
Tanner NA, Hamdan SM, Jergic S, Schaeffer PM, Dixon NE, van Oijen AM (2008) Single-molecule studies of fork dynamics in Escherichia coli DNA replication. Nat Struct Mol Biol 15:170–176
Oshige M, Kawasaki S, Takano H, Yamaguchi K, Kurita H, Mizuno T, Matsuura S, Mizuno A, Katsura S (2011) Direct observation method of individual single-stranded DNA molecules using fluorescent replication protein A. J Fluoresc 21:1189–1194
Sugimoto N (2000) DNA recognition of a 24-mer peptide derived from RecA protein. Biopolymers 55:416–424
Nishinaka T, Doi Y, Hashimoto M, Hara R, Shibata T, Harada Y, Kinosita K Jr, Noji H, Yashima E (2007) Visualization of RecA filaments and DNA by fluorescence microscopy. J Biochem 141:147–156
Acknowledgments
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 (B: 24770164).
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Takahashi, S., Kawasaki, S., Yamaguchi, K. et al. Direct Observation of Fluorescently Labeled Single-stranded λDNA Molecules in a Micro-Flow Channel. J Fluoresc 23, 635–640 (2013). https://doi.org/10.1007/s10895-013-1210-1
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10895-013-1210-1