Abstract
Single-molecule studies of protein–DNA interactions continue to yield new information on numerous DNA processing pathways. For example, optical microscopy-based techniques permit the real-time observation of proteins that interact with DNA substrates, which in turn allows direct insight into reaction mechanisms. However, these experiments remain technically challenging and are limited by the paucity of stable chromophores and the difficulty of acquiring statistically significant observations. In this protocol, we describe a novel, high-throughput, nanofabricated experimental platform enabling real-time imaging of hundreds of individual protein–DNA complexes over hour timescales.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Hamdan, S.M., Loparo, J.J., Takahashi, M., Richardson, C.C., and van Oijen, A.M. (2009) Dynamics of DNA replication loops reveal temporal control of lagging-strand synthesis. Nature 457, 336–339.
van Oijen, A.M. (2007) Single-molecule studies of complex systems: the replisome. Mol Biosyst 3, 117–125.
Perumal, S.K., Yue, H., Hu, Z., Spiering, M.M., and Benkovic, S.J. (2010) Single-molecule studies of DNA replisome function. Biochim Biophys Acta 1804, 1094–1112.
Yao, N.Y., Georgescu, R.E., Finkelstein, J., and O’Donnell, M.E. (2009) Single-molecule analysis reveals that the lagging strand increases replisome processivity but slows replication fork progression. Proc Natl Acad Sci USA 106, 13236–13241.
Bai, L., Santangelo, T.J., and Wang, M.D. (2006) Single-molecule analysis of RNA polymerase transcription. Annu Rev Biophys Biomol Struct 35, 343–360.
Hodges, C., Bintu, L., Lubkowska, L., Kashlev, M., and Bustamante, C. (2009) Nucleosomal fluctuations govern the transcription dynamics of RNA polymerase II. Science 325, 626–628.
Herbert, K.M., Greenleaf, W.J., and Block, S.M. (2008) Single-molecule studies of RNA polymerase: motoring along. Annu Rev Biochem 77, 149–176.
Finkelstein, I.J., and Greene, E.C. (2008) Single molecule studies of homologous recombination. Mol Biosyst 4, 1094–2104.
Spies, M., Amitani, I., Baskin, R.J., and Kowalczykowski, S.C. (2007) RecBCD enzyme switches lead motor subunits in response to chi recognition. Cell 131, 694–705.
Gorman, J., Chowdhury, A., Surtees, J.A., Shimada, J., Reichman, D.R., Alani, E., and Greene, E.C. (2007) Dynamic basis for one-dimensional DNA scanning by the mismatch repair complex Msh2-Msh6. Mol Cell 28, 359–370.
Kwon, Y., Seong, C., Chi, P., Greene, E.C., Klein, H., and Sung, P. (2008) ATP-dependent chromatin remodeling by the Saccharomyces cerevisiae homologous recombination factor Rdh54. J Biol Chem 283, 10445–10452.
Visnapuu, M.L., and Greene, E.C. (2009) Single-molecule imaging of DNA curtains reveals intrinsic energy landscapes for nucleosome deposition. Nat Struct Mol Biol 16, 1056–1062.
Robertson, R.B., Moses, D.N., Kwon, Y., Chan, P., Zhao, W., Chi, P., Klein, H., Sung, P., and Greene, E.C. (2009) Visualizing the disassembly of S. cerevisiae Rad51 nucleoprotein filaments. J Mol Biol 388, 703–720.
Robertson, R.B., Moses, D.N., Kwon, Y., Chan, P., Chi, P., Klein, H., Sung, P., and Greene, E.C. (2009) Visualizing the disassembly of S. cerevisiae Rad51 nucleoprotein filaments. Proc Natl Acad Sci USA 106, 12688–12693.
Prasad, T.K., Yeykal, C.C., and Greene, E.C. (2006) Visualizing the assembly of human Rad51 filaments on double-stranded DNA. J Mol Biol 363, 713–728.
Gorman, J., Fazio, T., Wang, F., Wind, S., and Greene, E.C. (2009) Nanofabricated racks of aligned and anchored DNA substrates for single-molecule imaging. Langmuir 26, 1372–1379.
Visnapuu, M.L., Fazio, T., Wind, S., and Greene, E.C. (2008) Parallel arrays of geometric nanowells for assembling curtains of DNA with controlled lateral dispersion. Langmuir 24, 11293–11299.
Fazio, T., Visnapuu, M.L., Wind, S., and Greene, E.C. (2008) DNA curtains and nanoscale curtain rods: high-throughput tools for single molecule imaging. Langmuir 24, 10524–10531.
Graneli, A., Yeykal, C.C., Prasad, T.K., and Greene, E.C. (2006) Organized arrays of individual DNA molecules tethered to supported lipid bilayers. Langmuir 22, 292–299.
Visnapuu, M.L., Duzdevich, D., and Greene, E.C. (2008) The importance of surfaces in single-molecule bioscience. Mol Biosyst 4, 394–403.
Groves, J.T., Ulman, N., and Boxer, S.G. (1997) Micropatterning fluid lipid bilayers on solid supports. Science 275, 651–653.
Richter, R.P., Bérat, R., and Brisson, A.R. (2006) Formation of solid-supported lipid bilayers: an integrated view. Langmuir 22, 3497–3505.
Jaiswal, J.K., Mattoussi, H., Mauro, J.M., and Simon, S.M. (2003) Long-term multiple color imaging of live cells using quantum dot bioconjugates. Nat Biotechnol 21, 47–51.
Medintz, I.L., Uyeda, H.T., Goldman, E.R., and Mattoussi, H. (2005) Quantum dot bioconjugates for imaging, labeling and sensing. Nat Mater 4, 435–446.
Ebenstein, Y., Gassman, N., Kim, S., Kim, Y., Ho, S., Samuel, R., Michalet, X., and Weiss, S. (2009) Lighting up individual DNA binding proteins with quantum dots. Nano Lett 9, 1598–1603.
Pinaud, F., Michalet, X., Bentolila, L.A., Tsay, J.M., Doose, S., Li, J.J., Iyer, G., and Weiss, S. (2006) Advances in fluorescence imaging with quantum dot bio-probes. Biomaterials 27, 1679–1687.
Rasnik, I., McKinney, S.A., and Ha, T. (2006) Nonblinking and long-lasting single-molecule fluorescence imaging. Nat Methods 3, 891–893.
Escude, C., Geron-Landre, B., Crut, A., and Desbiolles, P. (2009) Multicolor detection of combed DNA molecules using quantum dots. Methods Mol Biol 544, 357–366.
Thompson, R.E., Larson, D.R., and Webb, W.W. (2002) Precise nanometer localization analysis for individual fluorescent probes. Biophys J 82, 2775–2783.
Yildiz, A., and Selvin, P.R. (2005) Fluorescence imaging with one nanometer accuracy: application to molecular motors. Acc Chem Res 38, 574–582.
Gueroui, Z., Freyssingeas, E., Place, C., and Berge, B. (2003) Transverse fluctuation analysis of single extended DNA molecules. Eur Phys J E Soft Matter 11, 105–108.
Quake, S.R., Babcock, H., and Chu, S. (1997) The dynamics of partially extended single molecules of DNA. Nature 388, 151–154.
Carter, B.C., Shubeita, G.T., and Gross, S.P. (2005) Tracking single particles: a user-friendly quantitative evaluation. Phys Biol 2, 60–72.
Acknowledgments
We thank the many members of the Greene Laboratory who have worked on developing the DNA curtain experimental platform, in particular, Teresa Fazio for establishing the nanofabrication process. The Greene Laboratory is supported by the Howard Hughes Medical Institute, the National Institutes of Health, the National Science Foundation, the Susan G. Komen Foundation, and the Irma T. Hirschl Trust. IJF is supported by the NIH Fellowship #F32GM80864. We apologize to any colleagues whose work we were not able to cite due to length limitations.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2011 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Finkelstein, I.J., Greene, E.C. (2011). Supported Lipid Bilayers and DNA Curtains for High-Throughput Single-Molecule Studies. In: Tsubouchi, H. (eds) DNA Recombination. Methods in Molecular Biology, vol 745. Humana Press. https://doi.org/10.1007/978-1-61779-129-1_26
Download citation
DOI: https://doi.org/10.1007/978-1-61779-129-1_26
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-61779-128-4
Online ISBN: 978-1-61779-129-1
eBook Packages: Springer Protocols