Advertisement

Supported Lipid Bilayers and DNA Curtains for High-Throughput Single-Molecule Studies

  • Ilya J. Finkelstein
  • Eric C. Greene
Part of the Methods in Molecular Biology book series (MIMB, volume 745)

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.

Key words

Single molecule TIRF microscopy nanofabrication DNA curtains nucleosome DNA motors 

Notes

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.

References

  1. 1.
    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.PubMedCrossRefGoogle Scholar
  2. 2.
    van Oijen, A.M. (2007) Single-molecule studies of complex systems: the replisome. Mol Biosyst 3, 117–125.PubMedCrossRefGoogle Scholar
  3. 3.
    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.PubMedGoogle Scholar
  4. 4.
    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.PubMedCrossRefGoogle Scholar
  5. 5.
    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.PubMedCrossRefGoogle Scholar
  6. 6.
    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.PubMedCrossRefGoogle Scholar
  7. 7.
    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.PubMedCrossRefGoogle Scholar
  8. 8.
    Finkelstein, I.J., and Greene, E.C. (2008) Single molecule studies of homologous recombination. Mol Biosyst 4, 1094–2104.PubMedCrossRefGoogle Scholar
  9. 9.
    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.PubMedCrossRefGoogle Scholar
  10. 10.
    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.PubMedCrossRefGoogle Scholar
  11. 11.
    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.PubMedCrossRefGoogle Scholar
  12. 12.
    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.PubMedCrossRefGoogle Scholar
  13. 13.
    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.PubMedCrossRefGoogle Scholar
  14. 14.
    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.PubMedCrossRefGoogle Scholar
  15. 15.
    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.PubMedCrossRefGoogle Scholar
  16. 16.
    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.CrossRefGoogle Scholar
  17. 17.
    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.PubMedCrossRefGoogle Scholar
  18. 18.
    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.PubMedCrossRefGoogle Scholar
  19. 19.
    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.PubMedCrossRefGoogle Scholar
  20. 20.
    Visnapuu, M.L., Duzdevich, D., and Greene, E.C. (2008) The importance of surfaces in single-molecule bioscience. Mol Biosyst 4, 394–403.PubMedCrossRefGoogle Scholar
  21. 21.
    Groves, J.T., Ulman, N., and Boxer, S.G. (1997) Micropatterning fluid lipid bilayers on solid supports. Science 275, 651–653.PubMedCrossRefGoogle Scholar
  22. 22.
    Richter, R.P., Bérat, R., and Brisson, A.R. (2006) Formation of solid-supported lipid bilayers: an integrated view. Langmuir 22, 3497–3505.PubMedCrossRefGoogle Scholar
  23. 23.
    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.PubMedCrossRefGoogle Scholar
  24. 24.
    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.PubMedCrossRefGoogle Scholar
  25. 25.
    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.PubMedCrossRefGoogle Scholar
  26. 26.
    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.PubMedCrossRefGoogle Scholar
  27. 27.
    Rasnik, I., McKinney, S.A., and Ha, T. (2006) Nonblinking and long-lasting single-molecule fluorescence imaging. Nat Methods 3, 891–893.PubMedCrossRefGoogle Scholar
  28. 28.
    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.PubMedCrossRefGoogle Scholar
  29. 29.
    Thompson, R.E., Larson, D.R., and Webb, W.W. (2002) Precise nanometer localization analysis for individual fluorescent probes. Biophys J 82, 2775–2783.PubMedCrossRefGoogle Scholar
  30. 30.
    Yildiz, A., and Selvin, P.R. (2005) Fluorescence imaging with one nanometer accuracy: application to molecular motors. Acc Chem Res 38, 574–582.PubMedCrossRefGoogle Scholar
  31. 31.
    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.PubMedCrossRefGoogle Scholar
  32. 32.
    Quake, S.R., Babcock, H., and Chu, S. (1997) The dynamics of partially extended single molecules of DNA. Nature 388, 151–154.PubMedCrossRefGoogle Scholar
  33. 33.
    Carter, B.C., Shubeita, G.T., and Gross, S.P. (2005) Tracking single particles: a user-friendly quantitative evaluation. Phys Biol 2, 60–72.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Department of Biochemistry and Molecular BiophysicsColumbia UniversityNew YorkUSA
  2. 2.Howard Hughes Medical InstituteChevy ChaseUSA

Personalised recommendations