Assembly of Recombinant Nucleosomes on Nanofabricated DNA Curtains for Single-Molecule Imaging
Eukaryotic chromosomes are highly packed into chromatin, the basic unit of which is the nucleosome. The presence of nucleosomes and the resulting organization of the genome into higher-order chromatin structures has profound consequences for virtually all aspects of DNA metabolism, including DNA transcription, repair, and chromosome segregation. We have developed novel approaches for nanofabricating “DNA curtains” for high-throughput single-molecule imaging, and we have begun adapting these new research tools in an effort to begin studying chromatin biology at the single-molecule level. In this protocol, we describe procedures for assembly and real-time single-molecule visualization of DNA curtains bound by reconstituted nucleosomes made from recombinant histones.
Key wordsDNA curtain TIRF microscopy Lipid bilayer Nanofabrication Nucleosomes Chromatin
We thank the many members of the Greene laboratory who have worked on developing the DNA curtain experimental platform, and we extend special thanks to Mari-Liis Visnapuu for establishing procedures for nucleosome purification and assembly. The Greene laboratory is supported by the Howard Hughes Medical Institute, the National Institutes of Health, and the National Science Foundation. J. Y. Lee was supported in part by the Korea Research Foundation Grant funded by the Korean Government (KRF-2008-357-C00048). We apologize to any colleagues whose work we were not able to cite due to length limitations.
- 12.Greene, E. C., Wind, S., Fazio, T., Gorman, J., and Visnapuu, M. L. (2010) DNA Curtains for High-Throughput Single-Molecule Optical Imaging, Methods in Enzymology, Vol 472: Single Molecule Tools, Pt A: Fluorescence Based Approaches 472, 293–315.Google Scholar
- 13.Finkelstein, I. J., and Greene, E. C. (in press) Supported lipid-bilayers and DNA curtains for high-throughput single molecule studies. Methods in Molecular Biology.Google Scholar