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Exploring protein-DNA interactions in 3D using in situ construction, manipulation and visualization of individual DNA dumbbells with optical traps, microfluidics and fluorescence microscopy

Nature Protocols volume 8, pages 525–538 (2013)Cite this article

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Abstract

In this protocol, we describe a procedure to generate 'DNA dumbbells'—single molecules of DNA with a microscopic bead attached at each end—and techniques for manipulating individual DNA dumbbells. We also detail the design and fabrication of a microfluidic device (flow cell) used in conjunction with dual optical trapping to manipulate DNA dumbbells and to visualize individual protein-DNA complexes by single-molecule epifluorescence microscopy. Our design of the flow cell enables the rapid movement of trapped molecules between laminar flow channels and a flow-free reservoir. The reservoir provides the means to examine the formation of protein-DNA complexes in solution in the absence of external flow forces while maintaining a predetermined end-to-end extension of the DNA. These features facilitate the examination of the role of 3D DNA conformation and dynamics in protein-DNA interactions. Preparation of flow cells and reagents requires 2 days each; in situ DNA dumbbell assembly and imaging of single protein-DNA complexes require another day.

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Figure 1: Examples of techniques used to tether and extend DNA for single-molecule imaging.
Figure 2: Custom-fabricated flow cell that contains channels for laminar buffer flow and a flow-free reservoir.
Figure 3: DNA dumbbell captured in a dual optical trap showing a single fluorescent RecA-ssDNA nucleoprotein filament paired at the homologous locus.
Figure 4: Schematic diagram for a dual laser-trap microscope.
Figure 5: Steps involved in flow cell fabrication.
Figure 6: Attachment of the cover glass to the etched microscope slide with ultraviolet curing adhesive.
Figure 7: Flow cell installed in the microscope.
Figure 8: Custom-fabricated holder for a 50-μl reservoir syringe.
Figure 9: Summary of the protocol for the visualization of homologous ssDNA pairing with an individual optically trapped DNA dumbbell promoted by RecA.

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Acknowledgements

The authors are appreciative of members of the Kowalczykowski laboratory for their comments on this work. A.L.F. was funded by an American Cancer Society Postdoctoral Fellowship (PF-08–046–01-GMC); C.C.D. was supported by US National Institutes of Health (NIH) training grant T32 CA108459; and S.C.K. was supported by grants from the NIH (GM-62653 and GM-64745).

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Authors and Affiliations

  1. Department of Microbiology and Molecular Genetics, University of California, Davis, California, USA

    Anthony L Forget, Christopher C Dombrowski, Ichiro Amitani & Stephen C Kowalczykowski

  2. Department of Molecular and Cellular Biology, University of California, Davis, California, USA

    Anthony L Forget, Christopher C Dombrowski, Ichiro Amitani & Stephen C Kowalczykowski

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  1. Anthony L Forget
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  2. Christopher C Dombrowski
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  4. Stephen C Kowalczykowski
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Contributions

A.L.F. and C.C.D. conceived the reservoir flow cell design and fabrication process; I.A., C.C.D. and A.L.F. built the microscope; A.L.F. and S.C.K. conceived the RecA pairing experiments; A.L.F. carried out the RecA pairing experiments and A.L.F., C.C.D., I.A. and S.C.K. wrote and revised the manuscript.

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Correspondence to Stephen C Kowalczykowski.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Figure 1

The template used in the laser-etching step of the flow cell fabrication (PDF 2032 kb)

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Forget, A., Dombrowski, C., Amitani, I. et al. Exploring protein-DNA interactions in 3D using in situ construction, manipulation and visualization of individual DNA dumbbells with optical traps, microfluidics and fluorescence microscopy. Nat Protoc 8, 525–538 (2013). https://doi.org/10.1038/nprot.2013.016

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