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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

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

Authors

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.

Corresponding author

Correspondence to Stephen C Kowalczykowski.

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Competing interests

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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