Visualizing single DNA-bound proteins using DNA as a scanning probe

Abstract

Many biological processes involve enzymes moving along DNA. Such motion might be impeded by DNA-bound proteins or DNA supercoils. Current techniques are incapable of directly measuring forces that such 'roadblocks' might impose. We constructed a setup with four independently moveable optical traps, allowing us to manipulate two DNA molecules held between beads. By tightly wrapping one DNA around the other, we created a probe that can be scanned along the contour of the second DNA. We found that friction between the two polymers remains below 1 pN. Upon encountering DNA-bound proteins substantial friction forces are measured, allowing accurate localization of protein positions. Furthermore, these proteins remained associated at low probe tensions but could be driven off using forces greater than 20 pN. Finally, the full control of the orientation of two DNA molecules opens a wide range of experiments on proteins interacting with multiple DNA regions.

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Figure 1: Dual DNA manipulation assay.
Figure 2: DNA scanning scheme and reference scan.
Figure 3: Detection of individual DNA-bound restriction enzymes.
Figure 4: Nondestructive and destructive imaging.

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Acknowledgements

We thank E.J.G. Peterman and R.T. Dame for useful discussions. We acknowledge D.A. Hiller (Yale) and J.J. Perona (University of California Santa Barbara) for the kind gift of EcoRV. This work is part of the research program of the 'Stichting voor Fundamenteel Onderzoek der Materie (FOM)', which is financially supported by the 'Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO)' and was supported by a NWO-Vernieuwingsimpuls grant.

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Correspondence to Gijs J L Wuite.

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Supplementary Text and Figures

Supplementary Figures 1–3, Supplementary Note (PDF 185 kb)

Supplementary Movie 1

Trapping beads, catching two DNA molecules and wrapping of the probing DNA around the scanned DNA. The movie (sped up 3×) shows how two optically trapped DNA molecules are wound around each other. First, beads are captured in the four optical traps. Next, two DNA molecules are caught between these beads. The displacements of the beads out of their traps (indicated by crosshairs) are a signal that DNA is tethered between them. By moving one of the traps in the direction perpendicular to the field of view, the DNA molecules are then wound around each other. Finally, the DNA molecules are brought into a crossed configuration, required for the DNA scanning experiments. (MOV 5015 kb)

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Noom, M., van den Broek, B., van Mameren, J. et al. Visualizing single DNA-bound proteins using DNA as a scanning probe. Nat Methods 4, 1031–1036 (2007). https://doi.org/10.1038/nmeth1126

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