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Tethered particle analysis of supercoiled circular DNA using peptide nucleic acid handles

An Erratum to this article was published on 20 November 2014

This article has been updated

Abstract

This protocol describes how to monitor individual naturally supercoiled circular DNA plasmids bound via peptide nucleic acid (PNA) handles between a bead and a surface. The protocol was developed for single-molecule investigation of the dynamics of supercoiled DNA, and it allows the investigation of both the dynamics of the molecule itself and of its interactions with a regulatory protein. Two bis-PNA clamps designed to bind with extremely high affinity to predetermined homopurine sequence sites in supercoiled DNA are prepared: one conjugated with digoxigenin for attachment to an anti-digoxigenin-coated glass cover slide, and one conjugated with biotin for attachment to a submicron-sized streptavidin-coated polystyrene bead. Plasmids are constructed, purified and incubated with the PNA handles. The dynamics of the construct is analyzed by tracking the tethered bead using video microscopy: less supercoiling results in more movement, and more supercoiling results in less movement. In contrast to other single-molecule methodologies, the current methodology allows for studying DNA in its naturally supercoiled state with constant linking number and constant writhe. The protocol has potential for use in studying the influence of supercoils on the dynamics of DNA and its associated proteins, e.g., topoisomerase. The procedure takes 4 weeks.

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Figure 1: Flowchart of the entire procedure providing an overview of the main steps involved in developing the assay and performing tethered particle motion experiments.
Figure 2: Sketch of the naturally supercoiled DNA to which two PNA handles (yellow) are specifically attached.
Figure 3: Constructed plasmids.
Figure 4: Electrophoretic analysis of materials.
Figure 5: Preparation of perfusion chamber.
Figure 6: Image analysis.
Figure 7: Example of PCA.
Figure 8: r.m.s.d. of various plasmid constructs usable for troubleshooting the overall length of the tether.
Figure 9: Typical data resulting from TPM experiments with a supercoiled DNA plasmid.

Change history

  • 12 September 2014

     In the version of this article initially published, the source from which Figures 2, 4, 8 and 9 was adapted was not cited and credited correctly. The error has been corrected in the HTML and PDF versions of the article.

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Acknowledgements

The CI protein was a generous gift from D. Lewis and S. Adhya, Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, US National Institutes of Health. We acknowledge financial support from a University of Copenhagen center of excellence and from the Swedish Research Council.

Author information

Authors and Affiliations

Authors

Contributions

P.E.N., S.B. and L.B.O. designed the study; K.N., M.A. and S.B. performed the experiments; P.E.N. and S.B. contributed new reagents; K.N., M.A., S.B. and L.B.O. analyzed data; all authors wrote the paper.

Corresponding author

Correspondence to Lene B Oddershede.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Purification and identity of PNA product.

(a) HPLC analysis of purified digoxigenin labelled bis-PNA. (b) MALDI-TOF spectrum of digoxigenin labelled bis-PNA (calculated mass is 7187 and the found mass is within the accuracy of the instrument).

Supplementary information

Supplementary Figure 1

Purification and identity of PNA product. (PDF 499 kb)

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Norregaard, K., Andersson, M., Nielsen, P. et al. Tethered particle analysis of supercoiled circular DNA using peptide nucleic acid handles. Nat Protoc 9, 2206–2223 (2014). https://doi.org/10.1038/nprot.2014.152

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