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Single-molecule analysis of DNA-protein complexes using nanopores

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Abstract

We present a method for rapid measurement of DNA-protein interactions using voltage-driven threading of single DNA molecules through a protein nanopore. Electrical force applied to individual ssDNA-exonuclease I complexes pulls the two molecules apart, while ion current probes the dissociation rate of the complex. Nanopore force spectroscopy (NFS) reveals energy barriers affecting complex dissociation. This method can be applied to other nucleic acid–protein complexes, using protein or solid-state nanopore devices.

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Figure 1: Detecting ssDNA-ExoI complexes using a nanopore.
Figure 2: Monitoring ExoI digestion of DNA using a nanopore.
Figure 3: NFS of DNA-protein complexes.

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Acknowledgements

We thank D. Branton, D. Deamer and A. Marziali for thoughtful critiques of early drafts of this manuscript. K. Lieberman edited the final draft and provided important advice on presentation of our results. S. Benner and R. Abu-Shumays provided technical assistance. This work was supported by US National Human Genome Research Institute Grant HG003703 (M.A.), National Institute of General Medical Sciences Grant GM075893 (A.M.), National Science Foundation Grant NIRT-26384 (A.M.) and Human Frontier Science Program Award RGP0036/2005 (A.M.). Concentrated stocks of Exonuclease I were a gift from New England Biolabs.

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Author notes

  1. Richard D Whitaker & Stephen J Picone

    Present address: Present address: Enzymatics, 100 Cummings Center, Suite 336H, Beverly, Massachusetts 01915, USA.,

Authors and Affiliations

  1. Departments of Chemistry & Biomolecular Engineering, MS SOE2, University of California, Santa Cruz, 95064, California, USA

    Breton Hornblower, Amy Coombs & Mark Akeson

  2. Departments of Biomedical Engineering & Physics, Boston University, 44 Cummington St., Boston, 02215, Massachusetts, USA

    Breton Hornblower & Amit Meller

  3. New England Biolabs, 240 County Road, Ipswich, 01938, Massachusetts, USA

    Richard D Whitaker & Stephen J Picone

  4. Department of Chemistry, MS60 Rice University, Houston, 77005, Texas, USA

    Anatoly Kolomeisky

Authors
  1. Breton Hornblower
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  2. Amy Coombs
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  3. Richard D Whitaker
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  4. Anatoly Kolomeisky
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  5. Stephen J Picone
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  6. Amit Meller
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  7. Mark Akeson
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Contributions

B.H. performed the majority of nanopore experiments and wrote the first draft of the manuscript; A.C. performed nanopore experiments in Santa Cruz; R.D.W. and S.J.P. contributed ExoI purification and ExoI biochemical assays; A.K. contributed theoretical models; A.M. and M.A. directed the research and are responsible for the overall quality of the work.

Corresponding authors

Correspondence to Amit Meller or Mark Akeson.

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Competing interests

R.D.W. and S.J.P. were employees of New England Biolabs when this work was done.

Supplementary information

Supplementary Fig. 1

ExoI-catalyzed hydrolysis of FAM-50mer or FAM-50mer-P substrates, in the absence or presence of 1 M KCl. (PDF 65 kb)

Supplementary Methods (PDF 100 kb)

Supplementary Note (PDF 100 kb)

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Hornblower, B., Coombs, A., Whitaker, R. et al. Single-molecule analysis of DNA-protein complexes using nanopores. Nat Methods 4, 315–317 (2007). https://doi.org/10.1038/nmeth1021

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