Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Replication of individual DNA molecules under electronic control using a protein nanopore

Abstract

Nanopores can be used to analyse DNA by monitoring ion currents as individual strands are captured and driven through the pore in single file by an applied voltage. Here, we show that serial replication of individual DNA templates can be achieved by DNA polymerases held at the α-haemolysin nanopore orifice. Replication is blocked in the bulk phase, and is initiated only after the DNA is captured by the nanopore. We used this method, in concert with active voltage control, to observe DNA replication catalysed by bacteriophage T7 DNA polymerase (T7DNAP) and by the Klenow fragment of DNA polymerase I (KF). T7DNAP advanced on a DNA template against an 80-mV load applied across the nanopore, and single nucleotide additions were measured on the millisecond timescale for hundreds of individual DNA molecules in series. Replication by KF was not observed when this enzyme was held on top of the nanopore orifice at an applied potential of 80 mV. Sequential nucleotide additions by KF were observed upon applying controlled voltage reversals.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: The nanopore device.
Figure 2: Blocking oligomer inhibition of DNAP-catalysed DNA synthesis.
Figure 3: Blocking oligomer inhibition of bulk-phase T7DNAP binding and voltage-promoted deprotection of individual DNA substrate molecules.
Figure 4: Nucleotide addition may occur above the nanopore orifice (before the probing step) or at the nanopore orifice (during the probing step).
Figure 5: T7DNA replication of individual DNA substrate molecules deprotected and tethered in the nanopore.
Figure 6: KF replication of individual DNA substrate molecules deprotected and tethered in the nanopore.

References

  1. Benner, S. et al. Sequence-specific detection of individual DNA polymerase complexes in real time using a nanopore. Nature Nanotech. 2, 718–724 (2007).

    Article  CAS  Google Scholar 

  2. Cockroft, S. L., Chu, J., Amorin, M. & Ghadiri, M. R. A single-molecule nanopore device detects DNA polymerase activity with single-nucleotide resolution. J. Am. Chem. Soc. 130, 818–820 (2008).

    Article  CAS  Google Scholar 

  3. Hurt, N., Wang, H., Akeson, M. & Lieberman, K. R. Specific nucleotide binding and rebinding to individual DNA polymerase complexes captured on a nanopore. J. Am. Chem. Soc. 131, 3772–3778 (2009).

    Article  CAS  Google Scholar 

  4. Wilson, N. A. et al. Electronic control of DNA polymerase binding and unbinding to single DNA molecules. ACS Nano 3, 995–1003 (2009).

    Article  CAS  Google Scholar 

  5. Gyarfas, B. et al. Mapping the position of DNA polymerase-bound DNA templates in a nanopore at 5 Å resolution. ACS Nano 3, 1457–1466 (2009).

    Article  CAS  Google Scholar 

  6. Song, L. et al. Structure of Staphylococcal alpha-hemolysin, a heptameric transmembrane pore. Science 274, 1859–1866 (1996).

    Article  CAS  Google Scholar 

  7. Kasianowicz, J. J., Brandin, E., Branton, D. & Deamer, D. W. Characterization of individual polynucleotide molecules using a membrane channel. Proc. Natl Acad. Sci. USA 93, 13770–13773 (1996).

    Article  CAS  Google Scholar 

  8. Branton, D. et al. The potential and challenges of nanopore sequencing. Nature Biotechnol. 26, 1146–1153 (2008).

    Article  CAS  Google Scholar 

  9. Moffitt, J. R., Chemla, Y. R., Smith, S. B. & Bustamante, C. Recent advances in optical tweezers. Annu. Rev. Biochem. 77, 205–228 (2008).

    Article  CAS  Google Scholar 

  10. Lechner, R. L., Engler, M. J. & Richardson, C. C. Characterization of strand displacement synthesis catalyzed by bacteriophage T7 DNA polymerase. J. Biol. Chem. 258, 11174–11184 (1983).

    CAS  Google Scholar 

  11. Lechner, R. L. & Richardson, C. C. A preformed, topologically stable replication fork. Characterization of leading strand DNA synthesis catalyzed by T7 DNA polymerase and T7 gene 4 protein. J. Biol. Chem. 258, 11185–11196 (1983).

    CAS  Google Scholar 

  12. Tabor, S. & Richardson, C. C. Selective inactivation of the exonuclease activity of bacteriophage T7 DNA polymerase by in vitro mutagenesis. J. Biol. Chem. 264, 6447–6458 (1989).

    CAS  Google Scholar 

  13. Canceill, D., Viguera, E. & Ehrlich, S. D. Replication slippage of different DNA polymerases is inversely related to their strand displacement efficiency. J. Biol. Chem. 274, 27481–27490 (1999).

    Article  CAS  Google Scholar 

  14. Asseline, U. et al. Nucleic acid-binding molecules with high affinity and base sequence specificity: intercalating agents covalently linked to oligodeoxynucleotides. Proc. Natl Acad. Sci. USA 81, 3297–3301 (1984).

    Article  CAS  Google Scholar 

  15. Asseline, U., Bonfils, E., Dupret, D. & Thuong, N. T. Synthesis and binding properties of oligonucleotides covalently linked to an acridine derivative: new study of the influence of the dye attachment site. Bioconjug. Chem. 7, 369–379 (1996).

    Article  CAS  Google Scholar 

  16. Patel, S. S., Wong, I. & Johnson, K. A. Pre-steady-state kinetic analysis of processive DNA replication including complete characterization of an exonuclease-deficient mutant. Biochemistry 30, 511–525 (1991).

    Article  CAS  Google Scholar 

  17. Huber, H. E., Tabor, S. & Richardson, C. C. Escherichia coli thioredoxin stabilizes complexes of bacteriophage T7 DNA polymerase and primed templates. J. Biol. Chem. 262, 16224–16232 (1987).

    CAS  Google Scholar 

  18. Datta, K. & LiCata, V. J. Salt dependence of DNA binding by Thermus aquaticus and Escherichia coli DNA polymerases. J. Biol. Chem. 278, 5694–5701 (2003).

    Article  CAS  Google Scholar 

  19. Rothwell, P. J. & Waksman, G. Structure and mechanism of DNA polymerases. Adv. Protein Chem. 71, 401–440 (2005).

    Article  CAS  Google Scholar 

  20. Astatke, M., Grindley, N. D. & Joyce, C. M. How E. coli DNA polymerase I (Klenow fragment) distinguishes between deoxy- and dideoxynucleotides. J. Mol. Biol. 278, 147–165 (1998).

    Article  CAS  Google Scholar 

  21. Tabor, S. & Richardson, C. C. A single residue in DNA polymerases of the Escherichia coli DNA polymerase I family is critical for distinguishing between deoxy- and dideoxyribonucleotides. Proc. Natl Acad. Sci. USA 92, 6339–6343 (1995).

    Article  CAS  Google Scholar 

  22. Akeson, M., Branton, D., Kasianowicz, J. J., Brandin, E. & Deamer, D. W. Microsecond time-scale discrimination among polycytidylic acid, polyadenylic acid and polyuridylic acid as homopolymers or as segments within single RNA molecules. Biophys. J. 77, 3227–3233 (1999).

    Article  CAS  Google Scholar 

  23. Meller, A., Nivon, L., Brandin, E., Golovchenko, J. & Branton, D. Rapid nanopore discrimination between single polynucleotide molecules. Proc. Natl Acad. Sci. USA 97, 1079–1084 (2000).

    Article  CAS  Google Scholar 

  24. Kibbe, W. A. OligoCalc: an online oligonucleotide properties calculator. Nucleic Acids Res. 35, W43–W46 (2007).

    Article  Google Scholar 

Download references

Acknowledgements

The authors are grateful to P. Walker at Stanford University PAN for expert oligonucleotide synthesis, Y. Kolodji for help with data analysis, B. Gyarfas for making the Supplementary movies and for assistance with FSM implementation, D. Garalde for advice on the use of T7DNAP, and W. Kibbe (Northwestern University) for modifications to OligoCalc. This work was supported by grants from Oxford Nanopore Technologies and from NHGRI (1RC2HG005553-01) to M.A. and D.D.

Author information

Authors and Affiliations

Authors

Contributions

F.O. designed experiments and performed data analysis. K.R.L. co-authored the manuscript, and designed and conducted experiments. S.B., G.M.C. and J.M.D. conducted nanopore experiments, including FSM implementation. D.W.D. conceived the idea of coupling polymerases to nanopores and helped design experiments. M.A. co-authored the manuscript, conceived the blocking oligomer strategy, designed experiments and is responsible for the overall quality of the work.

Corresponding author

Correspondence to Mark Akeson.

Ethics declarations

Competing interests

M. Akeson and D. Deamer are consultants to Oxford Nanopore Technologies (Oxford, England). Oxford Nanopore Technologies has licensed rights to some of the inventions detailed in this manuscript and has sponsored some of the research presented here through a grant to the University of California.

Supplementary information

Supplementary information

Supplementary information (PDF 802 kb)

Supplementary information

Supplementary movie 1 (AVI 5643 kb)

Supplementary information

Supplementary movie 2 (AVI 27245 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Olasagasti, F., Lieberman, K., Benner, S. et al. Replication of individual DNA molecules under electronic control using a protein nanopore. Nature Nanotech 5, 798–806 (2010). https://doi.org/10.1038/nnano.2010.177

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nnano.2010.177

This article is cited by

Search

Quick links

Find nanotechnology articles, nanomaterial data and patents all in one place. Visit Nano by Nature Research