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Sequence-specific detection of individual DNA polymerase complexes in real time using a nanopore

Nature Nanotechnology volume 2, pages 718–724 (2007)Cite this article

Abstract

Nanoscale pores have potential to be used as biosensors and are an established tool for analysing the structure and composition of single DNA or RNA molecules1,2,3. Recently, nanopores have been used to measure the binding of enzymes to their DNA substrates4,5. In this technique, a polynucleotide bound to an enzyme is drawn into the nanopore by an applied voltage. The force exerted on the charged backbone of the polynucleotide by the electric field is used to examine the enzyme–polynucleotide interactions. Here we show that a nanopore sensor can accurately identify DNA templates bound in the catalytic site of individual DNA polymerase molecules. Discrimination among unbound DNA, binary DNA/polymerase complexes, and ternary DNA/polymerase/deoxynucleotide triphosphate complexes was achieved in real time using finite state machine logic. This technique is applicable to numerous enzymes that bind or modify DNA or RNA including exonucleases, kinases and other polymerases.

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Figure 1: Detection of DNA translocation events with the nanopore device.
Figure 2: Distinguishing DNA, DNA/KF complexes or DNA/KF/dNTP complexes in the nanopore device.
Figure 3: Detection of correct dNTP binding to the KF/primer-template complex.
Figure 4: Proposed mechanism for voltage-facilitated dissociation of DNA from KF/DNA or KF/DNA/dNTP complexes.
Figure 5: Recognition and control of DNA complexes in real time using FPGA.

References

  1. Deamer, D. W. & Branton, D. Characterization of nucleic acids by nanopore analysis. Acc. Chem. Res. 35, 817–825 (2002).

    Article  CAS  Google Scholar 

  2. Dekker, C. Solid-state nanopores. Nature Nanotechnol. 2, 209–215 (2007).

    Article  CAS  Google Scholar 

  3. Siwy, Z. et al. Protein biosensors based on biofunctionalized conical gold nanotubes. J. Am. Chem. Soc. 127, 5000–5001 (2005).

    Article  CAS  Google Scholar 

  4. Hornblower, B. et al. Single-molecule analysis of DNA–protein complexes using nanopores. Nature Methods 4, 315–317 (2007).

    Article  CAS  Google Scholar 

  5. Zhao, Q. et al. Detecting SNPs using a synthetic nanopore. Nano Lett. 7, 1680–1685 (2007).

    Article  CAS  Google Scholar 

  6. 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 

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

    Article  CAS  Google Scholar 

  8. Vercoutere, W. et al. Rapid discrimination among individual DNA hairpin molecules at single-nucleotide resolution using an ion channel. Nature Biotechnol. 19, 248–252 (2001).

    Article  CAS  Google Scholar 

  9. Vercoutere, W. A. et al. Discrimination among individual Watson–Crick base pairs at the termini of single DNA hairpin molecules. Nucleic Acids Res. 31, 1311–1318 (2003).

    Article  CAS  Google Scholar 

  10. 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 

  11. Ashkenasy, N., Sanchez-Quesada, J., Bayley, H. & Ghadiri, M. R. Recognizing a single base in an individual DNA strand: A step toward DNA sequencing in nanopores. Angew. Chem. Int. Edn 44, 1401–1404 (2005).

    Article  CAS  Google Scholar 

  12. Howorka, S., Cheley, S. & Bayley, H. Sequence-specific detection of individual DNA strands using engineered nanopores. Nature Biotechnol. 19, 636–639 (2001).

    Article  CAS  Google Scholar 

  13. Butler, T. Z., Gundlach, J. H. & Troll, M. A. Determination of RNA orientation during translocation through a biological nanopore. Biophys. J. 90, 190–199 (2006).

    Article  CAS  Google Scholar 

  14. Sauer-Budge, A. F., Nyamwanda, J. A., Lubensky, D. K. & Branton, D. Unzipping kinetics of double-stranded DNA in a nanopore. Phys. Rev. Lett. 90, 23801 (2003).

    Article  Google Scholar 

  15. Storm, A. J. et al. Fast DNA translocation through a solid-state nanopore. Nano Lett. 5, 1193–1197 (2005).

    Article  CAS  Google Scholar 

  16. Nakane, J., Akeson, M. & Marziali, A. Evaluation of nanopores as candidates for electronic analyte detection. Electrophoresis 23, 2592–2601 (2002).

    Article  CAS  Google Scholar 

  17. Li, J. et al. Ion-beam sculpting at nanometre length scales. Nature 412, 166–169 (2001).

    Article  CAS  Google Scholar 

  18. Gu, L.-Q., Cheley, S. & Bayley, H. Capture of a single molecule in a nanocavity Science 291, 636–640 (2001).

    Article  CAS  Google Scholar 

  19. Iqbal, S. M., Akin, D. & Bashir, R. Solid-state nanopore channels with DNA selectivity. Nature Nanotechnol. 2, 243–248 (2007).

    Article  CAS  Google Scholar 

  20. Joyce, C. M. & Steitz, T. A. Function and structure relationships in DNA polymerases. Annu. Rev. Biochem. 63, 777–822 (1994).

    Article  CAS  Google Scholar 

  21. Joyce, C. M. & Benkovic, S. J. DNA polymerase fidelity: Kinetics, structure, and checkpoints. Biochemistry 43, 14317–14324 (2004).

    Article  CAS  Google Scholar 

  22. Beese, L. S., Derbyshire, V. & Steitz, T. A. Structure of DNA polymerase I Klenow fragment bound to duplex DNA. Science 260, 352–355 (1993).

    Article  CAS  Google Scholar 

  23. Li, Y., Korolev, S. & Waksman, G. Crystal structures of open and closed forms of binary and ternary complexes of the large fragment of Thermus aquaticus DNA polymerase I: Structural basis for nucleotide incorporation. EMBO J. 17, 7514–7525 (1998).

    Article  CAS  Google Scholar 

  24. Johnson, S. J., Taylor, J. S. & Beese, L. S. Processive DNA synthesis observed in a polymerase crystal suggests a mechanism for the prevention of frameshift mutations. Proc. Natl Acad. Sci. USA 100, 3895–3900 (2003).

    Article  CAS  Google Scholar 

  25. Purohit, V., Grindley, N. D. F. & Joyce, C. M. Use of 2-aminopurine fluorescence to examine conformational changes during nucleotide incorporation by DNA polymerase I (Klenow fragment). Biochemistry 42, 10200–10211 (2003).

    Article  CAS  Google Scholar 

  26. Sanger, F., Nicklen, S. & Coulson, A. R. DNA sequencing with chain-terminating inhibitors. Proc. Natl Acad. Sci. USA 74, 5463–5467 (1977).

    Article  CAS  Google Scholar 

  27. Steitz, T. A., Smerdon, S. J., Jager, J. & Joyce, C. M. A unified polymerase mechanism for nonhomologous DNA and RNA polymerases. Science 266, 2022–2025 (1994).

    Article  CAS  Google Scholar 

  28. Bates, M., Burns, M. & Meller, A. Dynamics of DNA molecules in a membrane channel probed by active control techniques. Biophys. J. 84, 2366–2372 (2003).

    Article  CAS  Google Scholar 

  29. Winters-Hilt, S. et al. Highly accurate classification of Watson–Crick basepairs on termini of single DNA molecules. Biophys. J. 84, 967–976 (2003).

    Article  CAS  Google Scholar 

  30. Gill, A. Introduction to the Theory of Finite-State Machines (McGraw-Hill, 1962).

  31. Trimberger, S. M. Field-Programmable Gate Array Technology (Springer, 1994).

  32. Smeets, R. M. M. et al. Salt dependence of ion transport and DNA translocation through solid-state nanopores. Nano Lett. 6, 89–95 (2006).

    Article  CAS  Google Scholar 

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Acknowledgements

We wish to thank D. Branton, V. Tabard-Cossa, M. Wiggin, D. Krapf and R. Mathies for reading early versions of this manuscript. We also thank A. Kottas for guidance on statistical analysis. This work was supported by National Institutes of Health grants GM073617-01A1 (M.A.), HG003703-01 (M.A., D.D.) and HG004035-01 (W.D.).

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Authors and Affiliations

  1. Department of Biomolecular Engineering, Baskin School of Engineering, University of California, Santa Cruz, 95064, California, USA

    Seico Benner, Robin Abu-Shumays, Kate R. Lieberman, David W. Deamer, William B. Dunbar & Mark Akeson

  2. Department of Chemistry & Biochemistry, University of California, Santa Cruz, 95064, California, USA

    Roger J. A. Chen, Nicholas Hurt & David W. Deamer

  3. Department of Computer Engineering, University of California, Santa Cruz, 95064, California, USA

    Noah A. Wilson & William B. Dunbar

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  1. Seico Benner
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Correspondence to William B. Dunbar or Mark Akeson.

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Supplementary figures S1, S2 and supplementary table 1 (PDF 125 kb)

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Benner, S., Chen, R., Wilson, N. et al. Sequence-specific detection of individual DNA polymerase complexes in real time using a nanopore. Nature Nanotech 2, 718–724 (2007). https://doi.org/10.1038/nnano.2007.344

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