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Sequence-specific detection of individual DNA strands using engineered nanopores

Abstract

We describe biosensor elements that are capable of identifying individual DNA strands with single-base resolution. Each biosensor element consists of an individual DNA oligonucleotide covalently attached within the lumen of the α-hemolysin (αHL) pore to form a “DNA–nanopore”. The binding of single-stranded DNA (ssDNA) molecules to the tethered DNA strand causes changes in the ionic current flowing through a nanopore. On the basis of DNA duplex lifetimes, the DNA–nanopores are able to discriminate between individual DNA strands up to 30 nucleotides in length differing by a single base substitution. This was exemplified by the detection of a drug resistance–conferring mutation in the reverse transcriptase gene of HIV. In addition, the approach was used to sequence a complete codon in an individual DNA strand tethered to a nanopore.

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Figure 1: A single DNA oligonucleotide attached to the αHL pore.
Figure 2: A single-base mismatch abolishes the binding of an oligonucleotide to a tethered DNA strand within a DNA–nanopore.
Figure 3: A DNA–nanopore detects a common mutation, which confers resistance to the drug nevirapine in the reverse transcriptase gene of HIV.

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References

  1. Eigen, M. & Rigler, R. Sorting single molecules: application to diagnostics and evolutionary biotechnology. Proc. Natl. Acad. Sci. USA 91, 5740–5747 (1994).

    Article  CAS  Google Scholar 

  2. Strunz, T., Oroszlan, K., Schafer, R. & Guntherodt, H.J. Dynamic force spectroscopy of single DNA molecules. Proc. Natl. Acad. Sci. USA 96, 11277–11282 (1999).

    Article  CAS  Google Scholar 

  3. Smith, S.B., Cui, Y. & Bustamante, C. Overstretching B-DNA: the elastic response of individual double-stranded and single-stranded DNA molecules. Science 271, 795–799 (1996).

    Article  CAS  Google Scholar 

  4. Rief, M., Clausen-Schaumann, H. & Gaub, H.E. Sequence-dependent mechanics of single DNA molecules. Nat. Struct. Biol. 6, 346–349 (1999).

    Article  CAS  Google Scholar 

  5. 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 

  6. Henrickson, S.E., Misakian, M., Robertson, B. & Kasianowicz, J.J. Driven DNA transport into an asymmetric nanometer-scale pore. Phys. Rev. Lett. 85, 3057–3060 (2000).

    Article  CAS  Google Scholar 

  7. 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 

  8. 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 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  11. Aboul-ela, F., Koh, D., Tinoco, I. Jr. & Martin, F.H. Base-base mismatches. Thermodynamics of double helix formation for dCA3XA3G + dCT3YT3G (X, Y = A,C,G,T). Nucleic Acids Res. 13, 4811–4824 (1985).

    Article  CAS  Google Scholar 

  12. Richman, D.D. et al. Nevirapine resistance mutations of human immunodeficiency virus type 1 selected during therapy. J. Virol. 68, 1660–1666 (1994).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Movileanu, L., Howorka, S., Braha, O. & Bayley, H. Detecting protein analytes that modulate transmembrane movement of a polymer chain within a single protein pore. Nat. Biotechnol. 18, 1091–1095 (2000).

    Article  CAS  Google Scholar 

  14. Braha, O. et al. Designed protein pores as components for biosensors. Chem. Biol. 4, 497–505 (1997).

    Article  CAS  Google Scholar 

  15. Gu, L.Q., Braha, O., Conlan, S., Cheley, S. & Bayley, H. Stochastic sensing of organic analytes by a pore-forming protein containing a molecular adapter. Nature 398, 686–690 (1999).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  17. Howorka, S. et al. A protein pore with a single polymer chain tethered within the lumen. J. Am. Chem. Soc. 122, 2411–2416 (2000).

    Article  CAS  Google Scholar 

  18. Stora, T., Lakey, J.H. & Vogel, H. Ion-channel gating in transmembrane receptor proteins: functional activity in tethered lipid membranes. Angew. Chem. Int. Edn. Engl. 38, 389–392 (1999).

    Article  CAS  Google Scholar 

  19. Cornell, B.A. et al. A biosensor that uses ion-channel switches. Nature 387, 580–583 (1997).

    Article  CAS  Google Scholar 

  20. Hulteen, J.C., Jirage, K.B. & Martin, C.R. Introducing chemical transport selectivity into gold nanotubule membranes. J. Amer. Chem. Soc. 120, 6603–6604 (1998).

    Article  CAS  Google Scholar 

  21. Schmidt, C., Mayer, M. & Vogel, H. A chip-based bisensor for the functional analysis of single ion-channels. Angew. Chem. Int. Edn. Engl. 39, 3137–3140 (2000).

    Article  CAS  Google Scholar 

  22. Quake, S.R. & Scherer, A. From micro- to nanofabrication with soft materials. Science 290, 1536–1540 (2000).

    Article  CAS  Google Scholar 

  23. Corey, D.R., Munoz-Medellin, D. & Huang, A. Strand invasion by oligonucleotide–nuclease conjugates. Bioconjug. Chem. 6, 93–100 (1995).

    Article  CAS  Google Scholar 

  24. Cheley, S., Braha, O., Lu, X., Conlan, S. & Bayley, H. A functional protein pore with a “retro” transmembrane domain. Protein Sci. 8, 1257–1267 (1999).

    Article  CAS  Google Scholar 

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Acknowledgements

The authors thank Stephen Cheley for providing the plasmid construct αHL-WT-RL-D4 and Orit Braha and Liviu Movileanu for helpful discussions. This work was supported by the US Department of Energy, NIH, the Office of Naval Research (Multidisciplinary University Research Initiative 1999), and the Texas Advanced Technology Program. S.H. is currently recipient of a fellowship from the Max-Kade Foundation and was supported by a postdoctoral scholarship from the Austrian Science Foundation (Fonds zur Förderung der wissenschaftlichen Forschung) during the earlier stages of the work.

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Correspondence to Stefan Howorka.

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Howorka, S., Cheley, S. & Bayley, H. Sequence-specific detection of individual DNA strands using engineered nanopores. Nat Biotechnol 19, 636–639 (2001). https://doi.org/10.1038/90236

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