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.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Rent or buy this article
Prices vary by article type
from$1.95
to$39.95
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Eigen, M. & Rigler, R. Sorting single molecules: application to diagnostics and evolutionary biotechnology. Proc. Natl. Acad. Sci. USA 91, 5740–5747 (1994).
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).
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).
Rief, M., Clausen-Schaumann, H. & Gaub, H.E. Sequence-dependent mechanics of single DNA molecules. Nat. Struct. Biol. 6, 346–349 (1999).
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).
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).
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).
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).
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).
Song, L. et al. Structure of staphylococcal alpha-hemolysin, a heptameric transmembrane pore. Science 274, 1859–1866 (1996).
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).
Richman, D.D. et al. Nevirapine resistance mutations of human immunodeficiency virus type 1 selected during therapy. J. Virol. 68, 1660–1666 (1994).
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).
Braha, O. et al. Designed protein pores as components for biosensors. Chem. Biol. 4, 497–505 (1997).
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).
Gu, L.Q., Cheley, S. & Bayley, H. Capture of a single molecule in a nanocavity. Science 291, 636–640 (2001).
Howorka, S. et al. A protein pore with a single polymer chain tethered within the lumen. J. Am. Chem. Soc. 122, 2411–2416 (2000).
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).
Cornell, B.A. et al. A biosensor that uses ion-channel switches. Nature 387, 580–583 (1997).
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).
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).
Quake, S.R. & Scherer, A. From micro- to nanofabrication with soft materials. Science 290, 1536–1540 (2000).
Corey, D.R., Munoz-Medellin, D. & Huang, A. Strand invasion by oligonucleotide–nuclease conjugates. Bioconjug. Chem. 6, 93–100 (1995).
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).
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.
Author information
Authors and Affiliations
Corresponding author
Supplementary information
Rights and permissions
About this article
Cite this article
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
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1038/90236
This article is cited by
-
Hyper-CEST NMR of metal organic polyhedral cages reveals hidden diastereomers with diverse guest exchange kinetics
Nature Communications (2022)
-
DNA structures embedded with functionalized nanomaterials for biophysical applications
Journal of the Korean Physical Society (2021)
-
Dimension-reconfigurable bubble film nanochannel for wetting based sensing
Nature Communications (2020)
-
Resolving repeat families with long reads
BMC Bioinformatics (2019)
-
Relaxation Effect on Intrapore Diffusivities of Highly Charged Colloidal Particles Confined in Porous Membranes
Transport in Porous Media (2018)