Summary
Nanopore analysis of single molecules can be performed by measuring the modulation in ionic current passing through the nanopore while an individual biomolecule such as DNA or RNA is resident in, translocating through, or otherwise interacting with the pore. The corresponding current signature has been shown to reveal properties of the biomolecule and information on its interactions with the pore. The α-hemolysin nanopore remains the pore of choice, particularly for single-molecule analysis of nucleic acids, because of its internal dimensions, hydrophilicity, and low-noise characteristics. In this chapter we present a detailed protocol for forming a robust α-hemolysin nanopore (or multiple nanopores) for single-molecule analysis.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Bhakdi, S., and Tranum-Jensen, J. (1991). Alpha-toxin of Staphylococcus aureus. Microbiol. Mol. Biol. Rev. 55, 733–751.
Song, L.Z., Hobaugh, M.R., Shustak, C., Cheley, S., Bayley, H., and Gouaux, J.E. (1996). Structure of staphylococcal alpha-hemolysin, a heptameric transmembrane pore. Science 274, 1859–1866.
Kasianowicz, J.J., Brandin, E., Branton, D., and Deamer, D.W. (1996). Characterization of individual polynucleotide molecules using a membrane channel. Proc. Natl Acad. Sci. U. S. A. 93, 13770–13773.
Akeson, M., Branton, D., Kasianowicz, J.J., Brandin, E., and Deamer, D.W. (1999). 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.
Nakane, J., Wiggin, M., and Marziali, A. (2004). A nanosensor for transmembrane capture and identification of single nucleic acid molecules. Biophys. J. 87, 615–621.
Howorka, S., Movileanu, L., Braha, O., and Bayley, H. (2001). Kinetics of duplex for individual DNA strands within a single protein nanopore. Proc. Natl Acad. Sci. U. S. A. 98, 12997–13001.
Sauer-Budge, A.F., Nyamwanda, J.A., Lubensky, D.K., and Branton, D. (2003). Unzipping kinetics of double-stranded DNA in a nanopore. Phys. Rev. Lett. 90, 238101-1–238101-4.
Ashkenasy, N., Sanchez-Quesada, J., Bayley, H., and Ghadiri, M.R. (2005). Recognizing a single base in an individual DNA strand: a step toward DNA sequencing in nanopores. Angew. Chem. Int. Ed. 44, 1401–1404.
Astier, Y., Braha, O., and Bayley, H. (2006). Toward single molecule DNA sequencing: direct identification of ribonucleoside and deoxyribonucleoside 5′-monophosphates by using an engineered protein nanopore equipped with a molecular adapter. J. Am. Chem. Soc. 128, 1705–1710.
Shin, S., Luchian, T., Cheley, S., Braha, O., and Bayley, H. (2002). Kinetics of a reversible covalent-bond-forming reaction observed at the single molecule level. Angew. Chem. Int. Ed. 41, 3707–3709.
Luchian, T., Shin, S., and Bayley, H. (2003). Kinetics of a three-step reaction observed at the single-molecule level. Angew. Chem. Int. Ed. 42, 1925–1929.
Jung, Y., Bayley, H., and Movileanu, L. (2006). Temperature-responsive protein pores. J. Am. Chem. Soc. 128, 15332–15340.
Oukhaled, G., Mathe, J., Biance, A.L., Bacri, L., Betton, J.M., Lairez, D., Pelta, J., and Auvray, L. (2007). Unfolding of proteins and long transient conformations detected by single nanopore recording. Phys. Rev. Lett. 98, 158101-1–158101-4.
Healy, K. (2007). Nanopore-based single-molecule DNA analysis. Nanomedicine 2, 459–481.
Nakane, J.J., Akeson, M., and Marziali, A. (2003). Nanopore sensors for nucleic acid analysis. J. Phys.: Condens. Matter. 15, 1365–1393.
Rhee, M., and Burns, M.A. (2006). Nanopore sequencing technology: research trends and applications. Trends Biotechnol. 24, 580–586.
Dekker, C. (2007). Solid-state nanopores. Nat. Nanotechnol. 2, 209–215.
Tropini C., and Marziali A. (2007). Multi-nanopore force spectroscopy for DNA analysis. Biophys. J. 92, 1632–1637.
Acknowledgments
We thank members of the University of British Columbia Applied Biophysics Laboratory, in particular, Vincent Tabard-Cossa and Dhruti Trivedi. Many thanks to Jonathan Nakane, Mark Akeson, and Carolina Tropini for their important contributions to this protocol. This work was supported by the US National Institutes of Health (NIH). Financial support for Matthew Wiggin was provided by the Natural Sciences and Engineering Research Council of Canada (NSERC).
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Jetha, N.N., Wiggin, M., Marziali, A. (2009). Forming an α-Hemolysin Nanopore for Single-Molecule Analysis. In: Foote, R., Lee, J. (eds) Micro and Nano Technologies in Bioanalysis. Methods in Molecular Biology™, vol 544. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-59745-483-4_9
Download citation
DOI: https://doi.org/10.1007/978-1-59745-483-4_9
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-934115-40-4
Online ISBN: 978-1-59745-483-4
eBook Packages: Springer Protocols