What is a Nanopore?

A nanopore is defined as a 1 – 100 nm hole in a thin membrane. This diameter is in the range of molecules and supramolecular structures that are biologically important at the cellular level. For instance, simple solutes such as amino acids, sugars and nucleotides are approximately 1 nm in diameter, the DNA double helix is ~2 nm in diameter, and globular proteins such as those in blood plasma are 2 - 6 nm in diameter. Ribosomes are 20 nm in diameter, and viruses range from 10 to 50 nm. This size relationship means that if a nanopore can be produced in a thin film or membrane, its size can be adjusted so that certain molecules in solution can pass through the pore, while others are excluded.

Biological nanopores include gramicidin, ion channels such as the potassium channel and the acetylcholine receptor, and porins of mitochondria and bacteria. A special case is the bacterial toxin alpha hemolysin, which forms a very stable transmembrane pore ~ 2nm in diameter. Synthetic pores include Nucleopores used in filtration (50 – 200 nm), etched nuclear tracks in mica (DeBlois et al. 1977), and nanosculpted nanopores in silicon nitride (Li et al. 2001)

A nanopore in action. Above image: A hemolysin channel has penetrated a lipid bilayer and conducts an open channel current of about 120 picoamperes, with a voltage of 120 millivolts. Potassium and chloride ions carry the current in 1.0 M KCl. Center image: When a DNA hairpin is captured by the nanopore, it partially blocks the ionic current, which is reduced by about half. Right image: A few milliseconds later the DNA hairpin spontaneously unfolds and is drawn through the limiting aperture of the hemolysin, producing a transient downward spike to about 10% of the open channel current. See Vercoutere et al. 2001.

What can Nanopores do?

If a nanopore is produced in a thin film that separates two compartments, ionic current can be driven through the pore when a voltage is imposed across the membrane containing the pore. Typical biological pores in cell membranes pass currents in the picoampere range when a 100 mV potential is applied and 0.1 M KCl is present. A large pore such as alpha hemolysin passes 120 picoamperes of ionic current at the same potential in 1.0 M KCl. Because typical molecules of biological relevance are in the nanometer size range, they can enter a nanopore of appropriate diameter and affect the ionic current flowing through the pore. An example is polyethylene glycol, a globular molecule which was shown to enter the hemolysin pore and produce measurable effects on the ionic current passing through the pore (Bezrukov et al 1994) . Linear polymers such as nucleic acids can also be drawn into the hemolysin pore. While they pass through the pore the molecule partially interrupts the ionic current to produce a signal called a blockade. Modulation of the ionic current blockade provides information about the concentration, length and composition of the molecule. ( Kasianowicz et al. 1996; Akeson et al. 1999; Meller et al. 2001). Similar measurements have been made with a synthetic SiN pore (Li et al 2001).

Both the size and motion of a molecule passing through a nanopore modulate the current in ways that give information about the molecule. To give a few examples obtained with the hemolysin pore, Kasianowicz et al. 1996 showed that the duration of an ionic current blockade was roughly proportional to the length of RNA strands ranging from 5 to 500 bases in length. Akeson et al. (1999) demonstrated that RNA homopolymers of purines (polyadenylic acid) and pyrimidines (polycytidylic acid) differed markedly in the blockade signal they produced, and that a block copolymer (A30C70G)produced a blockade with a distinct break that distinguished the A portion from the C portion. Vercoutere et al. (2001) found that DNA hairpins ranging from 3 to 9 base pairs could be distinguished with single base-pair resolution. Hworka et al (2002) demonstrated that a DNA sequence seven bases in length could be covalently bound to the hemolysin pore and gave a distinct signal when a complementary sequence passed through the pore.

Nanopores in the future

The state of the art in 2003 is to produce a synthetic nanopore that can emulate the desirable properties of the hemolysin pore, yet be far more robust so that it can be incorporated into a commercial instrument. An exciting possibility for such a nanopore instrument is that it has the potential to produce a vast improvement in sequencing rates. Because single stranded nucleic acids pass through the pore in strict linear sequence, in principle each base could be detected and read, much as the magnetic head of a tape recorder reads the magnetic signal embedded in the tape. If single base resolution becomes possible, nucleic acid sequences could be obtained at rates thousands of times faster than currently possible.

Other applications of nanopores do not depend on direct sequencing. For instance, synthetic nanopores should be able to serve as biosensors for DNA hybridization and SNP detection, identifying targeted encoded polymers as well as identifying proteins and viruses at with single molecule resolution.
The rest of this web site contains a history of the nanopore concept, links and information about laboratories working on nanopores, and a news page announcing meetings, publications, companies and grants related to nanopores.

References

Akeson, M., D. Branton, J.J. Kasianowicz, E. Brandin, and D.W. Deamer, Biophys. J. 77, 3227 (1999)

DeBlois, R.W., C.P. Bean and R.K.A.Wesley, J. Coll. Interface Sci. 61, 323 (1977)

Howorka, S., S. Cheley and H. Bayley, Nat Biotechnol 19, 636 (2001)

Kasianowicz, J., E. Brandin, D. Branton and D.W. Deamer, Proc. Natl. Acad. Sci. USA 93, 13770 (1996)

Li, J. D. Stein, C. McMullan, D. Branton, M. J. Aziz, and J. A. Golovchenko, . Nature 412, 166 (2001)

Meller, A., L. Nivon, and D. Branton, Phys. Rev. Lett. 86,3435 (2001)

Vercoutere, W., S. Winters-Hilt, H. Olsen, D. Deamer. D. Haussler and M. Akeson Nat. Biotechnol. 19, 248 (2001)