Nanopores and Single-Molecule Biophysics

Some proteins naturally form nanometer-scale pores that serve as channels for useful biochemical ions. Through this ionic communication, nanopores enable many functions in cells, such as allowing nerve cells to communicate (they are even responsible for twitching the frog leg in Galvani's famous discovery in the 1700s).

Nanopores can be destructive, too. When the proteins of bacteria and viruses attach to a cell, their nanopores can facilitate infection, for example by shooting viral DNA through them into the cell.

Last week, at the March Meeting of the American Physical Society in Baltimore, John J. Kasianowicz (National Institute of Standards and Technology, john.kasianowicz@nist.gov) showed how single biological nanopores can be used to detect and characterize individual molecules of RNA and DNA. He also demonstrated constructive uses for anthrax-related nanopores in diagnosing anthrax infections and testing anti-anthrax drugs.

Anthrax bacteria secrete a protein called "protective antigen" that attaches to an organic membrane such as a cell wall. The protein forms a nanopore that penetrates the membrane. When another anthrax protein, called "lethal factor," attaches to the protective antigen nanopore, it prevents ionic current from flowing through the pore and out of the organic membrane.

By monitoring animal blood samples for changes in ion current, Kasianowicz and his colleagues at the National Cancer Institute and the United States Army Medical Research Institute for Infectious Diseases electronically detected a complex of two anthrax proteins in less than an hour, as opposed to the existing methods which can take up to several days. Also, they demonstrated a method for screening potential therapeutic agents against anthrax toxins using the anthrax nanopore (see the NIST Web site for a picture and more information).

A Brown University group led by Sean Ling (Xinsheng_Ling@brown.edu) was among those reporting progress in developing a nanopore-based method for sequencing DNA faster and more cheaply than traditional biochemical techniques.

In one scenario, the change in ion current as DNA moves through the nanopore could yield the sequence of bases, or letters, in the DNA. However, the letters in DNA are so close to each other (about .4 nm), and the DNA moves so quickly through the nanopore, that researchers have had to come up with creative solutions for reading the individual letters. For example, the Brown group attaches complementary blocks of DNA, about six letters long, to the DNA sequence of interest, so that the researchers would read blocks of multiple letters at a time, while slowing down the passage of the DNA by attaching a magnetic bead to it.

Other researchers are finding value in developing nanopores for fundamental biology studies. Discussing his group's latest work with artificial, silicon-based nanopores, Cees Dekker of the Delft University of Technology in Delft, Holland (dekker@mb.tn.tudelft.nl), showed how lasers and other manipulations with the artificial pores are enabling new single-molecule biophysics studies on the properties of DNA, RNA, and proteins by studying how they pass through the pores.

Artist's rendering of DNA traversing through a nanopore at Physics News Graphics
NIST Web site with another picture and more information