# Nanopores Have a Zillion Uses, Researchers Say

Nanopores, the tiny holes formed by proteins, could be used for a variety of applications, including sequencing DNA and detecting anthrax. Researchers reported the latest developments on natural and artificial nanopores and their applications at the March Meeting in a number of sessions devoted to the topic.

Nanopores are nanometer scale holes formed naturally by proteins or cells, for instance to allow ions to pass between nerve cells. Single nanopores form the basis for nerve activity. Similarly sized holes can also be made artificially.

Sean Ling of Brown University is one of many researchers working on DNA sequencing using nanopores. The basic idea was first proposed 10 years ago as an alternative to the standard method of DNA sequencing, which requires making many copies of a strand of DNA, chopping it up into small pieces for sequencing and then reconstructing the genome. This method is slow and expensive, costing about $10 million to sequence the 109 bases in the entire human genome. Ling hopes nanopore sequencing could reduce that to about$1,000 per genome, and allow genomes to be sequenced in days.

Nanopore sequencing would work by looking at changes in ion flow as a single strand of DNA in a solution flows through a nanopore. Each nucleotide would affect the current in a characteristic way. One problem with this approach is that bases in a strand of DNA are too close together and move too quickly through the nanopore, making it difficult to identify individual nucleotides. Ling gets around this problem by attaching known probes, six or eight bases long, to single stranded strings of DNA, making it possible to read the strand in chunks of six or eight letters instead of one letter at a time. Also, by attaching a magnetic particle to one end of the DNA strand, he can slow down the rate at which the DNA traverses the pore, allowing for better resolution. In addition to the work with single nanopores, Ling also reported on developments of addressable nanopore arrays.

There are still some problems with nanopore DNA sequencing, said Ling, but as silicon nanopore technology becomes more reliable and affordable, fast DNA sequencing using nanopores will become a reality. There are challenges, but no show stoppers, Ling said.

Meanwhile, John Kasianowicz of NIST, who was one of the first to suggest that nanopores could be used for rapid DNA sequencing, is also working on other applications of nanopores. At the March Meeting, he described his recent work on a method for using nanopores for quickly detecting anthrax infections.

The anthrax bacterium secretes a protein, called “protective antigen,” that naturally forms into nanopores, which then penetrate cell walls, creating a hole. When a voltage is applied across the cell membrane, ions can flow through the pore. Anthrax also secretes other proteins, called “lethal factor” and “edema factor,” which can bind to the nanopore and prevent the flow of ions through the channel.

Kasianowicz can detect the presence of these toxic proteins in a sample of blood by measuring current flow through the nanopores. He has been able to measure these proteins in blood from guinea pigs, even at very low concentrations.

Previous methods of detecting active anthrax proteins relied on injecting live animals or cell cultures with samples for analysis, and required several days to work. This new method can reliably detect these anthrax proteins in about an hour.

In addition, the method could be used for screening potential therapeutic agents which would work by interfering with the binding of lethal factor and edema factor to the nanopore, Kasianowicz said.

While some scientists are using natural nanopores for these and other applications, other researchers at the meeting reported on developing artificial nanopores as important tools for biophysical studies. For instance, Cees Dekker of Delft University of Technology described advances in solid state nanopores, made from silicon oxide. Artificial nanopores are flexible, stable and adjustable, and can be used for a variety of studies, said Dekker. For instance, longer DNA strands take longer to travel through the pore, so one can use nanopores to measure the length of the strand.

In addition, DNA can go through a pore in either a folded or stretched state, making nanopores a potential tool for studying DNA or RNA folding and unfolding, or DNA-protein binding. Many other uses for nanopores are also being developed, said Dekker. “There’s like a zillion ways you can use it.”

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