Use of a Single Nanometer-Scale Pore to Rapidly Examine Individual DNA or RNA Strands
Mark Akeson*, a, Daniel Brantonb, John J. Kasianowiczc, Eric Brandinb, and David W. Deamera
aDepartment of Chemistry & Biochemistry, University of California, Santa Cruz
bDepartment of Molecular & Cellular Biology, Harvard University
cNational Institute of Standards and Technology
This is an abstract
for a presentation given at the
Foresight Conference on Molecular Nanotechnology.
There will be a link from here to the full article when it is
available on the web.
We have developed a technique that permits us to examine single nucleic acid strands on the millisecond time-scale. The key component of this new approach is a hydrated nanometer-scale pore embedded in a non-conducting support. This pore is required to be less than 2 nm in diameter so that only one nucleic acid strand can enter at once, and so that each base within the strand passes through the pore in single-file order. The pore is also required to have an integral detector that can translate the characteristic physical and chemical properties of a base (or sequence of bases) into an identifiable electrical signature.
The model pore we have used is -hemolysin, a bacterial toxin that forms a heptameric channel in biological membranes. Because the limiting diameter of this channel is large enough to translocate only a single strand of DNA or RNA (Kasianowicz et al., PNAS 93:13770-73, 1996; Song et al., Science 274:1859-66, 1996) the requirement of single-file, sequential base transport is fulfilled. The pore also serves as a detector. For example, in the absence of DNA or RNA, the -hemolysin channel exhibits a steady, open ionic current of 120 pA in 1M KCl at 120 mV potential. This current is reduced 95% when occupied by poly-cytosine, but only 85% when occupied by poly-adenine. Such measurements of ionic conductance have allowed us to unambiguously distinguish between segments of cytosine and segments of adenine within an individual RNA molecule. This technology has several possible applications including sequencing of individual DNA strands at very high rates.
This project is supported by the National Center for Human Genome Research and by DARPA
Chemistry Department, University of California, Santa Cruz, CA 95064