The essential idea behind nanopore sequencing is to electrophoretically drive DNA through a single, nanometer scale hole in a membrane. DNA is strongly negatively charged because of the phosphate groups that help form its backbone. The membrane can be either synthetic (usually silicon) or biological (a bilayer lipid membrane, similar to a cell membrane). In synthetic membranes, one uses lithography or similar techniques to create the hole, while in biological membranes, one can use a variety of bacterial porin proteins, such as &alpha-Hemolysin, which spontaneously self insert themselves into the membrane. We introduced the M. smegmatis outer membrane porin protein MspA to nanopore sequencing. MspA is one of four nearly identical outer membrane porins from this common, harmless cousin of the bacteria that causes tuberculosis.

Ideally, the identity of individual bases of a long single-stranded DNA (ssDNA) would be read out using the simultaneously passing ionic current. The bases do obstruct the pore so that while ssDNA is in the pore, the current passed is reduced. If each kind of base (cytosine, guanine, adenine and thymine) blocks the pore differently, this current signal can be used to "read" the DNA at high speed using just a single ssDNA molecule.

In comparison to &alpha-Hemolysin, MspA (PubMed list of MspA articles) has a structure uncannily suited to sequencing (see image below.) Instead of a long, narrow tube through which DNA must pass, as in &alpha-Hemolysin, MspA is shaped like a goblet, with an opening just slightly more than 1 nanometer in diameter at its base. Intuitively, this should provide single-base resolution. Early results are very promising--watch our Publications page for more articles soon!

(Left) MspA (Right) &alpha-Hemolysin. Colors indicate classes of amino acid side chains: hydrophobic aliphatic (yellow) or aromatic (orange); polar (purple); and negatively (blue) or positively (red) charged residues.

We have been surprised how well MspA appears to be suited for sequencing. However, initially MspA was a bit of a dissapointment because DNA could not be convinced to go through it. We had to mutate MspA a bit to alter its inner charge distribution to allow DNA to pass through the pore. We now work with our collaborators Michael Niederweis and Alek Aksimentiev to design new "mutant" pores based on MspA. Michael Niederweis isolated MspA, then crystalized it to detemine its exact shape via x-ray crystallography and he is the expert in mutating it. Alek Aksimentiev is well known for his molecular dynamics simulatons, specializing in new techniques for simulating pores and DNA transport through pores. By bringing our three distinct backgrounds together we formed a "dream team" focused on developing a nanopore that sequences DNA.

Please check out our Publications page, look for the recent Nature Nanotechnology review written collaboratively by several groups in the field and please feel free to chime in at the Nature discussion forum at Nature Network.