Moderator’s note: Make sure to read the forum intro and the Nature Biotech review and commentary linked from there!

The primary challenges to sequencing with a protein nanopore such as alpha hemolysin (a-HL) fall into two broad categories: stability of the pore and its support, and single base resolution. The latter includes control of translocation velocity, signal-to-noise in the system, blurring when multiple bases contribute to the signal, and errors introduced by molecular motion.
Bacterial protein pores are remarkably stable, surviving many environmental extremes without denaturing. The major instability is in the fluid lipid bilayer support. The usual bilayer of diphytanoylphosphatidylcholine lasts ~one hour. The setup can be tedious, so a reusable pore lasting a week or more would be especially helpful. Bayley’s, White’s, and Winterhalter’s labs have all developed some solutions to these problems.
a-HL has a 5 nm long, 2 nm diameter pore extending through its stem to the trans side of the channel. The stem accommodates ~10 – 12 bases, all of which contribute to the ionic current blockade during translocation. The challenge is to detect and identify a single base from the current modulation. One approach has been to modify the limiting aperture and to limit measurement to a single base in the pore at one time. Bayley’s team has shown how cyclodextrin can be attached to aHL to form a small constriction, and that the resulting current blockades can be used to unambiguously identify mononucleotides. An exonuclease can be used to release single mononucleotides from DNA, but one must then ensure that these interact with the pore strictly sequentially.
Questions to get us started:

• We’ve mentioned how the “adapter” cyclodextrin can be used with aHL; how else can we modify the pore, for both recognition/resolution and to control translocation, as discussed above?
• Are there other “hidden” problems in the ideas presented that we must address?
• Are there other pores/adapter molecules that we should know about?
• What methods from protein pores can be adapted to solid-state pores? Vice-versa?