Nanopore sequencing: from imagination to reality.

DNA sequencing and hence genomics have been transformed over the last decade by the commercialization of inexpensive, massively parallel, short-read sequencing technology. Nonetheless, a new generation of singlemolecule DNA sequencers, which uses nanopore technology, is initiating a further upheaval in genomics. These instruments are portable, capable of reads of 100 kb, cheap, and fast. Nanopore sequencing, a huge technical challenge, took 25 years to develop. Today’s availability of a commercial nanopore sequencing device can be traced to the 1990s, when nucleic acid translocation through nanopores was first observed, stochastic sensing was developed, and the high-resolution structure of a protein nanopore was solved. The nanopore platform that has been developed is also capable of the singlemolecule detection of a wide variety of additional analytes of medical interest, ranging from small molecules to posttranslationally modified proteins. When my group began work on the -hemolysin ( HL) pore in the 1980s (1 ), the possibility of nanopore sequencing was not on our agenda. Following the molecular characterization of the pore by pioneers including Sidney Harshman and Sucharit Bhakdi, we sought to investigate its mechanism of assembly. HL is secreted by Staphylococcus aureus as a monomeric water-soluble 293–amino acid protein that forms an oligomeric pore in lipid bilayers. To us, this appeared to be a relatively simple system from which basic principles in membrane protein assembly might be learned. Over the years, this has indeed proved to be the case. In particular, the prepore concept, in which an oligomer forms on a membrane surface before penetrating the bilayer, has proved to be generally applicable to pore-forming proteins (2 ). Nonetheless, in the late 1980s, we began to think about applications of protein pores in biotechnology. Our ideas included the incorporation of nanopores into filters for rapid purifications and separations, the permeabilization of cells both to introduce reagents for applications in basic research and for drug delivery, and the use of reversible poreforming proteins to transport molecules into cells for protection during preservation by freezing or desiccation. Of these early efforts, only our work on molecular sensing has been sustained. We were most fortunate to obtain funding for these speculative endeavors from the US Department of Energy and the Office of Naval Research. Our general approach to sensing was founded on knowledge about the interactions of channel blockers with natural ion channels, which had been investigated for many years. Currents carried through channels by aqueous ions can be measured by electrophysiological techniques. In the presence of blockers, which generally bind within the lumen of a channel, the ionic current is reduced. The current is restored when the blocker is removed. In early work, we used mutagenesis to build a binding site into the lumen of the HL pore on the basis of educated guesswork (in the absence of a structure) and used the mutant pores to detect divalent metal ions by macroscopic (many pores) current recording. The extent of current block revealed the concentration of the blocker. Even so, it was clear then that electrical recordings from individual pores (single-channel recordings) would reveal far more about the nature of a blocker. At the same time, the structure of the heptameric HL pore was solved by Eric Gouaux and his colleagues (3 ), which allowed the placement of designed binding sites within the lumen of the pore, opening up the possibility of “stochastic sensing” (4 ), a single-molecule detection technique that allows the identification of analytes.