Nanopore sequencing: from imagination to reality.

Abstract

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 single-molecule 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 single-molecule 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 α-hemolysin (αHL)2 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. αHL 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 …