Engineering a membrane-adapted viral helicase nanopore

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Background. Nanopore sequencing devices read the bases in atranslocating single-stranded DNA molecule using a protein nanoporeembedded in an electro-resistant membrane, with the DNA basesidentified by changes in the current across the membrane. Presentday devices couple a DNA translocating helicase with a separate poreprotein. Although the technology is capable of long reads (megabases) and high accuracy (~99%) these could be improved. Thecritical parameters include translocation speed, stability of themembrane-protein-DNA complex and pore size, which determinesnucleotide resolution. The direct membrane-insertion of hexamericreplicative helicases, that are by nature processive and have a centralpore optimised for ssDNA translocation, could lead to improveddevices. Having a single pore/translocase could also simplify sampleDNA preparation, obviating the need to pre-attach DNA to the helicaseand then the pore, as in current protocols. This project will usemolecular biology and biochemical approaches to re-engineer thehexameric replicative helicase E1 from papillomavirus, to a membrane-bound nanopore suitable for DNA sequencing.