Engineering a membrane-adapted viral helicase nanopore

Background. Nanopore sequencing devices read the bases in a
translocating single-stranded DNA molecule using a protein nanopore
embedded in an electro-resistant membrane, with the DNA bases
identified by changes in the current across the membrane. Presentday devices couple a DNA translocating helicase with a separate pore
protein. Although the technology is capable of long reads (mega
bases) and high accuracy (~99%) these could be improved. The
critical parameters include translocation speed, stability of the
membrane-protein-DNA complex and pore size, which determines
nucleotide resolution. The direct membrane-insertion of hexameric
replicative helicases, that are by nature processive and have a central
pore optimised for ssDNA translocation, could lead to improved
devices. Having a single pore/translocase could also simplify sample
DNA preparation, obviating the need to pre-attach DNA to the helicase
and then the pore, as in current protocols. This project will use
molecular biology and biochemical approaches to re-engineer the
hexameric replicative helicase E1 from papillomavirus, to a membrane
-bound nanopore suitable for DNA sequencing.