Exploring Structures of Membrane-Inserted Helicases for Advancing Nanopore Sequencing Revolution

Nanopore-based technology is revolutionising DNA sequencing. Modern devices read DNA sequences using protein nanopores embedded in a membrane, detecting base changes via electric current. Devices developed at Oxford Nanopore Technologies(ONT) couple a DNA translocating helicase with a separate protein containing a narrow channel.

Integration of the membrane pore and helicase into a single protein is expected to significantly advance sequencing, by rectifying translocation speed and improving mechanical stability of the pore. To achieve this, this project would explore the direct membrane insertion of hexameric helicases. Objectives:

(1) Probe addition of transmembrane peptides to optimise insertion of helicases into lipid bilayers (2) Determine cryo-EM structures for nano-disc inserted chimeric helicases (3) Enhance pore characteristics with site-directed mutagenesis Experimental approach:

We aim to obtain integral nanopore-translocases suitable for nucleic acid sequencing. This project will generate multiple constructs of helicases featuring outer hydrophobic surfaces to facilitate their insertion into membranes. Cryo-EM structures of nanodisc-inserted helicases will be determined, and used to inform further design.

This project is a collaboration with Dr.Cyril Sanders' lab from Sheffield. Dr Sanders' laboratory will optimize helicase activity of selected DNA/RNA machines, which will be structurally characterized at York.