Engineering a membrane-adapted viral helicase nanopore
Lead Research Organisation:
University of Sheffield
Department Name: Clinical Oncology
Abstract
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. Present day 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.
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. Present day 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.
People |
ORCID iD |
C Sanders (Primary Supervisor) | http://orcid.org/0000-0001-6006-4530 |
Studentship Projects
Project Reference | Relationship | Related To | Start | End | Student Name |
---|---|---|---|---|---|
BB/T007222/1 | 01/10/2020 | 30/09/2028 | |||
2741315 | Studentship | BB/T007222/1 | 01/10/2022 | 30/09/2026 |