The sequencing of individual DNA strands with nanopores is being developed as a rapid, low-cost platform in which bases are identified in order as a DNA strand is transported through a pore under an electrical potential. Several challenges remain and this thesis focuses on one major area, the base identification properties of the a-hemolysin (aHL) nanopore. Under the potentials required for threading, DNA translocates too fast for single bases to be identified. However, immobilization of the DNA within the pore increases the residence time and therefore improves the precision of the electrical current reading and allows for the small differences in current flow, associated with different sequences, to be observed. DNA molecules with a 3'-terminal biotin-tag were complexed with streptavidin. Streptavidin is too large to be transported through the aHL pore and therefore the DNA-btnestreptavidin complex is not fully translocated; thus, the DNA strand is immobilized within the pore. Using this approach the nucleobase recognition properties of the aHL pore were mapped. The data suggest that the transmembrane 13 barrel domain of the pore contains at least three nucleobase recognition sites, termed R1, R2 and R3. Additional sequence information can be gained when multiple recognition sites are employed within a single aHL pore, as compared to the simple case of a single recognition site. Recognition site R1, which is located near the central constriction, can be modified by site-directed mutagenesis of Met-113. It was observed that amino acids with related side chains produce similar patterns of nucleobase recognition. Amino acids that provide an energy barrier to ion flow (e.g. bulky or hydrophobic residues) strengthen base identification, while amino acids that lower the barrier, weaken identification. Deletion and site-directed mutagenesis were used to remove one recognition site and generate an αHL pore. With truncated β barrel domain that contains only two recognition sites.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:558397 |
| Date | January 2011 |
| Creators | Stoddart, David |
| Contributors | Bayley, Hagan |
| Publisher | University of Oxford |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://ora.ox.ac.uk/objects/uuid:597b217c-c995-401d-b772-4cb373d195af |
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