Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.698214
Title: Nanoscale patterning of complex DNA structures with the bacterial protein Recombinase A
Author: Corbett, Sybilla Louise
ISNI:       0000 0004 5989 9866
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2016
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Abstract:
The use of DNA as a structural material has been intensively developed since its inception in the early 1980s. The potential of DNA structures in the field of materials science is hampered by current approaches to augmentation. It is not currently possible to alter the targeting of heterogenous additional elements to structures once they have been made. The post hoc patterning of DNA architectures is therefore of great importance. The bacterial protein Recombinase A (RecA) may be able to provide this function. This thesis will discuss the patterning of DNA structures with RecA. RecA has been shown to pattern linear dsDNA strands with high levels of efficiency. To test the potential of RecA to pattern more complex DNA, novel strategies for creating DNA topologies have been explored. This work has produced DNA strands containing regions of base pair mismatching and with terminal three-way junctions. A method has also been developed for the creation of a 200 base product with unpaired branched junctions, using four synthetic oligomers in a scaffolded cycling ligation reaction with a heat stable ligase. A method to create longer DNA strands with three-way junctions at the termini has also been developed. RecA patterning of a structure with internal mismatches was carried out. Mismatches proximal to the patterning area led to an increase in patterning efficiency with an increase in mismatch length. When the mismatch was separated from the patterning region a more complex relationship was observed, with intermediate-length mismatches resulting in a decrease in pattering efficiency. The introduction of a nick in the phosphate backbone proximal to the patterning region also increased patterning efficiency. Two further DNA structures were produced on which patterning did not prove possible. The ligase chain reaction was shown to produce DNA strands that could be incorporated into a structure with central base pairing and terminal single stranded DNA regions. Attempts to create three-way junctions from these structures were not successful. A second structure was created through treatment of double stranded DNA from the polymerase chain reaction. Single strands of DNA were produced that could be annealed to produce terminal three-way junctions. Atomic force microscopy demonstrated the correct annealing of this structure. However, it did not prove possible to pattern these structures with RecA. Recombinant RecA production through bacterial induction produced soluble protein at a high yield. There was some evidence of DNA contamination and the purified protein showed low activity.
Supervisor: Walti, Christoph ; Davies, A. Giles Sponsor: EPSRC
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.698214  DOI: Not available
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