Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.680751
Title: Characterisation of a DNA tetrahedron and its interaction with sequence-specific ligands
Author: Carter, Michael
Awarding Body: University of Southampton
Current Institution: University of Southampton
Date of Award: 2015
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Abstract:
Nanostructures engineered from DNA are becoming increasing popular in interdisciplinary research laboratories globally due to their ease to design and build, as well as their huge potential as devices for applications such as drug delivery and biosensors. Although many studies have used these structures alongside molecules that interact with DNA, little has been done to understand the effects of nanostructured DNA on ligand binding, and vice versa. In this thesis, a simple DNA tetrahedron, folded from four oligonucleotides, is used as a model in which to assess ligand interactions for a variety of DNA-binding agents. We characterised two DNA tetrahedron designs by band shifts, cleavage assays, chemical probing and melting studies. One of these tetrahedra, based on a previous design, was modified to incorporate single binding sites on each edge for a number of sequence-specific DNA-binding ligands: minor groove binders Hoechst 33258 and distamycin, intercalators actinomycin D, echinomycin, TANDEM and nogalamycin, and major groove binding triplex forming oligonucleotides. Band shifts, DNase I footprinting, global melting with UV and SYBR green I, as well as location-specific melting with fluorescent beacons were used to assess the interaction of these ligands with the DNA tetrahedron compared to linear duplex DNA. The results found actinomycin was able to inhibit folding of the structure, whereas other ligands did not. Sequence-selectivity was generally consistent across linear duplex and nanostructured DNA, though some changes in secondary binding sites were observed, however, Hoechst, distamycin and actinomycin showed reduced affinity to the nanostructure. Global thermal melting studies showed that all the ligands enhance stability, however, fluorescence beacons placed at specific locations found that many of the ligands were able to stabilise regions of the tetrahedron remote from their binding sites, including across junctions. The work described helps present a clearer understanding of ligand interactions with DNA nanostructures, as well demonstrates a model in which to assess structure stability at specific locations within a DNA nanostructure. This will aid the design and function of future DNA-based devices.
Supervisor: Fox, Keith Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.680751  DOI: Not available
Keywords: QH301 Biology
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