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Title: Functional DNA nanotechnology
Author: Benn, Florence
ISNI:       0000 0004 6497 0911
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2016
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This thesis sets out to further the field of functional DNA nanotechnology through the design of novel functional DNA scaffolds, and investigates their applications and efficacy. The work presented here comprises two parts: The design of a chiral DNA nanotube that acts as a scaffold for motor motion and for an enzyme cascade; and the design of two different tetrahedral scaffolds for selection of a combination of three ligands, which together have a greater binding effect than the sum of the individual components. It begins by proposing the design of a DNA origami nanotube which distinguishes between the inside and outside face of the tube at the design stage, which most previous designs reported do not. The previous designs in the literature result in a distribution of 50:50, of one face forming the inside surface on one tube and the same face forming the outside surface of a different tube. In the design presented in this thesis, this distinction results from making the tube chiral, which forces it to roll up in a predetermined manner. Chirality is introduced by varying the positions of staple crossovers and this process is explained. The chiral tubes may stack end-to-end to form long polymers, or exist in monomeric form with stacking suppressed, by inclusion of different sets of staples at the ends of the tubes. We confirm tube formation and right-handed chirality with AFM and CD respectively. The efficacy of the tube as a scaffold for an enzyme cascade is tested and discussed in context of the wider field. No significant enhancement is observed when enzymes are tethered to the inside of the tubes, compared to when they are tethered to the outside or are free in solution, although the same slight trend is always observed. Suggestions are made to better this experiment and further understand the underlying physics of such systems. We propose using the tube as a scaffold for a DNA track, upon which a DNA motor may walk. DNA motors are introduced and we attempt to observe micron-scale, inter-tube motion within the confines of our origami tube. Initial experiments show the motor moving and we propose methods of fluorescent labeling via PAINT to better the experimental set-up for TIRF microscopy, which currently is limited by photobleaching. The second part of this thesis proposes systems for selection of a combination of three ligands, which together have a greater binding effect than the sum of the individual components. Here we design two tetrahedral systems where either three ligands or three aptamers are brought together at a vertex of the tetrahedron to form a binding domain. The aptameric system allows for selection, amplification and reassembly of the strongest binders, because the functional and structural sequences are on one strand of DNA, following ligation. This design betters the initial tetrahedral system, where the coding/record strands for amplification are separate from the functional binding domain strands the ligands are attached to. This means it is not possible to reassemble this particular structure after amplification of the record strand.
Supervisor: Turberfield, Andrew Sponsor: Engineering & Physical Sciences Research Council ; Biotechnology and Biological Sciences Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available