Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.730466
Title: Novel fluorescence and fluorine labelling methods for viruses and virus-like particles
Author: Leung, Lok Chun Rogen
ISNI:       0000 0004 6497 4488
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2016
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Abstract:
Molecular imaging involves the development of probes which can specifically label a certain object in the body at cellular or subcellular level. This thesis consists of three parts, each involving the development of novel labelling methods for viruses or virus-like particles with specific applications. Virus-like particles (VLP) derived from the E. coli bacteriophage Qβ are widely employed as a nano-carrier for drugs and vaccines, but a powerful method for tracing its circulation without affecting its structure is yet to be developed. In the first part of the thesis, the electrophilic fluorine source 19F-SelectfluorTM was employed for introducing single fluorine atoms on Qβ VLPs. For the 'tag-and-modify' approach, site-selective electrophilic C-F bond formation was achieved on the dehydroalanine (Dha) amino acid tag of VLPs under aqueous conditions. Chemoselective electrophilic aromatic fluorination on tyrosine residues were also achieved using the same reagent by manipulating the amino acid sequence. Similar results were observed in conditions required for 18F-Selectfluor™ reaction, indicating the potential of this technique for positron emission tomography (PET) imaging. In addition, there is a lack of in situ technique for tracking the functional status of Qβ VLPs and hence the release of cargos. In the second part of the thesis, a simple way to monitor the disassembly of 19F-labelled Qβ VLPs by 19F NMR spectrosocpy is reported. Analysis of resonances, using experiments under a range of conditions, allowed determination not only of the intact particle but also the disassembled multimeric species and even smaller peptides upon digestion by cells. This in turn allowed mutational redesign of disassembly and testing in both bacterial and mammalian systems as a strategy for the creation of putative, targeted-VLP delivery systems. In the third part of the thesis, a new type of rhodamine B fluorescent dye functionalised with a 2-imino-2-methoxyethyl (IME) group is reported. The amidine linkage formed between the IME group and lysine residue retains the pKaH of the original side chain, which cannot be achieved using commercially available conjugating dyes. This in turn minimises the change in net charge hence virus infectivity following virus labelling. By employing adenovirus (AV) as an example, the IME dye was shown to be a better choice in retaining virus infectivity compared to dyes linked with other coupling groups. In addition, preliminary experiments on dengue virus with the synthesised dyes were also performed.
Supervisor: Davis, Benjamin G. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.730466  DOI: Not available
Keywords: Virus-like particles ; Fluorine NMR ; Fluorescent labelling
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