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Title: Fluorescence studies of influenza RNA and RNA polymerase
Author: Tomescu, Alexandra Iulia
ISNI:       0000 0004 5920 9642
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2014
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The influenza A virus genome consists of eight single-stranded segments of negativesense viral RNA (vRNA) with highly conserved, partially complementary termini. These termini associate in a double-stranded RNA structure, known as a panhandle, which is bound by the viral RNA-dependent RNA polymerase and can serve as a promoter in both viral transcription and replication. In part A of this thesis, I use a combination of classical biochemistry techniques and fluorescence techniques (both at the ensemble and single-molecule level) for a quantitative investigation of the interaction between purified influenza A RNA polymerase and the individual 5' and 3' conserved termini of the vRNA segments, as well as the double-stranded vRNA promoter. Furthermore, I report the first direct, real-time observation of the promoter changing its structure when bound by the polymerase and show that the structure assumed agrees best with the corkscrew model. In part B of this thesis, I use fluorescence to detect RNA: I design and test a singlemolecule biosensor aimed at probing the presence of influenza A RNA in a sample, on the one hand, and I use click-chemistry to fluorescently label very shorty RNAs (3-25nt) that have been generated in an in vitro transcription reaction, on the other. The biosensing assay I propose can be further developed for diagnostic purposed, while click-chemistry labelling of short RNAs can be optimised and extended such that it becomes a reliable alternative to the use of radiolabels.
Supervisor: Kapanidis, Achilles Sponsor: Medical Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Molecular biophysics (biochemistry) ; Biophysics ; influezna ; RNA-dependent RNA polymerase ; polymerase ; microscopy ; fluorescence