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.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:686944 |
| Date | January 2014 |
| Creators | Tomescu, Alexandra Iulia |
| Contributors | Kapanidis, Achilles |
| Publisher | University of Oxford |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://ora.ox.ac.uk/objects/uuid:77f33f82-76f7-4154-912e-5c92bd4bf9c6 |
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