Fluorescence studies of influenza RNA and RNA polymerase
<p>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 p...
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| Format: | Thèse |
| Langue: | English |
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2014
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| _version_ | 1826280002991161344 |
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| author | Tomescu, A |
| author2 | Kapanidis, A |
| author_facet | Kapanidis, A Tomescu, A |
| author_sort | Tomescu, A |
| collection | OXFORD |
| description | <p>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.</p> <p>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.</p> <p>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.</p> |
| first_indexed | 2024-03-07T00:07:15Z |
| format | Thesis |
| id | oxford-uuid:77f33f82-76f7-4154-912e-5c92bd4bf9c6 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T00:07:15Z |
| publishDate | 2014 |
| record_format | dspace |
| spelling | oxford-uuid:77f33f82-76f7-4154-912e-5c92bd4bf9c62022-03-26T20:27:33ZFluorescence studies of influenza RNA and RNA polymeraseThesishttp://purl.org/coar/resource_type/c_db06uuid:77f33f82-76f7-4154-912e-5c92bd4bf9c6Molecular biophysics (biochemistry)BiophysicsEnglishOxford University Research Archive - Valet2014Tomescu, AKapanidis, A<p>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.</p> <p>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.</p> <p>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.</p> |
| spellingShingle | Molecular biophysics (biochemistry) Biophysics Tomescu, A Fluorescence studies of influenza RNA and RNA polymerase |
| title | Fluorescence studies of influenza RNA and RNA polymerase |
| title_full | Fluorescence studies of influenza RNA and RNA polymerase |
| title_fullStr | Fluorescence studies of influenza RNA and RNA polymerase |
| title_full_unstemmed | Fluorescence studies of influenza RNA and RNA polymerase |
| title_short | Fluorescence studies of influenza RNA and RNA polymerase |
| title_sort | fluorescence studies of influenza rna and rna polymerase |
| topic | Molecular biophysics (biochemistry) Biophysics |
| work_keys_str_mv | AT tomescua fluorescencestudiesofinfluenzarnaandrnapolymerase |