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Title: Dark quenchers and novel fluorescent thymidine monomers for single-molecule detection
Author: Le Reste, Ludovic
ISNI:       0000 0004 2722 1938
Awarding Body: Oxford University
Current Institution: University of Oxford
Date of Award: 2010
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Constant improvements of single-molecule techniques over the past two decades have lead to multiple new insights in various biological processes. In particular, single- molecule Forster Resonance Energy Transfer (smFRET), a fluorescence-based method capable of measuring distances in the 2-10nm range, is extensively used in the study of diverse biomolecular interactions. In this thesis, I discuss novel assays for single-molecule fluorescence detection. In the first part, the main single-molecule experimental setup built in the course of my doctorate is described. In the second part, the use of dark quenchers is investigated. One of the main hurdles in single-molecule fluorescence experiments is the difficulty of working at high concentrations of labelled molecules, thus preventing the study of biomolecular interactions with high dissociation constants. Dark quenchers, used as acceptors in a smFRET pair, can circumvent this problem as they can be used at high concentration. However, very few studies using dark quenchers at the single- molecule level have been reported in the literature and little is known about their photophysical properties. This thesis demonstrates that dark quenchers, used in combination with Alternating Laser Excitation (ALEX) spectroscopy, can be used in a 3-probe system (two fluorophores and one dark quencher) to monitor multiple distances and interactions. Their basic photophysical properties are also investigated. Finally, dark quencher detection is applied to study interactions of DNA Polymerase I (Klenow Fragment) with DNA. In the third part of this thesis, fluorescent properties of novel thymidine monomers labelled with Cyanine dyes (CydT monomers) that were used as building blocks for DNA synthesis are characterised. CydT monomers open the possibility of attaching many Cy3 and Cy5 fluorophores site specifically on a DNA strand, thus allowing more complex labelling schemes than currently possible. Their performance as FRET pairs is also investigated. The methods and assays discussed in this thesis can be used to study a broad range of biomolecular interactions, including protein-protein interactions and protein-DNA complexes, and have potential applications in real-time DNA sequencing.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available