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Title: Confocal single-molecule fluorescence as a tool for investigating biomolecular dynamics in vitro and in vivo
Author: Torella, Joseph Peter
ISNI:       0000 0004 2728 5931
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2011
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Confocal single-molecule fluorescence is a powerful tool for monitoring conformational dynamics, and has provided new insight into the enzymatic activities of complex biological molecules such as DNA and RNA polymerases. Though useful, such studies are typically qualitative in nature, and performed almost exclusively in highly purified, in vitro settings. In this work, I focus on improving the methodology of confocal single-molecule fluorescence in two broad ways: (i) by enabling the quantitative identification of molecular dynamics in proteins and nucleic acids in vitro, and (ii) developing the tools needed to perform these analyses in vivo. Toward the first goal, and together with several colleagues, I have developed three novel methods for the quantitative identification of dynamics in biomolecules: (i) Burst Variance Analysis (BVA), which unambiguously identifies dynamics in single-molecule FRET experiments; (ii) Dynamic Probability Density Analysis (PDA), which hypothesis-tests specific kinetic models against smFRET data and extracts rate information; and (iii) a novel molecular counting method useful for studying single-molecule thermodynamics. We validated these methods against Monte Carlo simulations and experimental DNA controls, and demonstrated their practical application in vitro by analyzing the “fingers-closing” conformational change in E.coli DNA Polymerase I; these studies identified unexpected conformational flexibility which may be important to the fidelity of DNA synthesis. To enable similar studies in the context of a living cell, we generated a nuclease-resistant DNA analogue of the Green Fluorescent Protein, or “Green Fluorescent DNA,” and developed an electroporation method to efficiently transfer it into the cytoplasm of E.coli. We demonstrate in vivo confocal detection of smFRET from this construct, which is both bright and photostable in the cellular milieu. In combination with PDA, BVA and our novel molecular counting method, this Green Fluorescent DNA should enable the characterization of DNA and protein-DNA dynamics in living cells, at the single-molecule level. I conclude by discussing the ways in which these methods may be useful in investigating the dynamics of processes such as transcription, translation and recombination, both in vitro and in vivo.
Supervisor: Kapanidis, Achilles N. Sponsor: University of Oxford, Clarendon Award
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Life Sciences ; Biochemistry ; Molecular biophysics (biochemistry) ; Biology ; Cell Biology (see also Plant sciences) ; Mathematical biology ; Probability theory and stochastic processes ; Biophysics ; Biosensors ; Chemical kinetics ; Enzymes ; Microscopy ; Spectroscopy and molecular structure ; Condensed Matter Physics ; Stochastic processes ; Single molecule fluorescence ; confocal fluorescence ; DNA polymerase ; DNA structure ; single molecule fluorescence in vivo