Light-induced interactions of DNA, synthetic fluorophores and solid-state nanopores
Di Fiori, Nicolas
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In the first part of my thesis I studied the effect of dye-dye interactions in labeled double-stranded DNA molecules on the Forster Resonance Energy Transfer (FRET) efficiency. FRET is a spectroscopic process by which energy is passed nonradiatively between molecules over long distances (10-100 A). The donor molecule, a fluorophore, transfers part of its energy to an acceptor, usually a second fluorophore, with an efficiency that depends strongly on their separation. I have found that when some fluorophores are covalently bounded to DNA, a second process which directly competes with FRET can be observed. I have studied these novel interactions by immobilizing the molecules on a surface and probing them one at a time, in this way avoiding ensemble averages that mask this phenomenon. By comparing four different donor-acceptor pairs I found that these interactions depend on the nature of the fluorophores. These results show for the first time that when dye-dye interactions are accounted for, single-molecule FRET can be used to accurately measure absolute inter-dye distances, even at the shortest separations. Secondly, these results are useful when deciding which dye pairs to use for nucleic acid analyses using FRET. In the second part, I found a novel effect between visible light and solid-state nanopores which allows us to slow down the speed at which nucleic acid threads through the pore (i.e. translocates). Solid-state nanopores are nanometer-sized holes drilled in thin ceramic films, and they hold the potential of achieving ultra-fast and cost-effective whole genome sequencing. A major deterrent to this goal is the lack of a way to modulate the translocation speed of nucleic acids without compromising the electrical signal. I have found that light enhances the pore conductance and simultaneously produces a >10-fold increase in the translocation time. I propose a mechanism to account for these observations that involve light-induced charges on the silicon nitride pore surface. With this assumption, I then successfully modeled the conductance enhancement created by light, and suggested an underlying physical mechanism that can explain the production of transient charges in silicon nitride membranes using visible light.
Thesis (Ph.D.)--Boston University PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at firstname.lastname@example.org. Thank you.