Light-induced interactions of DNA, synthetic fluorophores and solid-state nanopores

Abstract

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.