Interactions between lipid membranes mediated by DNA oligonucleotides

Lipid membranes are the fundamental barriers in biological systems, and thus reactions involving membranes play an integral role in many crucial processes. My graduate studies have been inspired most directly by the example of synaptic vesicle fusion, during which neurotransmitter-containing vesicles fuse to the cellular membrane in a highly regulated process involving several integral membrane proteins. This thesis primarily presents work developing and implementing methods to use membranes presenting DNA as a model for biological fusion.;DNA-lipid conjugates can be used to tether vesicles to supported lipid bilayers in a sequence specific manner. Tethered vesicles were found to diffuse on the surface with an average diffusion constant, , of 0.2 mum2/s, which is roughly 5-times less than that of fluorescently-labeled lipids in the same surface. The reduction in the diffusion constant may be due transient interactions between the vesicle and supported membranes. Tethered vesicles can be brought to a complete stop in a reversible manner by introducing polymers such as poly(ethylene glycol) or divalent cations to the bulk solution. The addition of these agents gives rise to depletion effects or chelation (when both membranes present anionic lipid headgroups), respectively, which increases non-specific membrane-membrane interactions and inhibits free diffusion of the vesicles.;The kinetics of the specific recognition and binding events between tethered vesicles presenting complementary DNA were measured as a reaction analogous to that of synaptic vesicle docking, i.e. the localization of the vesicle to the neuronal membrane preceding fusion. In these studies, DNA-vesicles were tethered in spatially separated regions of a supported bilayer using a microfluidic device. The docking of pairs of vesicles results in tandem diffusion, and simulation analysis of the diffusion-reaction showed that the efficiency of docking scales quadratically with the average number of DNA per vesicle. Docking kinetics are also much faster when using a repeating sequence rather than a fully-overlapping sequence. These results led to the development of a geometric reaction model which predicts that the probability of docking is enhanced by increasing the number of DNA per vesicle and by increasing the vesicle-vesicle separation distance at which hybridization can successfully occur.;To more closely approximate the orientations of fusion proteins in membranes, a new method for synthesizing DNA-lipid conjugates was implemented to allow coupling of the lipophilic tailgroup to either the 3'- or 5'-end of an oligonucleotide. Thus, a membrane presenting 5'-coupled DNA reacting with a membrane presenting 3'-coupled DNA would be brought into close apposition, mimicking the hypothesized function of SNARE proteins which have been shown to be able to mediate fusion in in vitro studies. In this orientation, DNA mediates vesicle fusion as characterized by both lipid mixing and content mixing assays. The DNA system is very attractive for probing fundamental questions of membrane fusion, as variables including hybridization energetics and distance parameters can easily be controlled by changing the DNA sequence, and potential directions for future work are explored.;Supported lipid bilayers are typically formed by vesicle fusion to a silica substrate, and the adsorption of vesicles to a surface leads to transient pore formation and content leakage. Increasing the amount of a fluorescently labeled lipid increases the probability of leakage, and smaller vesicles tend to leak a higher percentage of content.