Individual Vesicle-Vesicle and Vesicle-Planar Bilayer Fusion Events Mediated by DNA

Abstract

We have previously reported that DNA-lipid conjugates, when inserted into lipid vesicles, can mediate interactions between vesicles such as docking and fusion∗. These DNA-mediated interactions are a model for the biological machinery (SNARE proteins) employed in synaptic vesicle fusion. We can also use DNA-lipids to build model membrane platforms, such as tethered vesicles and tethered free-standing membranes, which are employed in these studies to observe individual fusion events using fluorescence microscopy. Two systems will be described: vesicle to vesicle fusion between mobile, tethered vesicles, and vesicle to planar bilayer fusion of small vesicles to a DNA-tethered free-standing bilayer. Fusion of individual vesicles is observed in real time using lipid and content mixing assays, as well as FRET upon hybridization of labeled DNA-lipids. These studies provide insight into the relative timescales and extents of docking, lipid mixing, and content exchange. In addition, using different sequences of fluorescently labeled DNA to mediate fusion gives insight into the importance of the rates and number density of hybrid formation at the fusion pore relative to the rate of fusion pore opening. This fusion machinery can also be used to deliver membrane proteins from small vesicles into GUVs or DNA-tethered planar bilayers, allowing membrane proteins to be studied in a free-standing bilayer. ∗Chan et al, PNAS 2007 and 2009; Chan et al, Biointerphases 2008.