Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes are the core molecular machinery of membrane fusion, a fundamental process that drives inter- and intracellular communication and trafficking. One of the questions that remains controversial has been whether and how SNAREs cooperate. Here we show the use of self-assembled DNA-nanostructure rings to template uniform-sized small unilamellar vesicles containing predetermined maximal number of externally facing SNAREs to study the membrane-fusion process. We also incorporated lipid-conjugated complementary ssDNA as tethers into vesicle and target membranes, which enabled bypass of the rate-limiting docking step of fusion reactions and allowed direct observation of individual membrane-fusion events at SNARE densities as low as one pair per vesicle. With this platform, we confirmed at the single event level that, after docking of the templated-SUVs to supported lipid bilayers (SBL), one to two pairs of SNAREs are sufficient to drive fast lipid mixing. Modularity and programmability of this platform makes it readily amenable to studying more complicated systems where auxiliary proteins are involved.

SNARE proteins are the core machinery to drive fusion of a vesicle with its target membrane. Inspired by the tethering proteins that bridge the membranes and thus prepare SNAREs for docking and fusion, we developed a lipid-conjugated ssDNA mimic that is capable of regulating SNARE function, in situ. The DNA-lipid tethers consist of a 21 base pairs binding segment at the membrane distal end that can bridge two liposomes via specific base-pair hybridization. A linker at the membrane proximal end is used to control the separation distance between the liposomes. In the presence of these artificial tethers, SNARE-mediated lipid mixing is significantly accelerated, and the maximum fusion rate is obtained with the linker shorter than 40 nucleotides. As a programmable tool orthogonal to any native proteins, the DNA-lipid tethers can be further applied to regulate other biological processes where capturing and bridging of two membranes are the prerequisites for the subsequent protein function.

The stability of model surfactant bilayers from the poly(ethylene glycol) mono-n-dodecyl ether (C12Ej) family was probed. The surfactant bilayers were formed by the adhesion of emulsion droplets. We generated C12Ej bilayers by forming water-in-oil (w/o) emulsions with saline water droplets, covered by the surfactant, in a silicone and octane oil mixture. Using microfluidics, we studied the stability of those bilayers. C12E1 allowed only short-lived bilayers whereas C12E2 bilayers were stable over a wide range of oil mixtures. At high C12E2 concentration, a two-phase region was displayed in the phase diagram: bilayers formed by the adhesion of two water droplets and Janus-like particles consisting of adhering aqueous and amphiphilic droplets. C12E8 and C12E25 did not mediate bilayer formation and caused phase inversion leading to o/w emulsion. With intermediate C12E4 and C12E5 surfactants, both w/o and o/w emulsions were unstable. We provided the titration of the C12E2 bilayer with C12E4 and C12E5 to study and predict their stability behavior.