Effects of linker sequences on vesicle fusion mediated by lipid-anchored DNA oligonucleotides

Abstract

Synthetic lipid-oligonucleotide conjugates inserted into lipid vesicles mediate fusion when one population of vesicles displays the 5 -coupled conjugate and the other the 3 -coupled conjugate, so that anti-parallel hybridization allows the membrane surfaces to come into close proximity. Improved assays show that lipid mixing proceeds more quickly and to a much greater extent than content mixing, suggesting the latter is rate limiting. To test the effect of membrane-membrane spacing on fusion, a series of conjugates was constructed by adding 2-24 noncomplementary bases at the membrane-proximal ends of two complementary sequences. Increasing linker lengths generally resulted in progressively reduced rates and extents of lipid and content mixing, in contrast to higher vesicle docking rates. The relatively flexible, single-stranded DNA linker facilitates docking but allows greater spacing between the vesicles after docking, thus making the transition into fusion less probable, but not preventing it altogether. These experiments demonstrate the utility of DNA as a model system for fusion proteins, where sequence can easily be modified to systematically probe the effect of distance between bilayers in the fusion reaction.DNA machine ͉ synthetic biology F usion of lipid membranes plays a central role in many biological processes. For example, in the case of synaptic transmission, the reaction is tightly regulated by a number of proteins of which the SNAREs [soluble N-ethylmaleimide sensitive factor (NSF) attachment protein receptors] have been shown to play a crucial role (1-3). In vitro studies have demonstrated the sufficiency of SNAREs to mediate fusion when reconstituted into a number of model systems, including freestanding lipid vesicles (4-7), supported lipid bilayers (8-10), and tethered vesicles (11). Generally these studies suggest that SNAREs are able to bring two membranes into close apposition by forming a trans complex in a docking reaction which is highly energetically favorable (12). This is illustrated in Fig. 1A, which is based on the x-ray structures of the soluble domains of SNARE proteins in complex (13). Several steps along the mechanism to achieve membrane fusion following this basic docking step have been proposed, and some of these are illustrated in Fig. 2, which also serves to illustrate assays commonly used to assess their role. In this step-wise mechanism, the bilayers first dock, then adopt an intermediate, hemifused state in which the outer leaflets have merged, but the inner leaflets and contents remain distinct. A transition into full fusion then involves mixing of both leaflets and content exchange.Despite substantial research on the role of SNARE proteins in the fusion process, many questions remain regarding the physical mechanism. Studies using simpler model systems can provide insight into the fundamental mechanisms of the rearrangement of bilayers and lead to a better understanding of the biological process (14, 15). We have prepared a series of DNA-lipid conjugates in which DNA o...

Application of nucleic acid–lipid conjugates for the programmable organisation of liposomal modules

“…More critical for the application of these systems for controlled chemical mixing between compartments is the prohibitively low contents mixing observed between the liposomes [57,65,124]. Contents mixing values as high as ~15% have been achieved [57,65], however efficiencies of less than 2% are more common [65,124], which may, in part, be explained by leakage of contents during the fusion process [57].…”

“…Non-hybridizing spacer groups between the membranes and the DNA zipper sequences fairly predictably enhance the docking rate between liposomes but systematically reduce fusion efficiency due to the liposome membranes not being brought into as close proximity [124]. Perhaps more surprisingly, there appears to only be a slight dependence of fusion efficiency on the length (and hence binding strength) of the DNA strands; while 27 base sequences are more efficient than short 12 base sequences, an increase to 42 base strands provides no significant enhancement [57].…”

“…Most studies of DNA-mediated liposome fusion have been conducted using a highly fusogenic liposome formulation of 2:1:1 DOPC:DOPE:cholesterol [57,65,122,124]; inner monolayer mixing is found to be considerably lower than total lipid mixing within these systems, suggesting that a significant proportion of zippering interactions stall at, or reverse after, formation of the hemifusion state. More critical for the application of these systems for controlled chemical mixing between compartments is the prohibitively low contents mixing observed between the liposomes [57,65,124].…”

“…Traditional fluorescence assays for membrane fusion have been employed to study total lipid mixing, inner monolayer lipid mixing and contents mixing between interacting liposomes [57,65,122,124]. Significant (up to ~80%) lipid mixing can be initiated between liposomes by the DNAzippering mechanism [65].…”

Sec17 can trigger fusion of trans -SNARE paired membranes without Sec18

“…Several factors can contribute to lowering the energy barrier for two apposed membranes to rearrange their bilayers for fusion: (i) the physical force of membrane deformation deriving from trans-SNARE complexes forming continuous α-helices between their SNARE and transmembrane domains (42) [the initial concept that the energy for fusion derives entirely from forming a continuous α-helix between SNARE and transmembrane domains has been questioned by observations that transmembrane domains can be replaced by lipidic anchors for yeast vacuole fusion (43) or neuronal fusion (44)]; (ii) the docking of membranes in close proximity (45,46); (iii) the membrane bending that surrounds an extended region of membrane contact, driven by tethers (47); (iv) the enrichment of small headgroup lipids that can more readily fit into the nonbilayer lipidic structures of hemifusion and fusion intermediate states (25,34); and (v) localized (e.g., trans-SNARE-associated) additional proteins or other factors that can bind to lipids or insert into membranes to promote nonbilayer transitions. The latter includes Ca 2+ -triggered insertion of synaptotagmin at the neuronal synapse (48) and Sec17 for intracellular fusion.…”