DNA-Controlled Lipid-Membrane Fusion
Licentiate thesis, 2009
Membrane fusion is essential for nerve-cell communication, for protein transport between cell organelles and the cell-membrane and for enabling the merger between virus and host membranes during virus infection. We have demonstrated that short DNA oligonucleotides, membrane-attached via CH in an orientation that mimics the overall zipperlike architecture of fusion-inducing proteins, induce fusion of both suspended vesicles and vesicles site-specifically tethered to SLBs. The site-specific surface-based assay is attractive for membrane-protein array applications.
Model systems for membrane fusion have been generated from lipid vesicles, which are artificial spherical cell membranes encapsulating liquid compartments. In the bulk assay, two vesicle populations were decorated with complementary DNA strands which hybridize in a zipper-like fashion to bring about membrane fusion. The DNA strands were anchored in the lipid membranes of the vesicles via a hydrophobic moiety, cholesterol (CH), covalently attached at one end of the DNA strands. The lipid rearrangements taking place as a consequence of the forced bilayer contact and subsequent fusion were investigated using fluorescence resonance energy transfer (FRET) between donor and acceptor dyes. Total lipid mixing, inner leaflet mixing as well as content mixing was monitored. In order to design a DNA zipper with improved fusion properties, we have assessed how parameters such as length of the DNA strands, anchoring strategy and DNA surface coverage affect the ability of DNA to induce fusion. The results reveal that the use of two CH anchors is essential to prevent complementary DNA strands from shuttling between differently modified lipid vesicles. A surface coverage of 6-13 DNA strands per lipid vesicle was a precondition for efficient fusion, whereas fusion was insensitive to DNA length within the tested range.
Implementation of the DNA-controlled fusion concept into a site-specific surface-based assay, by fusing the vesicles to a SLB, brings the concept towards site-specific delivery of membrane constituents and thus further towards the realization of a protein array bio-sensor. The geometry of the surface-based system resembles that of native vesicles fusing with the cell membrane better than do the vesicle-vesicle fusion assay. The surface-based fusion assay also provides details and heterogeneities of the fusing vesicle population, as it allowed us to study single vesicle fusion events. From the results we learn that fusion is observed for a specific range of 10-16 DNA strands per vesicle, and from studies of the diffusion of the tethered vesicles prior to fusion, together with the vesicle docking time prior to fusion, a possible scenario for the zipper-DNA induced vesicle-SLB fusion machinery is proposed. Future applications and upcoming projects are also discussed.
fluorescence resonance energy transfer
total internal reflection fluorescence microscopy