We propose the following procedure to avoid these problems associated with cholesterol-tagged membrane-anchored DNA-DNA-hybridization. The processes of vesicle formation and their functionalization are separated by a constant modification of the lipid bilayer during vesicle formation followed by a two-step functionalization process. By incorporating biotinylated phospholipids into the lipid bilayer during vesicle formation a functionalization process independent of the formation process is provided. After formation and incorporation of biotinylated phospholipids to the lipid bilayer the vesicles are functionalized by decoration of their surface with (strept)avidin first followed by a decoration with biotinylated DNA (btnDNA). Thus, as shown in Figure 1 linking of vesicles is established via a complex of biotinylated phospholipid-(strept)avidin-btnDNA hybrid-(strept)avidin- biotinylated phospholipid.The anchorage of DNA strands using biotinylated phospholipid-(strept)avidin-btnDNA is stronger than cholesterol-tagged ssDNA. Thus, we expect no loss of specificity due to spontaneously leaving the lipid bilayer of the linkers and incorporate randomly into (other) lipid bilayers.
Concept of how to equip vesicles with specific linkers stickers. The linking was done with biotinylated phospholipids, streptavidin and biotinylated DNA.
Biotinylated DNA strands bind to the surface of some of the vesicles, but to rarely to allow reliable self-assembly of vesicles. The fluorescence signal of Streptavidin Alexa Fluor 488 conjugate was detected on the surface of all vesicles (data not shown) the fluorescence signal of the Cy3-labeled ssDNA was found rarely (see Fig. 5). Different hypothesis for this fact were developed : unspecific binding of DNA to surfaces, stoichiometric interactions of the different molecules etc.
Although a fluorescence signal of the Cy3-labeled ssDNA is found in certain cases, such signals occurred very rarely. Therefore, the encounter probability of linking vesicles would be impractically low. We introduced Streptavidin Agarose beads to simplify the system (no vesicular interactions) in order to isolate the mechanisms responsible for the reduction of the fluorescent signals on the vesicular membrane. We performed the same experiments with streptavidin-coated beads.
The above figure shows the results that one could link the fluorescent DNA linked to the bead. To exclude the effect of an unspecific binding of the DNA-strands to the vesicular membrane or containment surfaces (DNA is know to stick to surfaces), fluorescent atto-biotin was incubated with a membrane-bound PEG-streptavidin construct visualized in Figure 7. As these experiments also showed a fluorescence singal of the Streptavidin Alexa Fluor 488 conjugate but no fluorescent signal of atto-biotin on the vesicular membrane, we had to consider the stoichiometric interactions of the different molecules.
DNA replaced by atto-biotin gave also a negative result: no binding to the linker. In order to get a reliable signal on the vesicles, we had to study the kinetics of the different molecules involved (biotin, PEG, DNA, streptavidin, phospholipids, membrane) and set up a dilution scheme to test different concentrations in the micro-well plates. We systematically tested the kinetic partners by reducing the types of possible interactions and measured afterwards the fluorescent signals in the micro-well plates. depending on the concentration of free biotin present during incubation. The results are shown in the graph – the fluorescence signal of the Streptavidin Alexa Fluor 488 conjugate depends on the concentration of free biotin present during incubation. By these experiments we were then able to set up a system where the fluorescent signal due to the binding of atto biotin to the surface of biotinylated vesicles coated with Streptavidin was detectable and distributed over the whole population of the vesicles
After having studied the kinetics of the involved molecules for the building of the linkers and different dilution series experiments in mico-well plates. In comparison to prior results (Fig. 4) the fluorescent signal on the vesicles could be remarkably increased. In the next step the atto-biotin was then replaced by DNA-DNA hybrids. DNA-DNA binding needs NaCl concentration of about 50 mM. Although we got a signals in certain cases (data not shown), the presence of NaCl induced leakage and/or lysis vesicles within few minutes of observation. Therefore, we had to modify the observation technique: The glass surfaces had to be pre-coated with salmon sperm DNA to avoid leakage and/or lysis.
Detection of a fluorescence signal on the vesciluar membrane due to the binding of Alexa Fluor 488 labled antisense-ssDNA binding to the biotinylated sense-ssDNA-Streptavidin conjugate coating the vesicular surface. In the primordial important step of linking specifically vesicles was finally achieved. All the steps were only possible by a combined effort between SDU and UZH groups. In Figure 11 an aggregate of two spontaneously linked vesicles are shown.
Specific linkers binding vesicles together: a crucially important step for controlled self-assembly of programmable vesicular structures.
Concept of how to equip vesicles with specific linkers stickers. The linking was done with biotinylated phospholipids, streptavidin and biotinylated DNA.
DNA directly linked with phospholipids can exchange places with their neighbors. This exchange of linkers will lead to unspecific clustering of vesicles due to the distribution of the linkers on all partner vesicles (as discussed for cholesterol-tagged membrane-anchored DNA-DNA-hybridization Beales and Vanderlick, 2007). Therefore the second method with biotin-streptavidin-DNA-linkers proved to be superior due to anchoring of the sticker with two phospholipids and therefore a diminished probability of an exchange of stickers between two vesicles jeopardizing the specificity of linking between them. In a first step to realize specific intervesicular interactions phospholipids with fluorescent carboxyfluorescein labeled polyethylene glycole (PEG2000) were incorporated into the membrane (see Figure 2). PEG2000 was used to diminish unspecific adhesions between vesicles. Similar techniques are also used to stabilize paint emulsions (Jones, 2002) . In Figure 2 fluorescent vesicles can be seen proving the insertion of PEG2000 into the membrane. A control experiment without the fluorescent dye was also performed.
PEGylated vesicles were incubated with fluorescent streptavidin. In the lower row of the picture the negative control experiment can be seen in order to exclude unspecific binding of the Streptavidin Alexa Fluor 488 conjugate to the membrane.
Biotinylated DNA strands bind to the surface of some of the vesicles, but to rarely to allow reliable self-assembly of vesicles. The fluorescence signal of Streptavidin Alexa Fluor 488 conjugate was detected on the surface of all vesicles (data not shown) the fluorescence signal of the Cy3-labeled ssDNA was found rarely (see Fig. 5). Different hypothesis for this fact were developed : unspecific binding of DNA to surfaces, stoichiometric interactions of the different molecules etc.
Although a fluorescence signal of the Cy3-labeled ssDNA is found in certain cases, such signals occurred very rarely. Therefore, the encounter probability of linking vesicles would be impractically low. We introduced Streptavidin Agarose beads to simplify the system (no vesicular interactions) in order to isolate the mechanisms responsible for the reduction of the fluorescent signals on the vesicular membrane. We performed the same experiments with streptavidin-coated beads.
The above figure shows the results that one could link the fluorescent DNA linked to the bead. To exclude the effect of an unspecific binding of the DNA-strands to the vesicular membrane or containment surfaces (DNA is know to stick to surfaces), fluorescent atto-biotin was incubated with a membrane-bound PEG-streptavidin construct visualized in Figure 7. As these experiments also showed a fluorescence singal of the Streptavidin Alexa Fluor 488 conjugate but no fluorescent signal of atto-biotin on the vesicular membrane, we had to consider the stoichiometric interactions of the different molecules.
DNA replaced by atto-biotin gave also a negative result: no binding to the linker. In order to get a reliable signal on the vesicles, we had to study the kinetics of the different molecules involved (biotin, PEG, DNA, streptavidin, phospholipids, membrane) and set up a dilution scheme to test different concentrations in the micro-well plates. We systematically tested the kinetic partners by reducing the types of possible interactions and measured afterwards the fluorescent signals in the micro-well plates. depending on the concentration of free biotin present during incubation. The results are shown in the graph – the fluorescence signal of the Streptavidin Alexa Fluor 488 conjugate depends on the concentration of free biotin present during incubation. By these experiments we were then able to set up a system where the fluorescent signal due to the binding of atto biotin to the surface of biotinylated vesicles coated with Streptavidin was detectable and distributed over the whole population of the vesicles
After having studied the kinetics of the involved molecules for the building of the linkers and different dilution series experiments in mico-well plates. In comparison to prior results (Fig. 4) the fluorescent signal on the vesicles could be remarkably increased. In the next step the atto-biotin was then replaced by DNA-DNA hybrids. DNA-DNA binding needs NaCl concentration of about 50 mM. Although we got a signals in certain cases (data not shown), the presence of NaCl induced leakage and/or lysis vesicles within few minutes of observation. Therefore, we had to modify the observation technique: The glass surfaces had to be pre-coated with salmon sperm DNA to avoid leakage and/or lysis.
Detection of a fluorescence signal on the vesciluar membrane due to the binding of Alexa Fluor 488 labled antisense-ssDNA binding to the biotinylated sense-ssDNA-Streptavidin conjugate coating the vesicular surface. In the primordial important step of linking specifically vesicles was finally achieved. All the steps were only possible by a combined effort between SDU and UZH groups. In Figure 11 an aggregate of two spontaneously linked vesicles are shown.
Specific linkers binding vesicles together: a crucially important step for controlled self-assembly of programmable vesicular structures.