Schematic representation of experimental setups A, B, and C.

<p>Numbers (0–3) indicate processes and small letters (a–e) indicate states. (Setup A) The incorporation of phospholipid-grafted PEG tethers into the vesicle membrane is analyzed. Vesicle populations (VPs) differ in the presence (VP A1) and absence (VP A2) of phospholipid-grafted fluorescently labeled PEG tethers (cfPEG2000-DSPE) during vesicle formation. (Setup B) To settle the specificity of membrane loading with streptavidin depending on the presence of anchoring sites, phospholipid-grafted biotinylated PEG tethers (bPEG2000-DSPE) are either present (VP B1) or absent (VP B2) during vesicle formation. Both VPs are subsequently incubated with fluorescently labeled streptavidin. Excess streptavidin is removed after incubation. (Setup C) To designate both the sequence-dependence and the dependence on the monovalent salt concentration of the vesicle self-assembly process two VPs either loaded with complementary (VP C1, VP C2) or noncomplementary (VP C3, VP C4) DNA single strands (ssDNA) are unified in solutions distinct in sodium iodide concentration. The streptavidin solutions were individually preincubated with biotin-ssDNA solutions prior to vesicle decoration (see microtubes holding the streptavidin/biotin-ssDNA solutions). After incubation of vesicles excess streptavidin/biotin-ssDNA is removed. DNA hybridization of complementary ssDNA causes accumulation of linkers and of the fluorescence signal (e.i) in the contact area over time (d to e) that is absent for noncomplementary ssDNAs (e.ii). (e.i) DNA-independent crystallization of streptavidin molecules on the surface of vesicles (*) that distributes stresses arising during/after DNA-mediated self-assembly may stabilize the linking system by compensating streptavidin molecules either incompletely equipped with biotin-ssDNA (**) or anchored only partially (***).