Figure 4: Membrane penetration mediated by biotin–streptavidin linkages.

(a) An alternative strategy to mediate DNA nanopore–membrane interactions based on streptavidin linkages between biotinylated staple strands at the bottom of the the T pore plate and biotinylated lipids. (b) TEM image of a non-functionalized T pore. (c) TEM image of a biotinylated T pore decorated with streptavidin. Two streptavidin proteins are highlighted in black circles. (d) TEM image of biotinylated T pores interacting with SUVs (90% Egg phosphatidylcholine, 10% biotinylated 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(biotinyl) (sodium salt) (DOPE) in the presence of streptavidin. All TEM images have a scale bar of 50 nm). (e) Time series of fluorescence images of an immobilized GUV obtained from a dye influx experiment using confocal fluorescence microscopy as described in Fig. 3. At t=0, biotinylated T pores are added to the solution (streptavidin already present on T pores, which can be seen in image c; cf. Supplementary Movie 5). Dye influx demonstrates successful penetration of the membrane, which is absent in control experiments without pores. Scale bar, 10 μm. (f) Fluorescence intensity inside of the vesicles in e. Blue circles: pore formation by biotinylated T pores, grey circles: control. Data are fit with the linearized influx model (cf. Supplementary Note 4). (g) Normalized influx rate k/k₀ plotted against the area A of the vesicles. Lack of aggregation is translated to slower influx rates compared with the hydrophobically modified pores.