Fig. 2: Insertion and interaction of LGC with lipid bilayers.

a 1.5% agarose gel analysis of the DNA pore and its binding activity to lipid membrane vesicles. Lanes from left to right: no lid LGC without cholesterol modifications (LGC-N-Chol) without SUVs, incubated with SUVs (DOPC/DOPE = 7:3, 0.5 mM total lipid), no lid LGC with 64 cholesterol modifications (LGC-N+Chol) incubated with SUVs (total lipid concentration 0, 0.01, 0.025, 0.05, 0.25, and 0.5 mM, respectively). Data are representative of more than three repeats. b Representative TEM images of LGC-N+Chol pores bound to lipid vesicles. Blue arrowheads pinpoint the pores. Scale bars: 50 nm. The data are representative of n = 2 independent experiments. c Scheme of the GUV-dye-influx assay and d a time series of corresponding confocal GUV images with Cy3-labeled LGC (magenta) and Atto633 dye (green). Top: LGC-N+Chol readily interacts with bilayer (magenta circle around GUVs). Their insertion leads to an influx of the Atto633 dye inside the GUV interior. Bottom: LGC-N-Chol does not interact with bilayer (no magenta circle around GUV) or insert into GUV, showing no dye influx over the course of 3 h. The data are representative of n = 3 independent experiments e Bar plot showing the percentage of GUVs with a filled interior after 3 h. The data summarize the average percentage of influx and error bars show the standard deviation of mean percentage influx counted from three independent experiments across n = 49 GUVs in case of LGC-N+Chol and n = 59 for LGC-N-Chol. Scale bars: 10 μm. f Current trace showing a single LGC-N+Chol channel inserted into a planar DPhPC membrane. The trace was recorded at an applied voltage of +80 mV for the first 5 s after which voltage potential was switched to −80 mV. g Conductance histogram of 19 individual LGC-N+Chol channels recorded at −20 mV. h Current-voltage (IV) plot showing the average current of 19 individual insertions ±SEM at membrane potentials ranging from −100 mV to +100 mV in 20 mV steps. All electrophysiological experiments were conducted in buffers composed of 1 M KCl, 10 mM HEPES pH 7.6. Source data are provided as a Source Data file.