Fig. 3: Construction of a membrane-spanning nanogatekeeper on GPMV to mimic cell membrane ion channel.
From: DNA-based artificial molecular signaling system that mimics basic elements of reception and response
a CLSM imaging of localization of Alexa Fluor 488-labeled chol-c-DNGK on the membrane of our giant membrane vesicles. Chol-DNGK anchored on the giant membrane vesicles before (left) or after (right) addition of 3 mM ATP. One micromolar chol-DNGK incubated with 400 μL giant vesicles at 37 °C for 30 min. Scale bar: 5 μm. b, c Flow cytometry studies of fluorescent intensity change after addition of 3 mM ATP and incubation for 30 min. Decreased fluorescence on the membrane demonstrated that ATP switches the closed state of chol-DNGK to the open state. d Statistical evaluation with CLSM. Each column represents the statistical sample population of 100 membrane vesicles corresponding to each group in a. e Three-dimensional (3D) schematic representation of an ATP-driven chol-DNGK on the membrane of GPMV. f Flow cytometry study of one cholesterol-labeled nanogatekeeper (1-chol-c-DNGK and 1-chol-o-DNGK) on GPMV membrane. g A 200 μL GPMV solution incubated with 3 μM FluoZin™-3 indicator, followed by adding 0.5 μM chol-DNGK s for 30 min and, finally, treating with 0.2 mM Zn2+. Each column represents the statistical sample population of 50 membrane vesicles. h Dynamic study of ATP response on chol-c-DNGK directly showing that the closed state switches to the open state after treatment with ATP. A 200 μL solution of GPMVs with 3 μM FluoZin™-3 indicator was incubated for 2 h. Then 0.5 μM nanogatekeeper was incubated for 30 min, followed by the addition of 0.2 mM Zn2+ and 3 mM ATP and incubation for 20 min. Each column represents the statistical sample population of 50 membrane vesicles. P-values were calculated by Student’s t-test. Source data are provided in the Source Data file.
