A Light‐Triggered Synthetic Nanopore for Controlling Molecular Transport Across Biological Membranes

Abstract Controlling biological molecular processes with light is of interest in biological research and biomedicine, as light allows precise and selective activation in a non‐invasive and non‐toxic manner. A molecular process benefitting from light control is the transport of cargo across biological membranes, which is conventionally achieved by membrane‐puncturing barrel‐shaped nanopores. Yet, there is also considerable gain in constructing more complex gated pores. Here, we pioneer a synthetic light‐gated nanostructure which regulates transport across membranes via a controllable lid. The light‐triggered nanopore is self‐assembled from six pore‐forming DNA strands and a lid strand carrying light‐switchable azobenzene molecules. Exposure to light opens the pore to allow small‐molecule transport across membranes. Our light‐triggered pore advances biomimetic chemistry and DNA nanotechnology and may be used in biotechnology, biosensing, targeted drug release, or synthetic cells.


Assembly of LP
All DNA sequences and 2D connectivity maps of the DNA nanopores are provided in Table S1 and Figure S1, respectively. For pore assembly, equimolar mixtures of DNA oligonucleotides (1 μL each, stock concentration of 100 μM) were diluted in 16 mM MgCl2 to a final volume of 100 μL. Folding was achieved with a BioRad PCR thermocycler using heating to 95 °C for 10 min, cooling for 0.5 °C per min to 60 °C, followed by cooling at 1°C per min to 20 °C.

PAGE
Folded LP nanopores and variant pores were analysed with commercially procured 10% and 4-20% polyacrylamide gels. For pores lacking cholesterol tags, the gel buffer was 1 x Tris-Borate-EDTA buffer (TBE, from 10 x TBE buffer,89 mM Tris-borate and 2 mM EDTA, pH 8.3)(ThermoFisher scientific) and for pores with cholesterol anchors the buffer was 1 x Tris/ Glycine/ Sodium Dodecyl Sulphate (1x TGS, from 10 x TGS (25 mM Tris, 192 mM glycine and 0.1% SDS, pH 8.6)(BioRad). After equilibrating the gels (30 min at 100 V and 30 min at 0 V), the samples were loaded with gel loading dye using a 100 bp marker as a reference standard. The gels were run for a desired time at 100 V. Afterwards, the gels were washed with water and stained with ethidium bromide. The bands were visualised with UV illumination (Azure Biosystems c Series Imagine System).

Agarose gel electrophoresis
Folded LP and variant nanopores were analysed with agarose gels (1-3%) in 0.5 x TBE supplemented with 10 mM MgCl2, and ethidium bromide at a dilution of 1:30,000 of the stock solution (10 mg/mL in H2O). The DNA samples were loaded with gel loading dye using a 1 kb/100 bp marker as reference. 1 The gel was run for a desired time at 65 V, and gel bands were visualised with UV illumination (Azure Biosystems c Series Imagine System).

Formation of small unilamellar vesicles (SUVs)
A solution of POPC or DPhPC in chloroform (100 µL, 10 mg/ mL) was added to a 2 mL screw-top vial, and the solvent evaporated using argon gas under reduced pressure while rotating the vial to yield a dry lipid film. The lipid film was resuspended in 1 X Phosphate buffered saline buffer (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4) (1 mL) and vortexed to fully dissolve the lipid. After 30 min of sonication, the vesicles were extruded 25 times through an Avanti Mini-Extruder with a size-specific filter (200 nm) to yield vesicles with the set diameter. The diameter of SUVs was confirmed by dynamic light scattering (DLS) with the Zetasizer Nano S Malvern.

Binding of DNA nanopores to SUVs
The binding of LP to vesicles was monitored by incubating DNA nanopores with POPC SUVs (1:1 volume ratio, LP concentration of 1 µM) at different lipid concentrations (2-200 µM). The mixture was incubated for 30 min at RT and analysed via a 1.5% agarose gel in 0.5 TBE supplemented with 10 mM MgCl2 and EthBr. 1 The gel was run for 90 min at 65 V and 4°C, and bands were visualised with UV illumination (Azure Biosystems c Series Imagine System).

Formation of giant unilamellar vesicles (GUVs)
POPC in chloroform (36 µL, 100 µM lipid concentration) was added within a rubber ring on an indium tin oxide (ITO) coated glass slide and allowed to dry. To the dried lipid film, 200 µL of 400 mM sucrose was added, another ITO slide was placed on top, and the assembly was inserted into Nanion Vesicle Prep Pro. GUVs were formed via electroformation using the conditions of 10 Hz, 4V amplitude, 1 min rise, 100 min main, and 10 min fall.

Melting point analysis via UV-Vis spectroscopy
The melting temperatures of LP variants carrying up to 4 cholesterol tags were determined using a Varian Cary 300 Bio UV-Vis spectrophotometer with a Peltier element and a quartz cuvette with a 1 cm path length. Changes in absorbance at 260 nm of 0.1 µM of LP were monitored by heating at a rate of 1 °C per min to yield a full melting profile from 20°C to 80°C. 1

Formation of fluorophore-filled SUVs
A solution of DPhPC in chloroform (100 µL, 10 mg/mL) was added to a 2 mL-screw top vial, and the solvent evaporated using argon gas under reduced pressure while rotating the vial to yield a dry lipid film. The film was resuspended in 1 mL of fluorescent buffer (50 mM SRB in 1 x PBS) and vortexed to fully dissolve. After 30 min of sonication, the vesicles were equilibrating at 4°C overnight. The next morning, vesicles were extruded 31 times through an Avanti Mini-Extruder with a size-specific filter (200 nm) to yield vesicles with the set diameter. The extruded vesicles were filtered with a NAP-10 column (GE Healthcare). The NAP-10 column was washed twice with 10 mL 1 x PBS, loaded with 400 µL of the extruded vesicles, and eluted with 20 mL of 1 x PBS. The fractions were collected, the vesicle size was determined via DLS, and the SRB content was quantified with a fluorescence spectrophotometer by adding 1% (v/v) solution of Triton X-100 (10 µL) to quantify the maximum amount of released fluorophores. 1

Dye flux
To assay dye release via membrane-embedded LP and pore variants, a quartz cuvette with a path length of 10 mm (Hellma Analytics) was filled with SRB-encapsulated and purified DPhPC vesicles (120 µL, 100 µM lipid concentration). The fluorescence was monitored at 586 nm during excitation at 565 nm. After 5 min incubation, LP pore (30 µL, 1 µM) was added, and the fluorescence was monitored for 30 min of equilibration. Successful pore insertion and dye release of LP was recorded by irradiating the sample at 365 nm at an intensity of 154 µW cm -2 for 1 h. 1 After 60 min of monitoring SRB release by fluorescence monitoring, a 1% (v/v) solution of Triton X-100 (10 µL) was added to quantify the maximum amount of released fluorophores of lysed vesicles. The maximum fluorescent emission after lysing and the fluorescence before prior the addition of the pore and irradiation were used to calculate the percentual release rates.

Current recordings
Single-channel current recordings were carried out using a parallel recording set-up based on an integrated chip (Orbit Mini and Orbit 16, Nanion Technologies) with multielectrode-cavity-array chips (IONERA) 23 . Bilayers were formed of DPhPC dissolved in octane to a final concentration of 10 mg/mL.
The mixture was applied to the cis chamber, and insertions monitored by increases in conductance steps. Current traces were acquired at 10 kHz using Element Data Recorder software (Elements).
Single-channel analysis was performed using Clampfit software (Molecular Devices). 6 Tables   Table S1. Names, modifications, and sequences of DNA oligonucleotides used for the assembly of the   ladder, (2) strand S1, (3) strands S1-2, (4) strands S1-3, (5) strands S1-4, (6) strands S1-5, (7) LP-          nm) restores the spectrum to the initial state due to isomerisation to the trans form. The spectra differ from the absorption readings in Figure S13 due the higher amount of nucleic acids.        for voltages ranging from −100 mV to +100 mV at 20 mV steps, and conductance histograms were obtained at +20 mV. For the recordings, the cholesterol-tagged nanopores were mixed with mild detergent OPOE, and the mixture was added to the cis chamber for membrane insertion. The recordings were acquired in 1 M KCl, 10 mM HEPES, pH 7.4. Figure S23. Single-channel current recordings of LPDA containing a lid without an azobenzene modification, after irradiation with UV light. Analysis using representative single-channel current traces recorded at +50 mV relative to the cis chamber. Average current-voltage graphs (±SEM) for voltages ranging from −100 mV to +100 mV at 20 mV steps, and conductance histograms were obtained at +20 mV. For the recordings, pores were irradiated for 30 minutes at 365 nm with an intensity of 145 µW cm -2 . The cholesterol-tagged nanopores were mixed with mild detergent OPOE, and the mixture was added to the cis chamber for membrane insertion. The recordings were acquired in 1 M KCl, 10 mM HEPES, pH 7.4.