A Biomimetic DNA‐Based Membrane Gate for Protein‐Controlled Transport of Cytotoxic Drugs

Abstract Chemistry is ideally placed to replicate biomolecular structures with tuneable building materials. Of particular interest are molecular nanopores, which transport cargo across membranes, as in DNA sequencing. Advanced nanopores control transport in response to triggers, but this cannot be easily replicated with biogenic proteins. Here we use DNA nanotechnology to build a synthetic molecular gate that opens in response to a specific protein. The gate self‐assembles from six DNA strands to form a bilayer‐spanning pore, and a lid strand comprising a protein‐binding DNA aptamer to block the channel entrance. Addition of the trigger protein, thrombin, selectively opens the gate and enables a 330‐fold increase inw the transport rate of small‐molecule cargo. The molecular gate incorporates in delivery vesicles to controllably release enclosed cytotoxic drugs and kill eukaryotic cells. The generically designed gate may be applied in biomedicine, biosensing or for building synthetic cells.


DNA assembly
Equimolar mixtures of DNA oligonucleotides (1 μL each, stock concentration of 100 µM) (Table S2 for composition of DNA pores) were dissolved at 1 µM in a buffer solution of 12 mM MgCl2 in 0.6x TAE (40 mM Tris, 20 mM acetic acid), pH 7.4 to a final volume of 100 μL. Folding was achieved on a BioRad PCR thermocycler (UK) using a programme including heating to 85°C for 10 min, cooling to 65°C within 5 min, cooling to 25 °C at a rate of 0.1 °C per 2 min, and cooling to 10 °C at a rate of 0.2 °C per min. Samples were stored at 4 °C.

SDS PAGE
The assembled DNA nanostructure and component DNA oligonucleotides were analysed with commercial 10% polyacrylamide gels (BioRad, UK) in 1x TGS (25 mM Tris, 192 mM glycine and 0.1% SDS, pH 8.6). For gel loading, a solution of the DNA nanopores (2 μL, 1 μM) was mixed with folding buffer (13 µL, 2 mM MgCl2 in 0.6x TAE, pH 7.4) and 6x gel loading dye (5 μL, New England Biolabs, UK). The gel was run at 60 V for 60 min at 4°C. The gel bands were visualised by staining with ethidium bromide and UV illumination. A 100 bp marker (New England Biolabs, UK) was used as a reference standard.

Thrombin-TBA electrophoretic mobility shift assay
TBA (10 μL, 4 μM) were mixed with thrombin (10 µM stock) in buffer A (0.3 M KCl, 15 mM Tris pH 7.4) yielding concentrations of 0 to 2.67 µM in a final volume of 50 μL. After incubation for 30 min at 30°C, and 6x gel loading dye (10 µL, New England Biolabs) was added, and the samples were loaded onto a thermally equilibrated 2% agarose gel. The gel was run in 1x TAE buffer, pH 8.3 at 60 V for 60 min at 4°C. Staining and molecular markers were as described above.
1.5. Thrombin-pNP electrophoretic mobility shift assay pNP (2 μL, 1 μM) was mixed with thrombin (10 µM) in buffer A yielding concentrations of 0 to 3.2 µM in a final volume of 20 μL. After incubation for 30 min at 30°C, 6x gel loading dye (5 µL) was added, and the samples were loaded onto thermally equilibrated 10% PAGE. The gel was run in 1x TBE buffer (89 mM Tris-borate and 2 mM EDTA, pH 8.3) at 60 V for 90 min at 4°C. Staining and molecular markers were as described above.

Preparation of fluorophore-filled LUVs and dye release assay
Solutions of the lipids DOPC (70 μL, 10 mM) and DOPE (30 μL, 10 mM) in chloroform were added to a 5 mL round bottom flask. The solvent was removed using a rotary evaporator (Buchi) to yield a thin film, which was further dried under high vacuum (Buchi) for 1 h. The lipid was re-suspended in PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4) containing SRB (50 mM). The solution was sonicated for 20 min at 30°C and then equilibrated for 3 h. LUVs were extruded 25 times through a 200 nm polycarbonate membrane (Avanti Polar Lipids, US) using the extruder kit (Avanti Polar Lipids, US). The non-encapsulated dye was removed using a NAP-25 column (GE Healthcare, UK), and LUVs were exchanged into buffer B (0.2 M KCl, 10 mM Tris pH 7.4). LUVs were then subjected to dynamic light scattering with a Malvern Zetasizer Nano S (UK) to confirm the vesicles' diameter. Purified LUVs were used within 48 h and gently resuspended immediately prior to use. For release assays, the LUV suspension with encapsulated SRB (10 μL), pNP (30 µL, 1 µM) and buffer B (95 µL, 102.5 µL, 108.5 µL) were added to a 10 mm quartz cuvette (Hellma Analytics). Fluorescence was monitored at 586 nm and excited at 565 nm. After 5 min, thrombin (15 µL, 7.5 µL, 1.5 µL; 20 µM in buffer A) was added to give a ratio of 1:1, 1:5 or 1:10 (pNP:thrombin) in final volume of 150 µL. After 60 min of monitoring fluorescence, samples were mixed with a 1% solution of Triton X-100 (10 µL) to lyse all vesicles to identify maximum SRB release. Maximum fluorescence emission and the fluorescence prior to addition of thrombin were used to calculate the kinetics of release as %. For the kinetic analysis of efflux, the first 5 min of three 2 µM thrombin-mediated release traces were fitted with a linear line-ofbest-fit. For the 0 µM thrombin-mediated release trace (baseline), the first 10 min of seven traces obtained from different batches of fluorophore-filled LUVs were averaged to one trace which was fitted to the line-of-best-fit. From these lines-of-best-fits, the initial rate at 1 min, 1.5 min, and 2 min were calculated and averaged to minimize fluctuations in the data.

Cell-based assay
On day 0, HeLa cells were plated at a density of 10,000 cells per well in a 96-well plate and left to grow overnight. The next day (day 1), cells were supplanted with fresh culture medium and treated with either: thrombin (10 μL, 165 µM) in buffer A, pNP2 (10 μL, 1 μM); topotecan (10 μL, 3 μM), LUVs filled with 3 μM topotecan (10 μL, 0.1 mM, PC:PE 7:3 lipid ratio), pNP2 functionalized topotecan-filled LUVs (20 μL), and the latter in combination with thrombin (10 μL, 165 µM in buffer A). All wells were made up to final volume of 100 μL. The treated cells were maintained in a humidified atmosphere, containing 5% CO2 at 37°C, for 3 d. Brightfield images were captured at day 1, 2 and 3 using an inverted Nikon Eclipse microscope, 20x air objective. Images were processed using ImageJ. On day 3, a WST-1 colorimetric assay was used to quantify cell viability. WST-1 (10 μL) was added to the culture medium in each well (100 μL) [55] and incubated for 3 h. The absorbance was determined at 450 nm using a microplate reader (VICTOR multilabel plate reader, PerkinElmer), and the absorbance reading at 620 nm was used as a reference. The experiment was performed in triplicate. Tables   Table S1. Names, modifications and sequences of DNA oligonucleotides used for folding protein-gated nanopore (pNP) and variants.

Supplementary
X Cy3 = Cy3 fluorophore; X Cy5 = Cy5 fluorophore; X T = TAMRA fluorophore.  Figure S1. 2D DNA maps of protein-gated nanopore pNP, and variants pNP2 and pNP TAMRA . Strands are labelled 1-6. Squares denote the 5' end of DNA and triangles the 3' end. Orange asterisks indicate the position of cholesterol anchors, the red coloured section represents the TBA sequence in the lid, Green indicates oligonucleotide dye-ext. carrying the TAMRA-dye, and H and D1-D3. indicate the hinge region of the lid that remains bound to the pore, and the docking regions, respectively.