Fig.1 DLS showing the presence and size distribution of liposomes

A sample of newly-made liposomes was analysed using dynamic light scattering (DLS). Analaysis was performed using a scattering angle of 173°. The mean average size was found to be 68 nm, with a standard deviation of 27.77. These results confirm that the protocol we used for liposome preparation resulted in uniformly-distributed unilamellar vesicles. In subsequent experiments, we increased the extruder filter size to 200 nm, in order to encapsulate more molecules inside, as suggested by our model.

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Assaying nanopore assembly enabled understanding whether another basic building block of the design had been achieved. Each of the 6 DNA strands that form the DNA nanopore barrel should hybridise with their neighbour. Therefore, in theory, strands 1&2, 2&3, 3&4, 4&5, 5&6 should anneal to each other in pairs. Strands 1&6 should also anneal, resulting in a continuous barrel structure. When each of these combinations of DNA strands are run separately on an agarose gel, the resultant DNA bands are positioned such that they correspond to the expected molecular weights upon annealing (table below).
Furthermore, when all 6 bands of DNA are annealed together, the resulting band of DNA barely moves out of the well (FigXB). This is in accordance with the prediction of the molecular weight for the open nanopore. A similar result is also obtained for the closed nanopore, which additionally incorporates the lock DNA strand. However, simply incubating the 6 DNA strands together at room temperature, without annealing at 95°C, results in a smear of DNA. This smear occurs much farther away from the well and the position of the bands for the annealed closed and open nanopores. In fact, it appears within the range of much lower molecular weights for the pairs of DNA. This suggests that without annealing at 950C, the 6 DNA bands form varying combinations of hybrids but do not all anneal in the continuous barrel structure which would have resulted in a much higher molecular weight. Taken together, this data demonstrates that the closed and open nanopores are likely to have annealed in accordance with theoretical design and modelling predictions

Fig.4 2% agarose gel electrophoresis

The black box highlights the relative positions of the DNA bands when only two oligonucleotides are annealed together. The relative differences in these positions correspond to the differences in their molecular weights, as indicated in the table above. The black circles compare the positions of the DNA bands when all six DNA strands are incubated together, when annealed in a PCR thermocycler (lanes 2 and 3), and withouth annealing in thermocycler (lane 10).

Fig.5 Release of SRB from LUVs + open nanopore compared to LUVs

Fluorescence intensity over time of SRB-filled liposomes with (blue line) and without (green line) embedded open nanopores. Nanopores dissolved in buffer A were added to the phospholipid mixture prior to extrusion. The shaded region corresponds to standard deviation from 3 replicates. In the beginning the fluorescence levels remained close to zero in both samples due to fluorescence-quenching. However, when open nanopores are present in the membrane, SRB leaks from the vesicles over time. As it is no longer trapped inside in a high-concentration environment, the fluorescence of the sample increases until reaching a steady state after ca. 600 min. If no nanopores are embedded into the membrane, SRB stays inside the vesicle with minimal leakage (green curve). Taken together, these data demonstrates that the assembled nanopores can emben into the membrane of LUVs, allowing SRB to leak out.

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