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- The staple DNAs for the synthesis of DNA origami are purchased from Genenet. Co., Ltd. The M13mp18 single-stranded DNA for the scaffold of the DNA origami is purchased from New England Biolabs Japan. The buffer solution for the DNA is prepared by solvating 0.019 g of 2-amino-2-hydroxymethyl-1,3-propanediol, 0.12 g of ethylenediamine-N,N,N',N'-tetraacetic acid disodium salt (EDTA•2Na), and 0.119 g of MgCl2 in 100 mL of distilled water, followed by adding HCl aq to adjust the pH to 8.001. A Fluorescein-derivative is used as the fluorescence molecule.
Design and synthesis of the DNA weathercock
- The DNA origami is synthesized as follows. The DNA weathercock is designed by using the software caDNAo and the base sequence for the staple DNAs are determined. We mix 10 uL of the solutions of the scaffold DNAs and each staple DNAs. We then use the thermal cycler for annealing: the mixture is heated to 90 ℃, kept for 60 min, and then cooled down to 25 ℃ at the rate of 20 ℃ / h.
Synthesis of the nanoporous alumina substrate
- Al sheet (Wako Pure Chemical Industries Ltd., 99.99 %; 20×100 mm2) was electrochemically polished in a solution composed of perchloric acid and ethanol. The aluminum was then washed in ethanol and pure water. For the anodization, a carbon electrode was used as the cathodic electrode. The aluminum was anodized at 40 V in a 0.3 M oxalic acid solution at 16 °C. This long-period anodization step was performed for 3 h in order to obtain hexagonally well-ordered arrays of pores. The alumina layer was then dissolved in a mixed solution of 6 vol % phosphoric and 1.8 wt % chromic acids at 60 °C. The specimen was subsequently anodized for 5 min at 40 V in a 0.3 M oxalic acid. The produced holes were chemically etched for 30 min in a 5 vol-% phosphoric solution at 30 °C. The synthesized DNA origami is observed with atomic force microscope (AFM; Seico Instruments Inc. nanocute).
Mounting the DNA weathercock to the nanopore
- We then mount the DNA weathercock into the pore of the porous substrate. We drop the solution containing the DNA weathercock on to the substrate and evaporate the solvent. By tuning the components of the buffer solution, the interaction between the DNA shaft and the alumina pore surface is optimized. Thus we expect the shaft of the DNA weathercock is incorporated into the pores.(fig.3-1) For a future plan, we will sputter both inside and outside the pores with one material that have affinity with DNA, followed by sputtering the other material that have no affinity with DNA only onto the substrate surface (not in the pore) by tilting the substrate.(fig.3-2) The porous substrate modified by this way would be better for incorporation of the DNA shaft.
Applying a water flow and detection of the flow
- To apply the water flow, we put a silicon rubber spacer and a cover glass on the substrate mounted with the DNA weathercock (fig.3-3). We then slide the cover glass to give a flow to the DNA weathercock along a certain direction.
- We detect the motion of the weathercock in two ways (fig.3-4). In one simple method, we use a polarizer. If all the fluorescein attached to the weathercock is aligned in a flow direction, substrate will emit macroscopically polarized fluorescence under a uv irradiation. We can easily know the direction even by naked-eye observation of the fluorescence through a polarizer film.
In the other method, we use a confocal laser microscope (Nikon A1R+). Since fluorescein has excitation wavelength of 494 nm and emission wavelength of 525 nm, we use Ar laser (488 nm) for excitation. If the weathercock is rotated by 180º, the position of the fluorescein molecule will moves by ca. 100 nm. Considering the resolution of the microsope, the movement of 100 nm can be detected by a real-time observation.