Abstract

Recent years, the technology of DNA origami is attracting keen interest for the synthesis of various nanostructures.　But there are only few example of DNA origami with a rotating part, which are found in nature or industries: flagellum movement of Eugelena, turbines, and motors. Another problem is that the creation of the macroscopic-scale device only with DNA origami, which works with the macroscopic output or input signals is difficult because the synthesis of DNA-origami in large amount costs a lot. Here, we fabricate the device which detects the flow of surrounding water by combining inorganic nanoporous substrate with nanoscale weathercock made of DNA origami. The DNA weathercock consist of a blade and shaft parts are attached with a fluorescence molecule. We mount the DNA weathercock into the nanopore of the inorganic nanoporous substrate of size-tunable anodizing alumina, so that the DNA weathercock can freely rotate. When we give a flow, the DNA weathercock turns to the direction of the flow. We observe the behavior of fluorescence molecule with a fluorescence microscope or even by our eye through a polarizers set on the device.

Introduction

Background

There are many important objects which have rotating parts in nature, industries, and in our life. For example, we find rotation of the earth, flagellum movement of Euglena, turbine for power generations, motors of cars and locomotives, etc. Again, rotation is an important movement in many ways.(Figure 1)
If we could fabricate a rotating machinery in nanoscale, it will provide valuable applications such as nanorobotics. This may be achieved by using DNA nanotechnology (Figure 2). Recently, the DNA origami is attracting attentions. DNA-origami is the programmable nanostructure which is synthesized by weaving a very long single strand DNA with a large number of short single strand DNAs, just like the weft of the textile.

Problems and Motivations

Only a very few examples of a DNA-origami system with a rotational motion or even with a movable part have been reported so far. (Figure 3)It is also a problem that the creation of the macroscopic-scale device only with DNA origami, which works with the macroscopic output or input signal such as a mechanical one, is difficult due to high cost. (Figure 4)

Our Approach and Goals

As the start point to solve the problems, we here design the simplest nano-device that rotates: the weathercock. We fabricate the rotating weathercock to detect water flow by combining inorganic nanoporous substrate with the nanoscale weathercock made by DNA origami. (Figure 5)
We synthesize the DNA-origami-based “DNA weathercock” consist of the blade part which sense the water flow (instead of the wind in air) and the shaft part. (Figure 6) The shaft is mounted into the vertical nanopores of "anodizing alumina" which is synthesized by electrode reaction of an aluminiuma metal plate. (Figure 7) The size of the nanopore is tuned by changing the synthetic condition of the nanoporous alumina in the range of 20 nm - microns. We can further optimize the interaction between the DNA shaft and alumina surface by chemical modification of the alumina surface.
When the DNA weathercock catches the flow in the fluid, it turns to the direction of the flow. Since the head of the weathercock is attached with a fluorescence molecule, the flow direction is detected by the observation by confocal laser microsope or even by our naked-eyes through a polarizing film. (Figure 8)
The criteria for the success of this project is as follows:
- The synthesis and characterization of the nanoporous substrate
- The design, synthesis and characterization of DNA weathercock
- Mounting the DNA weathercock to the porous substrate
- Observation of the rotation of the weathercock induced by flow

Method

Materials

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.

Mounting the DNA weathercock to the nanopore

We then mount the DNA weathercock into the pore of the porous substrate by the following two methods (Figure 9. We drop the solution containing the DNA weathercock on to the substrate and evaporate the solvent. By tuning the components of the buffer solution, we expect the shaft of the DNA weathercock is incorporated into the pores. For a future plan, we will sputter the a material A that have affinity with DNA, followed by sputtering of the material B that have no affinity with DNA only onto the substrate surface (not in the hole) by tilting the substrate. 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 (Figure10). We then slide the cover glass to give a flow to the DNA weathercock along a certain direction(Figure11).

We detect the motion of the weathercock in two ways (Figure 12). 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.

Results and Discussion

The firstly synthesized anodizing alumina nanoporous substrate was observed by AFM and SEM, as shown in Fig. 12 and 13, respectively. In the AFM image, pores with the inner diameter of 100 nm are aligned in a honeycomb structure. The height image shows that the pore has a conical shape, but not the straight pore which may better fit to the shaft of the weathercock. However, we actually observe the straight pores in the SEM image as expected. The cross-sectional SEM image shows that the holes are 400 nm in depth. Thus the conical shape observed in AFM is an artifact. However, the problem is that the diameter of the pore is still too large to fit the DNA shaft. Because anodizing alumina with the pores of down to 20 nm can be generally fabricated, we changed the synthetic condition and the synthesis is now on the way to go.

We then designed the DNA weathercock by using a three-dimensional DNA-origami technique. We show the first design of the DNA weathercock in Figure 14. The concept of the first design is (1) arrow shaped blade which is optimal for sensing the flow and (2) the rigid shaft to firmly held in the pore. We prepared staple DNAs as shown in Table 15.

We observed the synthesized DNA weathercock by AFM as shown in Figure 16. The cross sectional image is shown in Figure 17. We compare the observed shape with the designed one. The objects of the shapes similar to the designed DNA-origami were observed. The Figure 18 is a cross-sectional view of for the green line in the Figure 19. Thus the size of the observed objects (30 nm x 17 nm x 21 nm) is also similar to the size of the designed one (28 nm x 17 nm x 20 nm), indicating the successful formation. However,the size of the weathercock may be too small for the optical confocal laser microscope to detect the rotational motion of the fluorescence head. Because the number of the staple is limited, we can not design a large enough weathercock if we adopt the 3-dimensinal DNA-origami technique. Also, we don't like this design because this is very different from the image of the weathercock !

So we redesigned the beautiful weathercock made of a simple 2-dimensional DNA-origami as shown Figure 20. However, unfortunately, this was still too small. So, we finally designed the DNA weathercock with the elongated body part of 56 nm (Figure 21), so that the movement could be detected by the microscope. This finally designed DNA weathercock consists of the M13 scaffold DNA bound by the 97 short staple DNAs as shown in Table 2. We are now synthesizing this weathercock by annealing the mixture of all the materials.