Biomod/2013/Komaba/Design

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Overall

A ring and cylinder is made of one scaffold and staples to make the distance between them close. DNA spiders are made of DNA strands, streptavidin, and biotin. We designed them based on the following papers. The cylinder: "Single-Step Rapid Assembly of DNA Origami Nanostructures for Addressable Nanoscale Bioreactors" by Yanming Fu et al. The ring: The spider: "Molecular robots guided by prescriptive landscapes" by Kyl Lund et al.

From the surface of the cylinder, 10mer long DNA strands, called probes, are jutting out and the probes bind footing DNAs. Two DNA spiders which have legs made of DNAzyme advance by cutting the footings. Those spiders are set in the opposite positions on the surface of the cylinder. The ring holds the cylinder and spiders inside of the ring sharing the same axis with the cylinder. Some strands coming out from the ring are hybridized with strands from the DNA spiders, which lets them connected each other. The detail is explained following.

Phase 1

The cylinder is put in the center of the DNA screw as an axis supporting the rotation. DNA strands of staples and a scafold are formed into a cylindrical shape using DNA Origami technology. It was designed with cadnano software, which "simplifies and enhances the process of designing three-dimensional DNA origami nanostructures". The diameter of the cylinder is 30.5 nm and the axial length 43.5 nm. To identify the left and the right, one side of cylinder is modified and we defined it as the right side. Not modified side is the left side. In order to bind footing DNAs on its surface spirally, 10mer long DNA strands, probes, are jutting out from the cylinder's surface.

The ring is also composed with DNA Origami technology (figure). We made the structure using cadnano. Two 10mer long strands come up from the inner side of the ring and are connected to the DNA spiders. The diameter of the ring is 62 nm and the thickness of ring is 12 nm in consideration of Atomic Force Microscope visibility (Figure).

After the cylinder with the probes is formed, anchor of DNA spiders is hybridized to a specific strand at the start point on the cylinder's surface. Then the footings get connected to the probes. The footing tracks, each of which consists of three lanes of the footings, enable DNA Spiders to orbit the cylinder. In detail, there are two tracks of DNA footings and two spiders walk on each track. In addition, in order to make it easy to observe the direction of spiders' walk, a small sub-cylinder is attached at the end of the footing tracks(Figure 2). The diameter of the cylinder is 25.4 nm and height is 43.5 nm. This is calculated considering that the cylinder can be observed with an Atomic Force Microscope and that the interval between the two tracks are wide enough for spiders not to jump to next footing track (Figure 3).

Phase 2

Our DNA screw rotates by using DNA spiders, which is being created based on the work of Lund, et al. Our DNA spider consists of a body, a double leg, and three walking legs. The double leg has two parts in one strand; head part and capture leg part. Here is the part that we modified from the original one in the original paper. The body is tetramer streptavidin and the sequence of other parts are listed below. Double Leg: 5′ - AGG CGC ACT T /iSp18//iSp18//3Bio//iSp18//iSp18/ TGA ACG CAG TCC AAG AGC CG - 3′ (The head part is AGG CGC ACT T /iSp18//iSp18/ and the capture leg part is /iSp18//iSp18/ TGA ACG CAG TCC AAG AGC CG) Walking Leg: 5′ - /5BioTEG//iSp18//iSp18/ TCT CTT CTC CGA GCC GGT CGA AAT AGT GAA AA - 3′

DNA spider's walking leg consists of DNAzyme and the spider advances by cutting common footings by the walking leg and utilizing Brownian motion, which is described in the figure. The sequence of the common footing is 5′- GGGTGAGAGG TTTTTCACTATrAGGAAGAG -3' and designed as it hybridized with the sequence of the walking leg. First Part A in a common footing is cut by walking leg. Second the partially cut common footing and the walking leg move by Brownian motion and one time walking leg hybridizes with Part A in next common footing. Finally, the walking leg is detached from the last common footing and the next common footing and the walking leg hybridize completely.

The cylinder is rounded from the rectangle shape in the figure. We operate two DNA spiders on the surface of cylinder so there are two tracks, each of which has three lines of the common footings. We designed the distance between the common footings taking into account the length of the common leg. The walking leg must be long enough for the DNA spider to move to next common footing but must be short as it does not cross the two tracks.

DNA spider is connected to the DNA ring by a “head” strand, and also DNA spider's legs are connected to strands on the surface of the DNA cylinder. DNA spider consists of a body, a head, an anchor, and three legs, all of which are going to be made by DNA. These legs advance by cutting strands with DNAzyme. DNAzyme is a DNA which cuts DNA strands like enzymes do. Because the spider's track is a spiral, DNA rings rotates as DNA spiders walk. The head is complementary to the strand stretching from a ring made of DNA.

In detail, legs and the footings are complementary strands. The footings are attached spirally to the surface of the DNA cylinder and function as a substrate of the DNA spider. DNA spiders normally move one way. Once DNAzyme cuts the tip of the footings, DNA spiders are disconnected to the footings. The footing is binding to the nearest track strand and the cutting process is occurring again.

We will use the same DNA spider’s sequences as the way Lund, et al. did; 5’ - TCT CTT CTC CGA GCC GGT CGA AAT AGT GAA AA – 3’. We are developing the body sequence of the DNA spider. When we decide them, we will order them as soon as possible.

DNA Screw (Combination of above parts)

The DNA screw is realized by assembling the above four parts: the cylinder, footings, DNA Spiders, and the rotary ring (Figure 6).