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Figure D1
Figure D1


With the scaffold, the cylinder has tracks made of unpaired staples on its surface. The ring also has unpaired staples to connect to the spiders. The spiders consist of a tetrameric streptavidin bound to 4 biotinylated ssDNA: 3 walking legs and one CL-H strand. The DNA spider was designed based on this paper[3]. From the surface of the cylinder, 10mer long DNA strands, called anchors, are jutting out and are hybridized with footing DNAs. Two DNA spiders, which have legs made of DNAzyme, advance by cleaving the footings. Those spiders are set in the opposite positions on the surface of the cylinder.

How to construct the DNA screw

Design of Cylinder

Figure　D2
Figure　D2

Some articles describing cylinder's designing methods were carefully read and the methods were tried with cadnano. Finally the following method was adopted; a rectangle, which is made of a scaffold and staples, are formed into a cylinder shape. This method has two advantages. This cylinder's design is rigid as well as flexible in designing. In addition, the yield is quite high (i.e.88%). However, it is not possible to designate which surface becomes the front surface. Anchors can come up from the back surface in this method. Also There is no separating it from the cylinder in which anchors grow up from the front surface.


Design of Ring

Figure D3
Figure D3


The ring is also composed of DNA Origami and designed using cadnano[4]. A scaffold winds in a spiral shape as shown in Figure D3. There are four loops in the ring and neighboring loops are connected by staples as these loops are as close as possible. The diameter of the ring is 61.8 nm and the thickness of ring is 12.0 nm in consideration of Atomic Force Microscope visibility. Two 10mer long strands come up from the inner side of the ring and are connected to the DNA spiders.

Design of DNA spider

Figure D4
Figure D4

The DNA screw rotates by using DNA spiders. Our DNA spider consists of a streptavidin tetramer body and biotinylated ssDNA as legs. There are two kinds of legs : the first is walking leg and the second is a CL-H strand (Figure D4). The CL-H strand has two parts; capture leg(CL) part and head strand(H strand) part. A biotin is fixed in the middle of the CL-H strand and attached to the DNA spider's body. This is the part that was modified from the one in the original paper[3]. The sequences of those strands are listed here. The capture leg has a function of connecting the DNA spider to the specific strand at the start point on the cylinder. Head strand connects the DNA spider to the Ring. Walking legs make the DNA spider move forward and this function is described in Figure D5. The cohesion of the spider relies on the strong streptavidin/biotin interaction.　The DNA spider with one CL-H strand and there walking legs are planned to be purified with electrophoresis。 v

<img src="DNAwalking.gif" width=120>

Motion of The DNA Screw

The function of DNA spider

DNA spider with one CL-H strand, and three walking legs, is the core of the rotary function. A DNA spider's walking leg consists of 8-17DNAzyme. The spider advances by cleaving common footings by the walking legs and utilizing Brownian motion. The sequence of the common footings is designed as they hybridizes with walking legs. The process is described in the Figure D5. First the tip of the common footing is cleaved away by a walking leg. Second the partially cleaved common footing and the walking leg move by Brownian motion and one time walking leg hybridizes with a tip of next common footing. Finally, the walking leg dissociates from the last common footing followed by binding to the nearest common footing. This cleaving process occurs again and again, and the spider advances down the track.

How the spider advances on the cylinder's surface

Figure D6
Figure D6

The cylinder is rounded from the rectangle shape.Two DNA spiders are operated on the surface of cylinder so there are two tracks, each of which consists of three lines of the common footings. The distance between the common footings was designed as 10.5nm taking it into account that the interval between the common footings on the same track is short enough for the walking leg to move to next common footing. In addition, the interval between the two tracks, 16.2nm, are wide enough to avoid spiders from jumping to next footing track(Figure D6).

The start point and the end point

In order to make the DNA spider start to advance from the same starting point, the start anchor is jutted out at left end side(Figure D6). This start anchor partially hybridizes with specific strand at the starting point, called a start footing, and then the start footing hybridizes with capture leg in the DNA spider. Also to stop the spider's walk, three end anchors are attached at the end side on each track. The end anchors partially hybridizes with end footings. The sequence of the end footing is slightly different from common footing in that the the end footing uses A instead of rA. These sequences are listed here.

Cutting the scaffold

To make the DNA screw rotate, the ring and the cylinder, both of which are made of the same one scaffold, had to be separated. Here two appropriate enzymes were carefully selected considering the temperature, content of buffer, and other factors; BbvCI and SbfI. Both enzymes were incubated at 37℃.

Overall process

The DNA screw was realized by assembling the above three parts: the cylinder, DNA Spiders, and the ring (Figure D7). The DNA screw was assembled in the following process.

Step 1. The cylinder and the ring were synthesized.
Step 2. The DNA spider was synthesized
Step 3. The cylinder-ring structure, the DNA spider, and the start footing were mixed
Step 4. The common footings and the end footings were mixed with the solution made in the Step 3 and hybridized to the common anchors and the end anchors respectively.
Step 5. The two enzymes were put to cut the scaffold.
Step 6. A trigger strand was put. This is complementary to starting strand, which initiate the spider walking.

Other Designed Structures

Cylinder(1st ver.)

Figure D8
Figure D8


Ring(1st ver.)&(2nd ver.)

Rings with two other designs, Ring(1st ver.) and Ring(2nd ver.), were designed besides the ring shown in the cylinder-ring structure.


The designing approach of Ring(2nd ver.) is that six-helix DNA bundle units, assembled from twelve single stranded DNAs arranged in networks of　contiguous and semicrossover strands, are connected into nano rings without scaffold. It was designed based on this paper[5]. The sequences of all the staples but one are the same as those in the original paper. The one is one mer longer than the counterpart in the paper. You can see the sequences from Ring(2nd ver.) sequences. These two rings were tried without being designed with a cylinder in one scaffold.










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