Biomod/2013/Komaba/Design
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Overview
Figure D1
Our project goal is to design the DNA screw in the Figure D1. It consists of three parts; a cylinder, a ring, and two DNA spiders. A ring and a cylinder are made of one scaffold and staples because if they are made of two different scaffolds their electrostatic interaction rejects each other. DNA spider is made of DNA strands, streptavidin, and biotin. We designed them based on some papers in the reference part[1][2][3].
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 to each other. The detail is explained following.
How to construct the DNA screw
Design of Cylinder
The cylinder is put in the center of the DNA screw as an axis supporting the rotation. A rectangular consisting of staples and a scafold is formed into a cylindrical shape using DNA Origami technology. It was designed with cadnano*4. The diameter of the cylinder is 30.5 nm and the axial length 43.5 nm. To identify the start and the end of probes, one side of the cylinder projects out and we defined it as the end side as it shown in FigureD2. Not modified side is the start 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 probes direct to the center of the cylinder from 5' end to 3' end.
Design of Ring
The ring is also composed with DNA Origami technology*4. We made the structure using cadnano*5. A scaffold winds in a spiral shape as showniin Figure D3. The diameter of the ring is 62 nm and the thickness of ring is 12 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
Our DNA screw rotates by using DNA spiders*5. Our DNA spider consists of a body, a CL-H strand, and three walking legs(FigureD4). The CL-H strand has two parts; capture leg(CL) part and head strand(H strand) part. A biotin is attached in the middle of the CL-H strand and attached to the DNA spider's body. Here is the part that we modified from the one in the original paper. 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 body is tetramer streptavidin and each monomer is strongly connected to a biotin. The sequence of other parts are listed in the reference*6.
How The Ring Rotates
The function of DNA spider
DNA spider with one CL-H strand, which means capture leg and head stand, and three walking legs, is the core of the rotary function. A DNA spider's walking leg consists of 8-17DNAzyme and the spider advances by cutting common footings by the walking leg and utilizing Brownian motion. The process is described in the FigureD5. The sequence of the common footing is written in the reference*7. It is designed as it hybridizes with the sequence of the walking leg. First the tip of the common footing is cut away by walking leg. Second the partially cut 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 cutting process occurs again and again, and the spider advances down the track.
How the spider advances on the cylinder's surface
The cylinder is rounded from the rectangle shape. We operated two DNA spiders on the surface of cylinder so there are two tracks, each of which consists of three lines of the common footings. We designed the distance between the common footings, taking 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 are wide enough for spiders not to jump 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 probe is jut out at left end side(Figure D6). This start probe 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 probes are attached at the right end side on each track. In addition, the end probes partially hybridizes with end footings. The sequence of the end footing is slightly different from common footing in that the the end footing uses rA instead of A. These sequences are listed in the reference*8.
Cutting the scaffold
To make the DNA screw rotate, the ring and the cylinder, both of which are made of the same one scaffold, have to be separated. Here we carefully selected two appropriate enzyme considering the temperature, content of buffer, and other factors; BbvCI and SbfI.
Overall process
The DNA screw is realized by assembling the above three parts: the cylinder, DNA Spiders, and the ring (Figure D7). The DNA screw is assembled in the following process.
Step 1. The cylinder and the ring are synthesized.
Step 2. The DNA spider is synthesized
Step 3. The cylinder-ring structure, the DNA spider, and the start footing are mixed
Step 4. The common footings and the end footings are mixed with the solution made in the Step 3 and hybridized to the common probes and the end probes respectively.
Step 5. Put the two enzyme to cut the scaffold.
Step 6. Put a trigger strand which is complementary to the start footing, which begins the spider to walk
