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=== Design of Cylinder===
=== Design of Cylinder===
We read articles cylinder's designing methods and tried in cadnano. We adopted the following method; a which is made of a scaffold and staples formed into a cylinder shape. This method has advantages. This cylinder's design is rigid as well as flexible in designing. In addition, the yield is 88% and quite high. However, we cannot designate which surface becomes the front surface. Probes come up from the back surface in this method and we separate it from the cylinder in which probes grow up from the front surface. The possibility would be 50:50.
The cylinder is put in the center of the DNA screw as an axis supporting the rotation. A rectangular DNA Origami (140 staples and a M13mp18 scaffold) is
The cylinder is put in the center of the DNA screw as an axis supporting the rotation. A rectangular DNA Origami (140 staples and a M13mp18 scaffold) is into a cylindrical shape from a design using Cadnano software*4. The two sides of the rectangular origami are connected by staples. 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 are designed for the 5' ends of the staples to be the probes' tips.
Revision as of 09:43, 26 October 2013
Our goal is to design the DNA screw in the Figure D1. It consists of three parts; a cylinder, a ring, and DNA spiders. Both ring and cylinder are made of DNA origami. We designed a ring and a cylinder in one scaffold to avoid the electrostatic interaction between them, which will cause them not to connect to each other. In that case, the cylinder and the ring stay keeping some distance and will have more possibility to connect to each other. However, with this condition, four difficulties exist. First, making the ring and cylinder within 7250 mer. Second, finding an enzyme to cut the ring and cylinder. Third, finding cylinder and ring with a compatible size in diameter. Fourth, proper designing which allows putting probes in an appropriate interval. We carefully adopted designs of a cylinder and a ring solving the these problems. The structure of the cylinder and the ring are designed based on two papers in the Supplementation*1*2.
With the scaffold, the cylinder has tracks on its surface which made of unpaired staples. The ring also has unpaired staples to connect to the spiders. The spiders consist of a tetrameric streptavidin core bound to 4 biotinylated ssDNA: 3 walking legs and one CL-H strand. The DNA spider was designed based on the paper in the Supplementation*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.
How to construct the DNA screw
Design of Cylinder
We read articles describing cylinder's designing methods and tried those methods in cadnano. We adopted the following method; 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 88% and quite high. However, we cannot designate which surface becomes the front surface. Probes come up from the back surface in this method and we cannot separate it from the cylinder in which probes grow up from the front surface. The possibility would be 50:50.
The cylinder is put in the center of the DNA screw as an axis supporting the rotation. A rectangular DNA Origami (140 staples and a M13mp18 scaffold) is bent into a cylindrical shape from a design using Cadnano software*4. The two sides of the rectangular origami are connected by staples. 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 is 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 are designed for the 5' ends of the staples to be the probes' tips.
Design of Ring
A Winding helical pattern was adopted as a ring's part of cylinder-ring structure. The reason of adopting it is that it is small enough for the cylinder and the ring to be designed within 7250 mer of M13mp18. Moreover, because there is no crossover, it is easy to grow probes. This designing method is not clearly written in the original paper*2 so it required some efforts to understand and duplicate.
The ring is also composed of DNA Origami*2 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 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
The DNA screw rotates by using DNA spiders. Our DNA spider consists of a streptavidin tetramer body, and biotinylated ssDNA as legs. We designed two kinds of legs : the first kind is walking leg, and then the second is a CL-H strand (FigureD4)*3. 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. . The sequences of other parts are listed in the reference*5. Here is the part that we modified from the one in the original paper*3. 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.
(We will put an animation which not only explains the relationship between a probe, a footing, and a DNA spider's leg, but also describes how the leg advances.)
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 and the spider advances by cutting common footings by the walking leg and utilizing Brownian motion. The process is described in the FigureD5.. 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, 10.5nm, 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, 16.5nm, 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*5.
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.
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
(We will put an animation here which describes how the DNA screw is constructed.)
Other Designed Structures
Before we designed the cylinder-ring structure, we made only a cylinder and confirmed that a cylinder designed based on the paper*1 was actually formed. The size is the diameter 38.2nm × the axial length 43.5nm. The interval of anchors of this cylinder was too wide for the DNA spiders to walk.
The other two rings, Ring A and Ring B, were designed than the ring shown in the cylinder-ring structure.
Ring A is a ring formed from a long tape using the same method as the cylinder*1.
The designing approach of Ring B 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*4. The sequences of the twelve staples are written in Reference*5. The sequences of all the staples but one are the same as those in the original paper and the one is one mer long than the counterpart in the paper. You can see the sequences from Ring B sequences.These two rings were tried only themselves without being designed with a cylinder in one scaffold.
FigureD9 Figure D10