(→The start point and the end point)
(→Cutting the scaffold)
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=== Cutting the scaffold ===
=== Cutting the scaffold ===
To make the DNA screw rotate, the ring and the cylinder, both of which are made of the same one scaffold,
To make the DNA screw rotate, the ring and the cylinder, both of which are made of the same one scaffold, to be separated. Here two appropriate were carefully selected considering the temperature, content of buffer, and other factors; BbvCI and SbfI. Both enzymes incubated at 37℃.
== Overall process ==
== Overall process ==
Revision as of 21:38, 26 October 2013
Our goal is to design the DNA screw in Figure D1. It consists of three parts; a cylinder, a ring, and DNA spiders. Both cylinder and ring are made of DNA origami. A cylinder and a ring were made in one scaffold to avoid the electrostatic interaction which would cause them to keep away from each other. With our design, 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 a cylinder and a ring with a compatible size in diameter. Fourth, a proper design which allows putting anchors in an appropriate interval. With careful selection, designs of a cylinder and a ring solving these problems were adopted. The structure of the cylinder and the ring were designed based on two papers.
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. 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
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.
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 the cylindrical shape. 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. The design was constructed using cadnano. To identify the start and the end of anchors, one side of the cylinder projects out and it was defined as the end side as shown in Figure D2. Not modified side is the start side. In order to bind footing DNAs on its surface spirally, 10mer long DNA strands, anchors, are jutting out from the cylinder's surface. The anchors are designed for the 5' ends of the staples to be the anchors' 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 anchors. This designing method is not clearly written in the original paper*2 so it required particular conditions to duplicate.
The ring is also composed of DNA Origami and designed using cadnano. 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. 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. 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. How to refine the DNA spiders is written in the Experiment part.
(We will put an animation which not only explains the relationship between an anchor, 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. 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
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 jut 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 rA instead of A. 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℃.
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 anchors and the end anchors 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 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.
Ring(1st ver.)&(2nd ver.)
The other two rings, Ring(1st ver.) and Ring(2nd ver.), were designed than the ring shown in the cylinder-ring structure.
Ring(1st ver.) is a ring formed from a long tape using the same method as the cylinder.
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. The sequences of all the staples but one are the same as those in the original paper. The one is one mer long than the counterpart in the paper. You can see the sequences from Ring(2nd ver.) sequences. These two rings were tried only themselves without being designed with a cylinder in one scaffold.
- Yanming Fu.et al., Single-Step Rapid Assembly of DNA Origami Nanostructures for Addressable Nanoscale Bioreactors, American Chemical Society, 2012
- Dongran Han et al., Unidirectional Scaffold-Strand Arrangement in DNA Origami
- Kyle Lund et al., Molecular robots guided by prescriptive landscapes, Nature Vol465, 2010
- Yang Yang et al., Self-Assembly of DNA Rings from Scaffold-Free DNA Tiles, Nano Letters 2013