Biomod/2013/Komaba/Discussion

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Cylinder

By the figure of AFM, it was figured out that a DNA structure with 60nm long × 90nm wide × 4nm thick was obtained. If this structure had been a flat rectangular, not a cylinder, it would have been 120nm long × 43.5nm wide × 2nm thick. Since the thickness of 4nm is twice of single DNA rectangular and there was no single layered structure near them, it is implied that this structure is a flattened cylinder squashed by cantilever of AFM. The original paper of the cylinder[1] also reported that the thickness of cylinders was around 4nm because they were flattened by pressure of cantilever. However, even if a cylinder gets flattened,the size will be about 60nm long × 43.5nm wide × 4nm thick, volume of which is about half of the observed structure. Therefore, it is considered that the dimers have formed dimers. These dimers are considered to be formed by pi stacking of DNA strands at the end of cylinders. Some cylinders were seen as forming into trimers. The number of trimers was smaller than that of dimers. This is becuase the sub-cylinder formed on one side of the cylinder disturbed the cylinders' connections. To see if they were actually dimers of cylinders, Observation with TEM is required but it is too expensive for us to afford.

On the other hand, the sub-cylinder structure could not be observed. The design was diameter 16.2nm × axial length 10.9nm. As it is considered that the sub-cylinder was squashed by cantilever, it would be 25nm long × 10.9nm wide × 4.0nm thick, but there was no structure with the size.

The possible reason why the dimer and trimer were formed as well as why the sub-cylinder was not observed is that it would be more stable to stack cylinders without forming sub-cylinders. In addition, the cylinder was annealed for four hours this time while the annealing time in the original paper[1] was 10 minutes. This difference might have affected the formation of cylinders. Image:dimerofcylinder.jpg

Ring(1st ver.)

In the design of Ring(1st ver.), some design mistakes were found. It is considered that the mistakes caused the ring to fail in formation. In detail, crossovers of scaffold and staples occurred in the same place, which would have made the ring move like a hing. We will retry this structure after the design mistake is fixed.

Ring(2nd ver.)

The Ring(2nd ver.) was designed with twelve strands, ten of which have exactly the same sequences as in the original paper while two of which are one mer longer than the counterparts in it. The result was that the ring did not form even by 2 hours and 72 hours annealing. The reason would be that the change in design may have affected the construction of the ring. In addition, PAGE refining was not conducted while the authors of the original paper of the ring[2] did. Both two reasons might have caused the ring not to be formed.


Cylinder-Ring structure

The cylinder in the original paper[1] was diameter 22nm × axial length 100nm and it was annealed for ten minutes from 60°C to 35°C. Also, the ring in the original paper[3] was diameter 7.9~9.0nm × axial length 113nm and it was annealed in the total length of 685 minutes in the following flow; The annealing temperature ramps are as follows: 94 to 86 °C at 4°C per 5 minutes; 85 to 70°C at 1 °C per 5 minutes; 70 to 40 °C at 1°C per 15 minutes; 40 to 25 °C at 1 °C per 10 minutes.

On the other hand, the cylinder in the cylinder-ring structure was designed as diameter 31nm × axial length 44nm. Also the ring in the cylinder-ring structure was designed as diameter 62nm × axial length 12nm. Two samples of the cylinder-ring structure were annealed for two hours and eleven hours respectively. In the sample annealed for two hours, some cylinders were formed and the size was close to the design, but there were not any ring-shaped structure. In the sample annealed for eleven hours, the yield of the cylinders decreased but a lot of ring-shaped structures were observed. The rings were not clear circular shapes and took a squashed shape. This reason is considered to be some pressure of cantilever of AFM as well as the Electrosorption of mica.

The change in the cylinder-ring structure by annealing time showed that short annealing time(around 10 minutes) forms cylinders well while rings require long annealing time(around 10 hours) to be formed. To see if this insight is correct, we are going to anneal it for six hours. If our hypothesis is correct, fewer rings and more cylinders will be found than in the case of 10-hour annealing, and more rings and fewer cylinders will be observed than in the case of ten-minute annealing. If so, the designing methods of the cylinder and the ring may not be compatible in annealing time. It would be better to construct both of the cylinder and the ring with the ring's designing approach of winding helix shape not only because appropriate annealing time of the two is expected to be similar but also because the designing approach allows us to design a cylindrical shape with a wide range of diameter.

Cylinder(first version) with footings and DNA spiders

Even though we used the same staple mix and protocol of synthesis as those on September 19th, both two samples failed to form into cylinder-shape. We have been considering every possible reason to find the cause of this result. The most possible reason is the staple mix is not good so after making a new staple mix, we will try this synthesis and AFM observation again. The uneven surface was considered to be caused by a lot of streptavidins.


How to Detect that the ring actually rotates

It is possible that the DNA spiders jump across tracks on the way and reach end footings. In the original paper of the DNA spider[4], in order to detect whether or not they actually advanced without crossing tracks, the fluorescence materials were attached to the surface of the walking ground and the walking legs of the DNA spider. In our design of the DNA screw, we did not put any fluorescence materials to it due to their high cost. Alternative method of detecting the spider's walk is to utilize Surface Plasmon Resonance(SPR). First, a solution of DNA screw is put on a substrate. When a footing is cleaved, the weight of a DNA screw decreases. The SPR measures how many times the weight changed. If the times of weight change are more than the number of DNA spiders, it is proved that the spiders actually advanced without crossing tracks.

Future Design

A new design in which the spider's body, streptavidin, is removed and the spider's walking legs are directly connected to the ring is planned to develop. With this design, the DNA screw could be more compact and less complex. It will be thoroughly examined which cylinder and ring designed are appropriate, whether the annealing temperatures of the ring and the cylinder are compatible, and whether the legs actually advance by cleaving the footings with this design.

Reference

  1. cylinder Yanming Fu.et al., Single-Step Rapid Assembly of DNA Origami Nanostructures for Addressable Nanoscale Bioreactors, American Chemical Society, 2012
  2. ring2 Yang Yang et al., Self-Assembly of DNA Rings from Scaffold-Free DNA Tiles, Nano Letters 2013
  3. ring3 Dongran Han et al., Unidirectional Scaffold-Strand Arrangement in DNA Origami
  4. spider Kyle Lund et al., Molecular robots guided by prescriptive landscapes, Nature Vol465, 2010
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