Design of Gate
Size / Structure
The Gate is the structure that connects inside and outside of the cell. We decided to apply a hexagonal cylinder nanostructure made of DNA origami for the Gate, We refer "A logic-gated nanorobot for targeted transport of molecular payloads" (SM Douglas, I Bachelet, GM Church - Science Signalling, 2012) for the hexagonal cylinder structure of DNA origami.
Next, we made a simulation in order to examine the size of the structure. The size of the cylinder must be small enough not to pass freely through anything. However, it must be large enough to pass through the desired product. The gate made of DNA origami has negative electric charge. So if the gate is too small, target DNA is not able to go through the Gate. Based on our simulation, the size of gate is determined as 24*24*33nm.
We used caDNAno to design the Gate.
Our Gate is made of DNA, so it has negative electric charge. Single stranded DNA has negative electric charge, too. Here is a graph at potential energy around the Gate. If the potential energy is high, it is difficult for single stranded DNAs to enter the Gate. If the radius of the Gate is 1.5 times larger than now design, potential energy decreases and to enter the Gate is easier.
Design of Porter
In the concept of Cell Gate, there are two problems. for making CELL-GATE.
- How to pull the target DNA into GATE ?
- How to pass the target through GATE ?
To solve these problems, we propose a nano-system made of ssDNA and named it Porter. Porter stands in line in GATE, pulls the target DNA, and transports it.
This idea is supported by GATE simulation, which shows that target DNA can not enter GATE by itself. So, the work of PORTER is to pull and bring the target DNA inside GATE.
We designed PORTER having some loop structures when it hybridizes with the target. Porter has some complementary sequences to the target here and there. So after the target attaches to Porter, Porter shrinks, or in other words it pulls the target DNA. Finally the target enter GATE.
The inner Porter has more complementary sequences to the target and has higher bonding energy than from the one at the entrance of Gate. This design enables the target to move to the inner Porter because of the combination stability. In experiment, we apply the sequences below.
These are the sequences for electrophoresis, and correspond to only a part of the actual Porter sequence. These are sequences that are complementary to the target. When planted in the gate, Porter has spacer sequences of 10 nucleotides length at its foot in addition to the sequences above; this is to reduce the Coulomb force produced by the wall of GATE.
We compared the ability to catch the target of Porter with that of toehold structure.
How to implement
We also have the aim of this GATE insert in the cell membrane, it can not sting in the cell membrane of normal hexagonal cylinder. However, We can create a cylinder with a different structure by exchanging some staple. We have designed a simple cylinder first,and I have to be attached anywhere in the structure to replace the staple. We can later add functionality to an existing structure using this method. We thought that to have an affinity for lipid membrane with the DNA that can be modified cholesterol on the side of the cylinder by this method. In addition, we have also designed DNA-like beard at the entrance of the cylinder. We expect the effect of electrostatically repel force with DNA which are not intended. Our cylinder is small,so we designed the cylinder to connect to each other and be long in order to easily confirmed using AFM. By replacing the staple, this structure is also removably.