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 called "Porter".
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.
PORTER has a loop structure when it hybridizes with the target. So when 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 higher affinity to the target. 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.
Coarse grained simulation in which one nucleotide is assumed as one bead indicates that long Porter can bind to the target, but toehold structure of the same affinity cannot catch the target.
Porter can binds to the target
Toehold structure cannot bind to the target
<A Href=" http://openwetware.org/wiki/Biomod/2012/TeamSendai/Simulation " Title="Simulation"> See detail in simulation page </A>
How to implement
To insert the Gate in cell membranes is essential for the CELL GATE. We used artificial lipid membrane, liposomes, as model cell membranes, to test implementation of our CELL GATE into membrane. As a preliminary step to insertion of the GATE into the liposome, we designed a smaller Gate named Mini-gate. We attempted to insert the Gate and Mini-gate into liposomes and we confirmed they inserted into liposomes by fluorescence microscopy or by SPR analysis.
To implement the Gate in membranes, we attached single-stranded DNA of 10 bases at the middle point of the GATE outside surface. A hydrophobic molecule, Cholesterol, was conjugated into the complementary DNA of the attached DNA. We expected that the GATE with cholesterol legs can be implemented into the hydrophobic portion of the liposome.
There is a possibility that the GATE with cholesterol legs lie on the membrane surface, and is not inserted. Thus, installing a module for insertion was required. For the aim, we designed that the Mini-gate remains a large amount of single stranded region of M13. We expected that this single stranded region of M13 breaks electrostatic symmetry of the Mini-Gate, and enables to stand vertically to penetrate the membrane by repulsion.