What structure is most suitable for the Gate? The Gate has to connect inside and outside of the cell. So we decided to apply a hexagonal tube nanostructure made of DNA origami. We refer "A logic-gated nanorobot for targeted transport of molecular payloads" (SM Douglas, I Bachelet, GM Church - Science Signalling, 2012) for the hexagonal tube structure of DNA origami.
Next, we made a simulation in order to examine the size of the structure. The size of the tube must be small enough not to pass freely through anything. However, it must be large enough to pass through the desired product. The gate which made of DNA origami has negative electric charge. So if the gate is too small, target can't enter the Gate. According to simulation, our Gate size determined 24*24*33nm. This size is suitable to transport the target.
We used caDNAno to design the hexagonal tube structure. This Gate tube is made from 6792bp M13mp18 and a lot of single stranded DNAs. And the Gate has double hexagonal structure because I think that is stronger than single hexagonal structure.
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 tube. GATE size means the length of 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.
You can see details in simulation page
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 ssDNAs called "Porter". Porter stands in line inside the GATE, selectively "pull" the target DNA.
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. So when the target attaches to Porter, Porter shrinks, or in other words it pulls the target DNA into the Gate. As a result the target enter GATE.
The inner Porter has longer complementary sequences to the target and thus higher bonding energy than from the one at the entrance of the Gate(Porter1). This design enables the target to move to the inner Porter(Porter2 and Porter3). In experiment, we designed and used the sequences below.
Blue:Target Red:This part is complementary with target Green:Spacer
※Porter3 use only electrophoresis.
We should note these described above are the sequences for electrophoresis experiments. Additional sequences to attach the GATE are included in the designed Porter sequences of the 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.
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.