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<h1>Cell-gate</h1>
[[Image: Designtopcellgate.png |left|340px]]
Our project "Cell-gate" is left.
Our project is divided major three parts, Gate, Porter, and Membrane.
Gate is Cell-gate itself.
　Porter is function to transport the target inside and outside cell membrane.
　Membrane is liposome which is model of cell membrane.
We divided our project into above three group and did experiment.
On this page, we descript how we determined our robot design.
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<h1>Design</h1>
<h1>Design</h1>
<h1>Design of Gate</h1>
<h2>Gate</h2>
<h2>Size / Structure</h2>
<h3>Size / Structure</h3>
The Gate has to connect inside and outside of the cell. So we decided to apply a hexagonal tube nanostructure made of DNA origami.
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.<br>
 
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.<br>
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.
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<h2>DNA origami</h2>
<h3>DNA origami</h3>
We used caDNAno to design the hexagonal tube structure.  
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.


[[Image: スライド15.jpg |340px]]
[[Image: スライド15.jpg |340px]]
[[Image: スライド16.jpg |340px]]
[[Image: スライド16.jpg |340px]]
[[Image: Cadnanogazou.png|680px]]
<youtube width="450" align="left">XMiheA1sWOA</youtube>
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<h3>Potential Barrier</h3>
[[Image: Potential_energy.png|340px]]
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.
[http://openwetware.org/wiki/Biomod/2012/TeamSendai/Simulation You can see details in simulation page ]
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<h2>Potential Barrier</h2>
 
[[Image: Potential graph.jpg|340px]]
<h2>Porter</h2>
Our Gate is made of DNA, so it has negative electric charge. Single stranded DNA has negative electric charge, too. Here is a graph about potential energy of the tube.(藤原さんに頂いた下のグラフ載せる。) 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. So our Gate is well designed.
<h3>Principle</h3>
[[Image:スライドGif.gif|left|400px]]
In the concept of Cell Gate, there are two problems. for making CELL-GATE.  
<ul>How to pull the target DNA into GATE ?</ul>
<ul>How to pass the target through GATE ?</ul>
 
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 [http://openwetware.org/wiki/Biomod/2012/TeamSendai/Simulation 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.
 
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[[Image: 配列.png|center|500px]]
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'''Blue''':Target '''Red''':This part is complementary with target  '''Green''':Spacer


<h1>Design of Porter</h1>
<h2>Principle</h2>
[[Image: Perportergifkoyama.gif|right|340px]]
When we consider making Cell Gate, there are two problems. One is how to pull the target oligonucleotide into Gate, and the other is how to pass the target. To solve these problems, we propose a nano structure of ssDNA and named it “Porter”. Porter stands in line in Gate, pulls the target, and transports it.


The Gate simulation shows that things by itself don’t come in Gate. So Porter has to pull and carry the target inside Gate.
※Porter3 use only electrophoresis.
----
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 at the end of Porter, Porter shrinks and pulls the target. Finally the target comes in Gate, otherwise unable to go through.
[[Image: スクリーンショット 2012-10-28 8.38.23.png|center|400px]]
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.  


The inner Porter has more complementary sequences to the target and has higher bonding energy from 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.


<DNA sequences>(実際の配列をお願いします) These are the sequences for electrophoresis, and only a part of the actual Porter sequence. These are the extracted region binding to the target. When planted in the gate, Porter has more spacer sequences of 10base at the root in addition to the sequences above, to reduce the Coulomb's force produced by the wall of Gate.
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<h3>Simulation</h3>
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.<br>
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Porter can binds to the target
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Toehold structure cannot bind to the target
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[http://openwetware.org/wiki/Biomod/2012/TeamSendai/Simulation See detail in simulation page]


<h2>Simulation</h2>
We compared the ability to catch the target of Porter with that of toehold structure.
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<h1>How to implement</h1>
<h2>Membrane: How to implement the GATE</h2>
<h2>Cell model</h2>
<h3>Cell model</h3>
<h2>Cholesterol</h2>
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.
We also have the aim of this GATE stab in the cell membrane, it can not sting in the cell membrane of normal hexagonal tube. However, We can create a tube with a different structure by exchanging some staple. We have designed a simple tube 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 tube by this method. In addition, we have also designed DNA-like beard at the entrance of the tube. We expect the effect of electrostatically repel force with DNA which are not intended. Our tube is small,so we designed the tube to connect to each other and be long in order to easily confirmed using AFM. By replacing the staple, this structure is also removably.
<h3>Cholesterol-leg</h3>
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.<br>
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.
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[[Image: スクリーンショット_2012-10-15_1.11.25.png |300px]]
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[[Image: スクリーンショット_2012-10-15_1.22.38.png |300px]]
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[[Image: みにげーと.png |300px]]
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Latest revision as of 19:43, 27 October 2012

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<body> <div id="Container"> <!-- Menu --> <ul id="menu"> <li><a href="http://openwetware.org/wiki/Biomod/2012/Tohoku/Team_Sendai ">Home</a></li> <li><a href=" http://openwetware.org/wiki/Biomod/2012/TeamSendai/Idea ">Project</a></li> <li><a href=" http://openwetware.org/wiki/Biomod/2012/TeamSendai/Design">Design</a> </li> <li><a href=" http://openwetware.org/wiki/Biomod/2012/TeamSendai/Simulation">Simulation</a> </li> <li> <a href=" http://openwetware.org/wiki/Biomod/2012/TeamSendai/Experiment ">Experiment</a> </li> <li> <a href=" http://openwetware.org/wiki/Biomod/2012/TeamSendai/Future Application">Future Application</a> </li> <li> <a href=" http://openwetware.org/wiki/Biomod/2012/TeamSendai/Diary">Diary</a> </li> <li> <a href=" http://openwetware.org/wiki/Biomod/2012/TeamSendai/Team ">Team</a> </li> <li> <a href=" http://openwetware.org/wiki/Biomod/2012/Tohoku/Team Sendai/header"></a> </li>

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Design

Gate

Size / Structure

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.

DNA origami

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.


Potential Barrier

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


Porter

Principle

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.



Simulation

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.

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Porter can binds to the target

Toehold structure cannot bind to the target



See detail in simulation page


Membrane: How to implement the GATE

Cell model

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.

Cholesterol-leg

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.

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