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<li><a href="http://openwetware.org/wiki/Biomod/2012/Tohoku/Team_Sendai ">Top</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/Simulation">Simulation</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/Experiment ">Experiment</a>
<ul>
<li><a href=" http://openwetware.org/wiki/Biomod/2012/TeamSendai/Method">Method</a>
<ul>
<li>
<a href=" http://openwetware.org/wiki/Biomod/2012/TeamSendai/Result#Porter">Porter</a>
<li>
<a href=" http://openwetware.org/wiki/Biomod/2012/TeamSendai/Result#Cylinder">Cylinder</a> </li>
<li> <a href=" http://openwetware.org/wiki/Biomod/2012/TeamSendai/Result# Vesicle">Vesicle</a>
</li>
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<a href=" http://openwetware.org/wiki/Biomod/2012/TeamSendai/Result">Result</a>
<ul>
<li>
<a href=" http://openwetware.org/wiki/Biomod/2012/TeamSendai/Result#Porter">Porter</a>
<li>
<a href=" http://openwetware.org/wiki/Biomod/2012/TeamSendai/Result#Cylinder">Cylinder</a> </li>
<li> <a href=" http://openwetware.org/wiki/Biomod/2012/TeamSendai/Result# Vesicle">Vesicle</a>
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<a href=" http://openwetware.org/wiki/Biomod/2012/TeamSendai/Achievement">Achievement</a>
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<a href=" http://openwetware.org/wiki/Biomod/2012/TeamSendai/Diary">Diary</a>
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<li>
<a href=" http://openwetware.org/wiki/Biomod/2012/TeamSendai/Team ">Team</a>
</li>
<li>
<a href=" http://openwetware.org/wiki/Biomod/2012/TeamSendai/FAQ">FAQ</a>
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<!-- コンテンツ -->
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<div id="Content">
<div id="Content">
<h1>Three experiment parts</h1>
<p>
We divided our project into 3 parts, and did experiment.
</p>
<img src=" http://openwetware.org/images/f/f1/Format_cellgate_kakudai.jpg
" align="right" width="927px" height="398px">


<h1>Design</h1>
<h2>Gate</h2>
<h3>Size / Structure</h3>
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>
{{-}}
<h3>DNA origami</h3>
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.


<h1>Gate</h1>
[[Image: スライド15.jpg |340px]]
<p>
[[Image: スライド16.jpg |340px]]
<youtube width="450" align="left">XMiheA1sWOA</youtube>
{{-}}


</br>
<h3>Potential Barrier</h3>
We decided to use the structure of the hexagonal tube.The reason is because We think there is a reference on the hexagonal structure of DNA origami（ショーンの論文を紹介）, and can take advantage of that knowledge.Next, We made a simulation in order to examine the size of the structure.Must not the size that it is the tube through anything.It must be large enough to be passed through the desired product, however.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.
[[Image: Potential_energy.png|340px]]
<img src=" http://openwetware.org/images/e/ec/Tsutsucadnano1.png
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.
" align="right" width="600px" height="398px">
[http://openwetware.org/wiki/Biomod/2012/TeamSendai/Simulation You can see details in simulation page ]
<img src=" http://openwetware.org/images/a/a4/TsutsucaDNAno2.png
{{-}}
" align="right" width="600px" height="398px">


</p>


<h1>Porter</h1>
<h2>Porter</h2>
<p>
<h3>Principle</h3>
We named the single stranded DNA “Porter” which stand in line in the GATE and transport the target. Porter is designed to be more complementary sequence to the target molecule as going back from the entrance. These designs enable to move to the inner porter because of the stability of combination. This way makes it possible to move the target. We did a simulation to find the best length and space of the porter. The result of simulation, we have found that not more than a certain distance away from the gate is the target DNA was negatively charged by electrostatic force so that the gate can be in the DNA. The solution of this phenomenon, we have to make the first Porter have enough length to beyond the original length and bring the target into the tube.
[[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>


So we design the Porter to take a loop structure when bound to the target  molecule. It was believed that the Porter would be able to go beyond the distance to the complementary sequences in the various parts of Porter, inaccessible electrostatically by target molecule is bound in a stepwise manner, pull the material into the Gate. In actual experiment, we compare the トーホールド and rope structure to use following sequence(配列載せる). And, this is for electrophoresis, so it’s the sequence to extract only the part related to the binding of the target. Actual Gate’s sequence integrate about 10bp spacer sequence to reduce the Coulomb force which affected by the wall when moving.
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.
</p>


<h1>Membrane</h1>
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.
<p>
We use liposomes as a model of cell membrane. We prepared a
preliminary step to that cell gate insert into the liposome. We designed
a smaller tube and attempted to insert into liposomes using it. Similar to
the cell gate, we stretched single-stranded DNA of 10 bases that can be
modified cholesterol from the side of this tube. We attached cholesterol
to the single-stranded DNA, and expected that these enter into the
hydrophobic portion of the liposome. Then, it is likely to that the tube
stick to the liposome. However, it is considered that the tube would be
in a state of lying on the liposome. So, when we designed the tube, we
made one side too much for the extra M13 deliberately. We expect that
the electrostatic symmetry of the tube is broken by this, and the tube
penetrate the membrane standing vertically by repulsion.


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.


</p>
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.
</br>
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{{-}}
[[Image: 配列.png|center|500px]]
{{-}}


'''Blue''':Target '''Red''':This part is complementary with target  '''Green''':Spacer




※Porter3 use only electrophoresis.
----
[[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.




{{-}}


<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>
<table border="0" align="left" vertical-align: middle;>
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<td>
<youtube width="450" align="left">NXo4cYKkrF0&border=1&color1=0x6699&color2=0x54abd6</youtube>
<html><div style="clear:both;"></div></html>
</td>
<td width="20">
</td>
<td>
<youtube width="450" align="left">qidZS1pI0lc&border=1&color1=0x6699&color2=0x54abd6</youtube>
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</td>
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Porter can binds to the target
</td>
<td width="20">
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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]


</div>


</body>
<h2>Membrane: How to implement the GATE</h2>
</html>
<h3>Cell model</h3>
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.
<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.
{{-}}
[[Image: スクリーンショット_2012-10-15_1.11.35.png |300px]]
[[Image: スクリーンショット_2012-10-15_1.22.38.png |300px]]
[[Image: みにげーと.png |300px]]
{{-}}

Latest revision as of 19:43, 27 October 2012

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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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