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<p class="sukima"> Design
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        <article>
        <h2>Design</h2>


<table id="toc" class="toc" summary="Contents"><tr><td><div id="toctitle"><h2>Contents</h2></div>
<ul>
<li class="toclevel-1"><a href="#chain">
<span class="tocnumber"></span> <span class="toctext">Project goal</span></a></li>
<ul>
<li class="toclevel-2"><a href="#Flower">
<span class="tocnumber"></span> <span class="toctext">First stage:Sensing system</span></a></li>
<li class="toclevel-2"><a href="#sensing">
<span class="tocnumber"></span> <span class="toctext">Second stage:Amplification system</span></a></li>
<ul>
<li class="toclevel-3"><a href="#5">
<span class="tocnumber"></span> <span class="toctext">DNA origami approach</span></a></li>
<li class="toclevel-3"><a href="#6">
<span class="tocnumber"></span> <span class="toctext">Flower DNA approach</span></a></li>
</li>


<article data-title="Egg-type initiator">
<h3 id="egg">Egg-type initiator</h3></br>
<a name="#egg"></a>


<img src="http://openwetware.org/images/6/64/Design-top-01.png" alt="example-tab2" border="0"></br>
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</li>
                    Fig.1 process of Egg-type initiator</div><br>
</ul>
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</td></tr></table>


Egg-type initiator consists of two layers.<br>
<h2 id=chain>Project goal</h2>
&nbsp;In Lipo-HANABI project, we need to develop the following two subsystems.<br><br>


<h4>The first layer: “alginate gel membrane”</h4>
i) Sensing system (First stage): liposome disruption by temperature control. <br>
The alginate gel membrane has a solution phase inside them, and resembles artificial salmon caviars (JINKOH-IKURA in Japanese).<br><br>


<h4>The second layer: “temperature-sensitive liposomes”</h4>
ii) Amplification system (Second stage): a chain-reactive disruption of the liposomes activated by the First stage. <br><br>
The temperature-sensitive liposomes contain PNIPAM lipids in their membrane. Temperature shift causing hydrophobicity change of the PNIPAM induces collapse of the liposomes (See


<a href="#Characters of the PNIPAM molecular">“Characters of the PNIPAM molecular”</a>
<h3 id=Flower>First stage: Sensing system </h3>
&nbsp;The purpose of First stage is to detect temperature change and release key molecules for the Second stage. This is achieved by temperature-sensitive liposomes containing &nbsp;the keys. To make the liposome, we used lipids conjugated with NIPAM polymer.<br>
&nbsp;This structural change of NIPAM induces stress on the surface of the liposome, and consequently disrupts them.<br>
<div align="center">
<Img Src="http://openwetware.org/images/9/95/NIPAM%E3%83%AA%E3%83%9D%E3%81%A1%E3%82%83%E3%82%933.png">
</div>
<div class="caption">Fig.1 Temperature-sensitive liposome</div>
<h3 id=sensing>Second stage: Amplification system </h3>
&nbsp;The purpose of Second stage is to accept the key from the First stage and release a lot of payload molecules in a chain-reaction. <br>
&nbsp;There are two different approaches to realize the Second stage.<br>
  A) DNA Origami approach<br>
  B) Flower DNA approach<br>


). The temperature-sensitive liposomes encapsulate a chelate compound (EGTA) and DNAs.<br><br>  
<h4 id=5>DNA origami approach </h4>


The two layers realize a dual disruption system as follows (Fig.1). The first is “liposome disruption” by increasing temperature. The second is “disruption of alginate membrane” by chelating reagent (EGTA) of calcium released from the liposomes disrupted. This system enables to release many trigger DNAs at a limited point.<br><br>


1.The alginate gel membrane encapsulates many temperature-sensitive liposomes.<br>
Please refer following link(
<a href="http://www.sigmaaldrich.com/etc/medialib/docs/SAJ/Brochure/1/j_recipedds2.Par.0001.File.tmp/j_recipedds2.pdf">pdf</a>
).<br>
2.
Warming the temperature-sensitive liposomes from room temperature to over 32 ºC causes disruption of the liposomes.<br>
3.
The liposome disruption release DNAs and EGTA (and urea) which chelate calcium. Lowering calcium concentration starts to melt the alginate gel membrane. During this process, DNA origami is formed when urea is added.<br>


4.
&nbsp;This approach is inspired by a paper about <a Href="http://www.ncbi.nlm.nih.gov/pubmed/19780639"> Membrane-bending proteins (Prinz WA, Hinshaw JE., Crit Rev Biochem Mol Biol., 2009)</a>.
As a result of the melting, trigger DNAs (DNA origami structure or DNA strands for the flower micelle approach (See design:
In this approach,　we use “Origami-anchor DNA” which connects DNA Origami with liposome membrane.
Chain-reactive burst
<!--
<a href="#chain-reactive burst"> Chain-reactive burst</a>
-->


<!--
A lot of DNA origamis are adsorbed on the surface of liposomes by using Origami-anchor DNA. DNA origami is supposed to be a stiff, straight board compared with liposome membrane, and as a result, liposome surface gets bending stress. At certain level of the absorbance, liposomes will burst. Also, DNA origamis on the surface repel each other because of negative charges on DNA backbones. This effect may add more stress on the membrane.<br>


 
<div align="center">
)) are released from the melted alginate membrane.<br>
<Img Src="http://openwetware.org/images/c/c5/%E8%86%9C%E3%80%80%E5%8F%8D%E7%99%BAdfhr.png">
 
</div>
<h4>An application to DNA origami formation using the dual disruption system</h4>
<div class="caption">Fig.2 Stress on liposome membrane</div>
This dual structure enables to spontaneous formation of DNA origami through environmental stimulation. Three components, Urea (a denature reagent of DNA nanostructure) and materials of DNA origami and EGTA, can be encapsulated in liposomes. It takes times (several hours) to melt the gel membrane by EGTA released from the temperature-sensitive liposomes. Because small molecules can diffuse in alginate gels, urea inside alginate is expected to be gradually diluted during the melting. DNA origami could be formed through the dilution of urea (See
 
 
 
<a href="#DNA origami formation through urea dilution">“DNA origami formation through urea dilution”</a>
 
 
).<br><br>
 
<h5>※Principle of this system</h5>
<h5>Alginate gels</h5>
Alginate is widely used in foods additives and drug stabilizers. <br>Sodium alginate is the neutral salt which an alginate carboxyl group coupled with a Na+. It dissolves in water well and becomes water solution with the high viscosity.
If Ca2+ is added to the sodium alginate solution, ion bridging happens instantly and gelation happen. And If Chelating reagent (for example EDTA, EGTA) is added to alginate hydrogel, Ca2+ is robbed and gel collapse.
<br>
<br>
&nbsp;From the reference, we learned that efficient structure design for destabilizing membranes should have the following properties: <br>
<ur><li>Having rigid scaffolds</li>
<li>Having large surface areas to maximize the effect of the scaffold on the membrane</li></ur>


<img src="http://openwetware.org/images/d/d2/Alginate-gel_2.png"><br>
<Design of DNA origami><br>
Fig.2 A image about Gelation and Solation of alginate gel<br><br>
&nbsp;DNA origami is known as a designable rigid structure made of DNA. We use DNA origami to make the rigid scaffolds. In order to meet the requirements, we designed a 2D rectangular DNA origami.<br>


<a name="Characters of the PNIPAM molecular"></a>
<div align="center">
<h5>Characters of the PNIPAM molecular</h5>
<Img Src="http://openwetware.org/images/4/45/Outsidefig8.png">
 
</div>
Hydrophobicity of NIPAM varies at temperatures. NIPAM is hydrophilic at less than 32 ºC, but it become hydrophobic and shrinks at > 32 ºC. Therefore, liposomes containing a modified NIPAM (poly(NIPAM-co-AA-co-ODA) in their membranes become unstable at high temperature (temperature-sensitive liposomes). Consequently, increasing temperature disrupt the liposomes.<br>
<div class="caption">Fig.3 Rectangular origami</div>
Reference(
<a href=
"http://www.sigmaaldrich.com/etc/medialib/docs/SAJ/Brochure/1/j_recipedds2.Par.0001.File.tmp/j_recipedds2.pdf">pdf</a>)
<br>
 
 
<div align="center"><img src="http://openwetware.org/images/d/df/PNIPAM-function-02.png" width="600"></div><br>
Fig.3 A schematic image how liposome containing PNIPAM disrupt at high temperature is shown.<br><br>
 
<a name="DNA origami formation through urea dilution"></a>
 
 
 
 
<h5>DNA origami formation through urea dilution</h5>
 
Polarity of water molecular becomes weak in the presence of urea. Thus, urea interrupts the hydrogen bond of DNA bases. For the reason, the melting point of DNA hybridization decreases. Thus, gradually decreasing the concentration of urea enables to form DNA origami structure under isothermal condition.<br>
 
<img src="http://openwetware.org/images/4/4b/Nyoso.png"><br>
Fig.4 A image about denaturation by urea
 
 
</article>
 
 
 
-->
 
 
 
  <article>
 
 
 
<h3 id="chain">Chain-reactive burst</h3></br>
<br>
We designed “chain-reactive burst” system as follows.<br>
Each liposome contains triggers and drugs inside, and aptamers for the trigger on its surface.  When liposomes are destroyed, new triggers and drugs are released. To achieve liposomal burst by outside triggers, we propose the following two approaches. <br>
<br>
<li> <h5>i) Bending approach</h5></li>
<li> <h5>ii) Flower micelle approach</h5></li>
<br>
First, we considered a theory to disrupt liposomes by a trigger DNA signal through calculation. If a liposome is destroyed, its size becomes smaller. We estimated the free energy gap between the two liposomal states: a large liposome and a small one. And  discuss which size of liposomes is more stable.<br>
Please see the details (Go to <a href="http://openwetware.org/wiki/Biomod/2013/Sendai/calcuation">Calculation</a>).<br>
 
<h4> i)Bending Approach</h4><br>
 
<Img Src="http://openwetware.org/images/f/f2/Design-bending-flow.png" Align="center" width="900px" ><br>
<div align="center">Fig.1 Process of bending approach</div><br>
<br>
Our bending approach consists of the following four steps.<br>
1.Cholesterol-conjugated DNA strands (in the rest of this document, referred to as “aptamers”) attach to the surfaces of liposomes.<br>
2. Then, DNA origami complementary to the aptamer is added as triggers.<br>
3. Triggers bind to the surfaces of liposomes and give a load on the membrane.<br>
4. Due to the load by triggers, liposomes are destroyed.<br>
<br>
 
<h5>a) Mechanism of bending membranes</h5>
To destroy liposomes, we focused on the mechanism the living things use to bend cell membranes. We consider that if we could make use of the mechanism of bending membranes (destabilizing membranes), it would lead to the collapse of membranes. The following three mechanisms have been proposed as of now (<A Href="http://www.ncbi.nlm.nih.gov/pubmed/19780639">Membrane-bending proteins</A>)<br>
 
<div class="caption-left">
<Img Src="http://openwetware.org/images/a/ae/Designfig2.png" width="280px" height="400px">
<span>Fig.2 Mechanism of bending membranes</span></div>
<br>
The mechanism A is that amphipathic molecules are inserted into the cell membrane and the bending is caused. The inner hydrophobic part of the lipid bilayer has a strong adhesive power for the two leaflets. Thus, once the amphipathic molecules are inserted into one leaflet of the membrane and expand it, the other leaflet bends according to it, making its surface area smallest.<br>
<br>
The mechanism B is that the molecule attached to the membrane becomes a rigid scaffold and distort the membrane under itself, or stabilize the already bended membrane.<br>
<br>
The mechanism C is that lipid molecules are clustered in one leaflet of the membrane and the inequality of lipid quantity makes the membrane bend.<br>
<div class="c-both"></div>
 
Most membrane bending proteins combine the above three mechanisms.<br>
In addition, a theory that protein crowding causes the bending of cell membranes ( <A Href="http://www.ncbi.nlm.nih.gov/pubmed/22902598">Membrane bending by protein- protein crowding</A>) has recently been suggested. This mechanism is that the collision of membrane proteins produces lateral pressure on membranes and distorts them.<br>
<br>
Due to the above reasons, the efficient design for destabilizing membranes is the structures that :<br>
<ur><li>have rigid scaffolds</li>
<li>have large surface areas to maximize the effect of the scaffold on the membrane</li>
<li>produce a large pressure by collisions</li></ur>
<br>
<h5>b) Rigid scaffolds</h5>
To make rigid scaffolds, we took note of DNA origami, because DNA origami is a method for making rigid structures of any shape. Moreover, we adopted a 2D structure to make the surface area largest.<br>
 
<br>
We also designed rectangle and triangle to make the pressure of the collision highest.<br>
<Img Src="http://openwetware.org/images/e/e7/Design-outside-fig3.png">
<br>
<div align="center">Fig.3 Rectangle origami and triangle origami</div><br>
We suppose that rectangle and triangle structures are most effective for the following reasons. <br>
Rectangle is expected to work as one scaffold in itself; triangle (the most efficient figure that covers a sphere) structures, to gather and work as one big rigid scaffold.<br>
<br>
The design of our rectangular DNA origami is as below.<br>
<Img Src="http://openwetware.org/images/b/b1/Design-outside-fig4.png">
<div align="center">Fig.4 Rectangular origami</div>
<br>
<br>
<div class="caption-right">
<div class="caption-right">
  <Img Src="http://openwetware.org/images/a/a7/Lipo5.png" ><span>Fig.5 DNA origami designed by caDNAno</span>
  <Img Src="http://openwetware.org/images/a/a7/Lipo5.png" style="padding-left:10mm"><span>Fig.4 DNA origami designed by caDNAno</span>
</div>
</div>
We used <A Href="http://cadnano.org/">caDNAno2</A> for our DNA origami design.<br>
&nbsp;We use <a href="http://cadnano.org/">caDNAno2</a> for our DNA origami design.  
The DNA origami has a rectangle shape of 67.6nm (26 helixes) by 127 nm (374 bases).<br>
The size of DNA origami is 67.6nm (26 helixes) in width and 127 nm (374 bases) in height.
We cut out a smaller rectangle of 10 helixes by 161 bases at one edge of this origami, so that we could distinguish the two sides during AFM (Atomic Force Microscope) observation.<br>
We cut out a smaller rectangle of 10 helixes (161 bases) at one of the corners,
Besides, to destabilize the membrane by inserting this origami, we designed 141 staples at the center of the origami to hybridize with aptamers (These aptamers give our origami amphipathicity), and enabled it to insert into the membrane.  
so that we could distinguish the two sides with AFM (Atomic Force Microscope) observation.  
Also, we put 141 staples sticking out from the bottom face of the origami.
Those staples hybridize with cholesterol-modified Origami-anchor DNA, which has high affinity with lipid membrane.<br>
<br>
<br>
<div class="c-both"></div>
<div align="center">
To sum up, the aptamer not only connects DNA origami and liposomes but also inserts into the membrane and destabilizes it.<br>
<Img Src="http://openwetware.org/images/a/a0/Outsidefig5rg.png"　width="450px" height="350px">
 
</div>
<Img Src="http://openwetware.org/images/c/c5/Outsidefig5.png">
<div class="caption">Fig.5 Unstable liposome</div>
<div align="center">Fig.6 Unstable liposome</div>
<br><br>
<br>  
<h4 id=6>Flower DNA approach</h4>
<h4>ii)Flower micelle approach</h4><br>
&nbsp;This approach is inspired by a paper about <a href="http://pubs.acs.org/doi/ipdf/10.1021/jp104711q">Polymer Flower-micelle (Yukio Tominaga, Mari Mizuse, Akihito Hashidzume, Yotaro Morishima and Takahiro Sato, J. Phys. Chem. B, 2010)</a>.
<Img Src="http://openwetware.org/images/8/8f/Design-flower-flow.png" Align="center" width="900px" ><br>
To adapt the Polymer Flower-micelle to our project, the followings are required.<br><br>
<div align="center">Fig.7 Process of flower micelle approach</div><br>
<ur><li>Embedding a lot of cholesterol-modified ss DNA on the liposome surface</li>
 
<li>Adding another ssDNA (complementary to the above DNA) which induces a structural change　by DNA hybridization</li>
<div class="caption-right">
<li>The induced structural change on the DNA results in disruption of the liposome</li>
<Img Src="http://openwetware.org/images/6/6f/Design-flowermicelle.png" style="width:425px;"><span>Fig.8 Flower micelle method</span></div>
There is a method called flower micelles for collapsing liposomes. <br>
In this method, we cover the surfaces of liposomes with many copolymer rings. The rings can be distorted by heating, place some stress on the liposomes, and collapse them.<br>
We tried to collapse liposomes by applying this mechanism of flower micelles.<br>
<div class="c-both"></div>
<br>
<br>
1. First, we mix aptamers (the same strands as used in i) Bending approach), loop strands, and liposomes.<br>
&nbsp;At first, we designed “Flower-anchor DNA”, which is a couple of ss DNAs both having cholesterol modified groups (Fig.6): Flower-anchor1 is 10nt ss DNA and Flower-anchor2 is 50nt ss DNA. Both are cholesterol-modified at their 3’ ends. <br>
Each of the loop strands is designed to have two complementary parts to aptamers at its both ends. So when it binds to the aptamers at the both ends, the middle part remains single-stranded and becomes a loop. <br>
&nbsp;In addition, the 5’ end of the Flower-anchor2 is complementary to Flower-anchor1. When they hybridize, the rest 40nt of Flower-anchor2 remains single-stranded.<br><br>
As an aptamer is cholesterol-conjugated and has high affinity for a liposome, it floats on a liposome and enables the aptamer-loop strand complex attach to the liposome. In other words, a loop strand hybridizes with a liposome via two aptamers.<br>
<div align="center">
<Img Src="http://openwetware.org/images/a/aa/Flower2.png">  
<Img Src="http://openwetware.org/images/3/3d/Flower-newfg.png" width="450px" height="350px" ></div><br>
<div class="caption">Fig.6 Liposome with Flower-anchor DNA</div>
<br>
<br>
<div align="center">Fig.9 Make loops on the surface of a liposome</div>
&nbsp;The key DNA released from stage 1 liposome is complementary to this single-stranded part. When the key hybridizes on it, a double-stranded section is formed. The length of the section is shorter than its persistence length; therefore it works as a rigid strut. The strut is anchored on the liposome at both ends, thus it extends the membrane. As a consequence, this may lead to drastic conformational change of the liposome, namely, disruption. <br><br>
<br>
<div align="center">
2. Next, we add a trigger strand corresponding (complementary) to the loop strand on the liposome. The trigger strand hybridizes with the loop part, making it change to be straight.<br>
<img src="http://openwetware.org/images/6/65/Flower3new8.png" width="70%" hight="800"><br>
<br>
<div class="caption">Fig.7 Process of flower DNA approach</div><br><br>
3. The double-stranded part keeps straight (though it was originally a loop part), because the trigger strand is designed to be shorter than its persistence length.<br>
<Img Src="http://openwetware.org/images/1/17/Flor4.png" width="70%" hight="800" ><br>
<img src="http://openwetware.org/images/0/03/Flower3.png"><br>
<div class="caption">Fig.8 How to disrupt a liposome</div>
<div align="center">Fig.10 How to straighten loop</div>
      </article>
4.In this process, some stress is placed on the liposome, and it is collapsed.<br>
</section>
<Img Src="http://openwetware.org/images/3/3b/Flower4.png"><br>
<div align="center">Fig.11 Liposomal burst</div>
<br>
It is considered if some triggers are kept inside a liposome, and the liposomal membrane is collapsed by the above i) and ii) methods from the outside, it would be much easy to trigger "Chain-reactive burst", because the released triggers serve as new triggers for neighbor liposomes. <br>
<br>
We designed the DNA sequences for this approach by <A Href="http://www.dna.caltech.edu/DNAdesign/">DNA design</A>, software for designing DNA sequences. <br>
We arranged three kinds of loop strands. <br>
Each loop strand has a 40nt, 20nt, or 10nt loop part (shown below in black and blue), which becomes a loop after the hybridization of the whole loop strand with aptamers.<br>
The blue part of a loop strand is complementary to a corresponding trigger strand (also shown in blue). So a loop strand and a trigger strand are expected to hybridize with each other, place some stress on a liposome, and collapse it. <br>
The red part of a loop strand is complementary to an aptamer (shown in red). Cooperating with aptamers, it enables the whole loop strand to attach to the surface of a liposome. <br>
Aptamers are the same strands as those used in i)Bending approach.<br>
<font size="-2">
Aptamer DNA<br>
<font color="red">CCAGAAGACG</font> -cholesterol<br>
40nt loop DNA<br>
<font color="red">CGTCTTCTGG</font>TTTTTTTTTT<font color="blue">GCGAACCACGGTTCCCAGCGTGACCTTCATGCTTAAGTTT</font><font color="red">CGTCTTCTGG</font><br>
Trigger DNA for 40 nt loop DNA<br>
<font color="blue">AAACTTAAGCATGAAGGTCACGCTGGGAACCGTGGTTCGC</font><br>
20nt loop DNA<br>
<font color="red">CGTCTTCTGG</font>TTTTTTTTTTTT<font color="blue">CATAACATGAGGCGCCGT</font><font color="red">CGTCTTCTGG</font><br>
Trigger DNA for 20 nt loop DNA<br>
<font color="blue">ACGGCGCCTCATGTTATGAA</font><br>
10nt loop DNA<br>
<font color="red">CGTCTTCTGG</font>TTTTTTTTTT<font color="blue">CTGTAACTAA</font><font color="red">CGTCTTCTGG</font><br>
Trigger DNA for 10 nt loop DNA<br>
<font color="blue">TTAGTTACAG</font><br>
</font>
 
        </article>
 
 
 
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        <h2>Design</h2>

<table id="toc" class="toc" summary="Contents"><tr><td><div id="toctitle"><h2>Contents</h2></div> <ul> <li class="toclevel-1"><a href="#chain"> <span class="tocnumber"></span> <span class="toctext">Project goal</span></a></li> <ul> <li class="toclevel-2"><a href="#Flower"> <span class="tocnumber"></span> <span class="toctext">First stage:Sensing system</span></a></li> <li class="toclevel-2"><a href="#sensing"> <span class="tocnumber"></span> <span class="toctext">Second stage:Amplification system</span></a></li> <ul> <li class="toclevel-3"><a href="#5"> <span class="tocnumber"></span> <span class="toctext">DNA origami approach</span></a></li> <li class="toclevel-3"><a href="#6"> <span class="tocnumber"></span> <span class="toctext">Flower DNA approach</span></a></li> </li>


</ul> </li> </ul> </td></tr></table>

<h2 id=chain>Project goal</h2> &nbsp;In Lipo-HANABI project, we need to develop the following two subsystems.<br><br>

i) Sensing system (First stage): liposome disruption by temperature control. <br>

ii) Amplification system (Second stage): a chain-reactive disruption of the liposomes activated by the First stage. <br><br>

<h3 id=Flower>First stage: Sensing system </h3> &nbsp;The purpose of First stage is to detect temperature change and release key molecules for the Second stage. This is achieved by temperature-sensitive liposomes containing &nbsp;the keys. To make the liposome, we used lipids conjugated with NIPAM polymer.<br> &nbsp;This structural change of NIPAM induces stress on the surface of the liposome, and consequently disrupts them.<br> <div align="center"> <Img Src="http://openwetware.org/images/9/95/NIPAM%E3%83%AA%E3%83%9D%E3%81%A1%E3%82%83%E3%82%933.png"> </div> <div class="caption">Fig.1 Temperature-sensitive liposome</div> <h3 id=sensing>Second stage: Amplification system </h3> &nbsp;The purpose of Second stage is to accept the key from the First stage and release a lot of payload molecules in a chain-reaction. <br> &nbsp;There are two different approaches to realize the Second stage.<br> 

  A) DNA Origami approach<br>
  B) Flower DNA approach<br>

<h4 id=5>DNA origami approach </h4>


&nbsp;This approach is inspired by a paper about <a Href="http://www.ncbi.nlm.nih.gov/pubmed/19780639"> Membrane-bending proteins (Prinz WA, Hinshaw JE., Crit Rev Biochem Mol Biol., 2009)</a>. In this approach,　we use “Origami-anchor DNA” which connects DNA Origami with liposome membrane.

A lot of DNA origamis are adsorbed on the surface of liposomes by using Origami-anchor DNA. DNA origami is supposed to be a stiff, straight board compared with liposome membrane, and as a result, liposome surface gets bending stress. At certain level of the absorbance, liposomes will burst. Also, DNA origamis on the surface repel each other because of negative charges on DNA backbones. This effect may add more stress on the membrane.<br>

<div align="center"> <Img Src="http://openwetware.org/images/c/c5/%E8%86%9C%E3%80%80%E5%8F%8D%E7%99%BAdfhr.png"> </div> <div class="caption">Fig.2 Stress on liposome membrane</div> <br> &nbsp;From the reference, we learned that efficient structure design for destabilizing membranes should have the following properties: <br> <ur><li>Having rigid scaffolds</li> <li>Having large surface areas to maximize the effect of the scaffold on the membrane</li></ur>

<Design of DNA origami><br> &nbsp;DNA origami is known as a designable rigid structure made of DNA. We use DNA origami to make the rigid scaffolds. In order to meet the requirements, we designed a 2D rectangular DNA origami.<br>

<div align="center"> <Img Src="http://openwetware.org/images/4/45/Outsidefig8.png"> </div> <div class="caption">Fig.3 Rectangular origami</div> <br> <div class="caption-right"> 

<Img Src="http://openwetware.org/images/a/a7/Lipo5.png" style="padding-left:10mm"><span>Fig.4 DNA origami designed by caDNAno</span>

</div> &nbsp;We use <a href="http://cadnano.org/">caDNAno2</a> for our DNA origami design. The size of DNA origami is 67.6nm (26 helixes) in width and 127 nm (374 bases) in height. We cut out a smaller rectangle of 10 helixes (161 bases) at one of the corners, so that we could distinguish the two sides with AFM (Atomic Force Microscope) observation. Also, we put 141 staples sticking out from the bottom face of the origami. Those staples hybridize with cholesterol-modified Origami-anchor DNA, which has high affinity with lipid membrane.<br> <br> <div align="center"> <Img Src="http://openwetware.org/images/a/a0/Outsidefig5rg.png"　width="450px" height="350px"> </div> <div class="caption">Fig.5 Unstable liposome</div> <br><br> <h4 id=6>Flower DNA approach</h4> &nbsp;This approach is inspired by a paper about <a href="http://pubs.acs.org/doi/ipdf/10.1021/jp104711q">Polymer Flower-micelle (Yukio Tominaga, Mari Mizuse, Akihito Hashidzume, Yotaro Morishima and Takahiro Sato, J. Phys. Chem. B, 2010)</a>. To adapt the Polymer Flower-micelle to our project, the followings are required.<br><br> <ur><li>Embedding a lot of cholesterol-modified ss DNA on the liposome surface</li> <li>Adding another ssDNA (complementary to the above DNA) which induces a structural change　by DNA hybridization</li> <li>The induced structural change on the DNA results in disruption of the liposome</li> <br> &nbsp;At first, we designed “Flower-anchor DNA”, which is a couple of ss DNAs both having cholesterol modified groups (Fig.6): Flower-anchor1 is 10nt ss DNA and Flower-anchor2 is 50nt ss DNA. Both are cholesterol-modified at their 3’ ends. <br> &nbsp;In addition, the 5’ end of the Flower-anchor2 is complementary to Flower-anchor1. When they hybridize, the rest 40nt of Flower-anchor2 remains single-stranded.<br><br> <div align="center"> <Img Src="http://openwetware.org/images/3/3d/Flower-newfg.png" width="450px" height="350px" ></div><br> <div class="caption">Fig.6 Liposome with Flower-anchor DNA</div> <br> &nbsp;The key DNA released from stage 1 liposome is complementary to this single-stranded part. When the key hybridizes on it, a double-stranded section is formed. The length of the section is shorter than its persistence length; therefore it works as a rigid strut. The strut is anchored on the liposome at both ends, thus it extends the membrane. As a consequence, this may lead to drastic conformational change of the liposome, namely, disruption. <br><br> <div align="center"> <img src="http://openwetware.org/images/6/65/Flower3new8.png" width="70%" hight="800"><br> <div class="caption">Fig.7 Process of flower DNA approach</div><br><br> <Img Src="http://openwetware.org/images/1/17/Flor4.png" width="70%" hight="800" ><br> <div class="caption">Fig.8 How to disrupt a liposome</div> 

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