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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>  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>  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  the keys. To make the liposome, we used lipids conjugated with NIPAM polymer.<br>  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>  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>  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>

 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>  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>  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>  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>  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>  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>  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>  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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Biomod/2013/Sendai/design: Difference between revisions

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-    </header>
+</header>
-<p class="sukima"> Design
-</p>
-   <section id="tabs">
- <article data-title="Egg-type trigger">
-<h3 id="designsubproject1">Egg-type trigger</h3></br>
-<img src="http://openwetware.org/images/5/5a/Alginate-design-01.png" alt="example-tab2" border="0"></br>
-<div align="center">Fig1 内側からアルギン酸膜を破壊し、トリガーとなるDNAオリガミを放出するシステム<br>
-                    Fig1 process of Egg-type trigger</div><br>
-<br>
-We designed the following system to break membrane of algin acid from inner side.(Fig1)</br>
-.<br>
-We made chelate agent of dissolving algin acid (EGTA),urea and liposome containing nipam and enclosing DNA strand and DNA origami, which can be triggers of breaking liposome lying outer the membrane of algin acid. nipam is hydrophile in the state of less than 32℃. However, in the state of more than 32℃, nipam constrict and become hydrophoby. Liposome modified by nipam is stable in the state of less than 32℃ due to hydration of nipam molecular, though it is unstable in the state of more than 32℃ because of hydrophoby of nipam molecular. So liposome will be bloken when the temperature of liposome is more than 32℃. We consult following URL.</br>
-http://www.sigmaaldrich.com/etc/medialib/docs/SAJ/Brochure/1/j_recipedds2.Par.0001.File.tmp/j_recipedds2.pdf</br>
-.<br>
-When the temperature of liposome is more than 32℃, it will be break down.</br>
-.<br>
-When liposome is broken, chelating agent enclosed in liposome broke down membrane of algin acid and urea solution is diluted. After that, DNA is annealed and formed DNA origami.</br>
-.<br>
-From broken membrane of algin acid, DNA origami fall out.</br>
-These system enable us to emit trigger on us own time when we increase heat at 37℃.</br>
-<br>
-内側からアルギン酸膜を破壊するために私たちは以下のようなシステムをデザインしました。（Fig1の数字と下記の説明の説明は対応している）</br>
-①内部にアルギン酸を溶かすキレート剤（ＥＧＴＡ）と尿素とトリガー（アルギン酸膜外にあるリポソームを割ることができるもの）となるＤＮＡオリガミ/ＤＮＡストランドを入れたニッパム分子付きのリポソームを作製します。ニッパムとは３２度以下では水和していて親水性だが、３２度以上では収縮して疎水性になる。ニッパムがリポソームに修飾されると、３２度以下ではニッパム分子の水和により安定な状態になるが、３２度以上ではニッパム分子が疎水性になって不安定な状態になるので、３２度以上になった時にリポソームが割れることになる。</br>
-参考</br>
-http://www.sigmaaldrich.com/etc/medialib/docs/SAJ/Brochure/1/j_recipedds2.Par.0001.File.tmp/j_recipedds2.pdf</br>
-</br>
-②温度を約３２度に上げることで、ニッパム分子が収縮しリポソームが破壊されます。</br>
-③リポソームが破壊されたことで内部のキレート剤がアルギン酸膜を破壊し、それと同時に尿素が希釈されることでＤＮＡがハイブリタイゼーションしてトリガーとなるＤＮＡオリガミが形成されます。</br>
-④破壊されたアルギン酸膜からトリガーのＤＮＡオリガミが外に出ていきます。</br>
-このようなシステムの設計によって、好きな時に温度を３７度に上げればトリガーを放出することが可能になります。</br></br>
-</article>
- <article data-title="Chain-reactive burst">
-<h3>Chain-reactive burst</h3></br>
-<!-- 計算式をコメントアウトここから -->
-<!--
-<h4 id="designsubproject2">Chain reaction</h4></br>
-<p>
-リポソームが割れるという事象は、以下のように表現できる。</br>The event breaking liposome means as following mathematical expression.
-</br>
-Calculation
-</br></br>
-・Thought</br>
-　Assume that a vesicle of large radius existing underwater changes to the plural vesicles of small radius (fig 1). The free energy of each sates F1: large radius, and F2: small radius, probably have following relationship (fig 2).  In this case, the vesicle is basically small states because being a small radius is more stable, but there is the energy gap “δ” that must exceed to change the size of vesicle. In this time we do not calculate δ, but calculate the free energy in each radius. From these energy and radius, we demand the relationship of them.</br>
-・Setting</br>
-　Surfactant molecules of the N0 units exist underwater, and N units participate in the formation of the vesicle. Assume that surface area of one molecule A=0.6 [nm^2] and the number of molecules to be included in one vesicle with n units. The volume of the water V=10^(-4) [m^3]. This time, we use DOPC (C44H84NO8P) so the molecular weight m=785 [g/mol]. The temperature of the system T=300[K], and Boltzmann’s constant and Planck's constant use the follows.</br>
-<div align="center"><img src="http://openwetware.org/images/2/29/Cal-00.png" width="200" height="100"></div>
-</br>
-・Calculation result</br>
+<div id="ttop">
-　Free energy “F” of the vesicle is given as follows.</br>
+<a href="#top" class="page_top" onfocus="this.blur();" onclick="scrollTo(0,0); return false;" title="Top"></a></div>
+ <section role="main">
+        <article>
+         <h2>Design</h2>
-<div align="center"><img src="http://openwetware.org/images/c/c8/Cal-01.png" width="200" height="100"></div>
+<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>
-In this time,</br>
+<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>
-<div align="center"><img src="http://openwetware.org/images/b/bf/Cal-02.png" width="200" height="100"></div>
+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>
-(pf)bulk is a partition function of the single discrete molecule underwater, and (pf)vesicle is a partition function of the vesicle.</br>
-Therefore, free energy “F”,</br>
+&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/6/6f/Cal-03.png" width="200" height="100"></div>
+<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>
-In this calculation, we assume that N, T, and V are constant,</br>
-<div align="center"><img src="http://openwetware.org/images/5/51/Cal-04.png" width="200" height="100"></div>
-α= 0 at the moment of N0 – N = 0, so simplify,</br>
-<div align="center"><img src="http://openwetware.org/images/6/6a/Cal-05.png" width="200" height="100"></div>
-We do not know the from of (pf)vesicle, so this time we assume,</br>
-<div align="center"><img src="http://openwetware.org/images/7/7c/Cal-06.png" width="200" height="100"></div>
-N/n=X means the number of the vesicle of the whole system. So we calculate free energy by using this,</br>
-<div align="center"><img src="http://openwetware.org/images/7/77/Cal-07.png" width="200" height="100"></div>
-Here, a vesicle which radius is 100μm changes to each 10 and 100 vesicles, calculate each free energy,</br>
-<div align="center"><img src="http://openwetware.org/images/6/6c/Cal-08.png" width="200" height="100"></div>
-From these results, we get the follow (fig 3).</br>
-From fig 3, free energy is smaller when the radius of vesicle is small.</br></br>
-・Consideration</br>
-　When all molecules participate in the formation of the vesicle, the free energy becomes a one-tenth time when the number of vesicle increase 10 times.  In addition, we think that the vesicle of the small radius is more stable.</br></br>
-</p>
-<br>
--->
-<!--計算式のコメントアウトここまで-->
-<br>
-<p>リポソームを割ることに必要な活性化エネルギーδを超えるために、私たちは以下の二つを考えた。</br>To exceed the activation energy to break liposomes, we propose the following two approaches.
-<br><ur>
-<li>i)膜を湾曲させるアプローチ</br>i)Bending membranes</li>
-<li>ii)フラワーミセルによるアプローチ</br>ii)Utilizing flower micelle</li></ur></p>
-<br>
-<h5>Ⅰ膜を湾曲させるアプローチ</br>ⅠBending membranes</h5><br>
-<Img Src="http://openwetware.org/images/d/d2/Bending-flow.png" Align="center" width="900px" ><br>
-<div align="center">Fig2 process of bending membranes</div><br>
-リポソームを割るため、私たちは生物が膜を湾曲させるメカニズムに着目した。膜の湾曲、すなわち不安定化を最大限に利用することが出来れば、膜の崩壊につながると考えたからである。膜を湾曲させるメカニズムには、以下の三つが提案されている。<br>
-To break 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>
-<br>
- <Img Src="http://openwetware.org/images/a/ae/Designfig2.png" Align="left" width="280px" height="400px">
-Aは、両親媒性基をもつ分子が細胞膜に挿入されることにより、膜が湾曲するというものである。脂質二重膜の内側の強い疎水性部分は、脂質両膜をくっつけて離さない性質をもっている。このため、両親媒性基が片方の膜内に入りこみ、その膜が広がると、もう片方の膜は、少ない表面積でも済むように、内側になるようつられて曲がる。
-<br>
-Bは、膜表面に付着した分子が固い足場となり、下の膜を変形したり、あらかじめ湾曲されていた膜を固定化(stabilize)するというものである。<br>
-Cは、片方の膜に脂質を群がらせることにより、脂質の量が両膜で不均等になることにより、膜が湾曲するというものである。
-<br>
-<br>
-<br>
-生体膜を湾曲させるタンパク質のほとんどは、A~Cのメカニズムを組み合わせて使っている。<br>
-また、近年、タンパク質同士が密集することで、膜が湾曲されるという考えも提唱されている(Membrane bending by protein-protein crowding). これは、膜結合タンパク質同士の衝突による、横方向の圧力により、膜が曲がるというものである。
-<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>
-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>
-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>
-<br>
-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>
-以上から、膜を不安定にさせるためには、<br>
-<ur><li>・固い足場となる</li>
-<li>・足場の影響を最大にするため、表面積が大きい</li>
-<li>・衝突により大きな圧力が生じる</li>
-構造が有効であると考えられる。
-<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>
-私たちは、固い足場となる分子を実現するために、DNAオリガミに着目した。DNAオリガミは任意の形に固い構造を作ることができるからである。
-そして、表面積の大きい構造として、平面構造を、
-<br>
-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>
-<!--
-<Img Src="http://openwetware.org/images/f/f9/Lipo2.png" Align="left">
-<br>
--->
 <br>
-衝突により横方向に最大の圧力が生ずるような構造として、長方形や三角形を考えた。<br>
+&nbsp;From the reference, we learned that efficient structure design for destabilizing membranes should have the following properties: <br>
-We also designed rectangle and triangle to make the pressure of the collision highest.<br>
+<ur><li>Having rigid scaffolds</li>
-<Img Src="http://openwetware.org/images/6/63/Outsidefig3.png" Align="left">
+<li>Having large surface areas to maximize the effect of the scaffold on the membrane</li></ur>
-<br>
-長方形はそれ自体で一つの足場として働き、三角形(球面をもっとも効率よく覆う図形)は沢山集合して一つの固い足場を作ればもっとも効率が良いと考えられる。<br>
-We suppose that rectangle and triangle structures are most effective for the following reasons. 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>
-長方形オリガミの設計は以下の様である。<br>
-The design of our rectangular DNA origami is as below.<br>
-<Img Src="http://openwetware.org/images/6/6e/Outsidefig4.png" Align="left">
- <Img Src="http://openwetware.org/images/a/a7/Lipo5.png" Align="right">
-<br>
-DNAオリガミは、縦67.6nm(26らせん)、横127nm(374塩基)の、長方形である。AFMでの観察時に裏表の区別が付けられるよう、右上で縦10らせん、横161塩基の長方形を切り取った形とした。設計はcaDNAno2で行った。<br>
-さらに、膜が不安定になるよう、このオリガミの中心部分のステイプル141本を、コレステロール付きDNAと結合させ(両親媒性基をもたせ)、膜に突き刺すことが出来るようにした。<br>
-つまり、このコレステロール修飾DNAは、リポソームと足場をつなぐ役割をするだけでなく、膜に突き刺さり、膜を不安定化する両親媒性分子としても働く。<br>
-We used caDNAno for our DNA origami design. The DNA origami has a rectangle shape of 67.6nm (26 helixes) by 127 nm (374 bases). 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. Besides, to destabilize the membrane by inserting this origami, we designed 141 staples at the center of the origami to hybridize with cc DNAs (in the rest of this document, referred to as ccDNA. It gives our origami amphipathicity) and enabled it to insert into the membrane. To sum up, the cc DNA not only connects DNA origami and liposomes but also inserts into the membrane and destabilizes it.<br>
-<Img Src="http://openwetware.org/images/c/c5/Outsidefig5.png" Align="right">
-<br>
-<br>
-<h5>Ⅱフラワーミセルによるアプローチ</br>ⅡUtilizing flower micelles</h5><br>
-<Img Src="http://openwetware.org/images/1/17/Designflowerflow.png" Align="center" width="900px" ><br>
-<div align="center">Fig2 process of flower miceles burst</div><br>
-<Img Src="http://openwetware.org/images/b/b2/Flower1.png" style="height:300px; width:425px; float:right;">
-リポソームを割るには、フラワーミセルという方法がある。これはミセルに隙間なくコポリマーによる輪を取り付け、その輪の温度による形状の変化によりミセルに負荷をかけ、割るというものである。<br>
-There is a method called flower micelles for breaking liposomes. In this method, we cover the surface of the micelles with many copolymer rings, heat and distort the rings, and produce pressure on the micelle and break them.<br>
+<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>
-今回はこのフラワーミセルの原理を応用しリポソームとＤＮＡによってリポソームを割ることを試みる。<br>
+<div class="caption-right">
-We tried to break liposomes by applying the basis of flower micelles.<br>
+ <Img Src="http://openwetware.org/images/a/a7/Lipo5.png" style="padding-left:10mm"><span>Fig.4 DNA origami designed by caDNAno</span>
+</div>
-<br>リポソームにコレステロール修飾されているDNA一本鎖ストランドと、これに相補なDNA一本鎖ストランドを加える。このＤＮＡは両端が相補に結合するように設計されているため、リポソーム表面でDNAのループが形成されるようになる。<br>
+&nbsp;We use <a href="http://cadnano.org/">caDNAno2</a> for our DNA origami design.
-First, we mixed cc DNAs, loop strands, and liposomes. The loop strand is designed to have two complementary parts to the cc DNAs at its both ends. So when it binds to the cc DNAs, it is expected to make a loop between its both ends. The complex of the cc DNAs and loop strand floats on the surface of the liposomes.<br>
+The size of DNA origami is 67.6nm (26 helixes) in width and 127 nm (374 bases) in height.
-<Img Src="http://openwetware.org/images/a/aa/Flower2.png">
+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>
-次にループを形成しているDNAに相補なトリガーストランドを加える。これとループDNAがハイブリタイゼーションし結合する。この際DNAが持続長以下の長さに設計してあるため、DNAはまっすぐに保とうとする。<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>
-Next, we added complementary trigger strand to the loop strand. The trigger strand hybridizes with the loop strand first, and then keeps straight, because we designed the trigger strand shorter than persistence length.<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>
-<img src="http://openwetware.org/images/0/03/Flower3.png">
+<div align="center">
-その際に生じる力でリポソームに負荷がかかり、リポソームが割れるはずである。<br>
+<Img Src="http://openwetware.org/images/3/3d/Flower-newfg.png" width="450px" height="350px" ></div><br>
-This process gives pressure on the liposome and breaks them.<br>
+<div class="caption">Fig.6 Liposome with Flower-anchor DNA</div>
-<Img Src="http://openwetware.org/images/3/3b/Flower4.png">
 <br>
-<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>
-リポソーム内部の溶液中にトリガーを入れておくことができるので、Ⅰ・Ⅱを使って、外側からリポソームを破壊し連鎖反応を引き起こすことも容易であると考えられる。<br>
+<div align="center">
-We consider if some triggers are kept inside the liposomes and the liposomal membrane is broken by the above Ⅰ and Ⅱ methods from the outside, it would be much easy to begin the chain reaction. <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>
-このシステムで使用するDNAの配列はDNAdesignを使って設計しました。<br>
+<Img Src="http://openwetware.org/images/1/17/Flor4.png"  width="70%" hight="800" ><br>
-プログラムのソースはこちら。ループの部分が40nt, 20nt,10ntのDNAを設計しました。<br>
+<div class="caption">Fig.8 How to disrupt a liposome</div>
-赤色の部分がリポソームから生えているコレステロール付きのDNAと相補になっています。青色の部分が相補的になっていてこれらがハイブリタイゼーションすることでリポソームに負荷がかかり、リポソームが割れます。<br>
+       </article>
-We designed the DNA sequence for this approach by <A Href="http://www.dna.caltech.edu/DNAdesign/">DNA design</A>, software for designing DNA sequences. <br>
+</section>
-We arranged three kinds of DNA strands that hybridize with the surface of liposomes via cc DNA. Each has 40nt, 20nt, and 10nt loop parts (shown below as blue parts). The blue parts are complementary to the blue trigger strands, and when they hybridize, they place some stress on the liposome and break it. <br>
- The red parts are for hybridizing with liposomes. They are complementary to the cc DNA on the surface of liposomes. The cc DNA is the same as that used in Ⅰ Approach by bending membrane (see <A Href="http://openwetware.org/wiki/Biomod/2013/Sendai/protocol">Protocol</A>). <br>
-<font size="-2">
-The sequence of cholesterol-conjugated DNA<br>
-<font color="red">CCAGAAGACG</font> -cholesterol<br>
-A loop is 40nt<br>
-<font color="red">CGTCTTCTGG</font>TTTTTTTTTT<font color="blue">GCGAACCACGGTTCCCAGCGTGACCTTCATGCTTAAGTTT</font><font color="red">CGTCTTCTGG</font><br>
-trigger Strand of coping in loop 40nt<br>
-<font color="blue">AAACTTAAGCATGAAGGTCACGCTGGGAACCGTGGTTCGC</font><br>
-A loop is 20nt<br>
-<font color="red">CGTCTTCTGG</font>TTTTTTTTTTTT<font color="blue">CATAACATGAGGCGCCGT</font><font color="red">CGTCTTCTGG</font><br>
-trigger Strand of coping in loop 20nt<br>
-<font color="blue">ACGGCGCCTCATGTTATGAA</font><br>
-A loop is 10nt<br>
-<font color="red">CGTCTTCTGG</font>TTTTTTTTTT<font color="blue">CTGTAACTAA</font><font color="red">CGTCTTCTGG</font><br>
-trigger Strand of coping in loop 10nt<br>
-<font color="blue">TTAGTTACAG</font><br>
-</font>
-        </article>
-<!--
-        <article data-title="B-Z">
-<h3>外側からリポソームを破壊するサブプロジェクト</h3><br>
-<h4 id="designsubproject3">ＢＺ班</h4><br>
-まずリポソームを割る方法としてフラワーミセルという方法がある。これはミセルに隙間なくコポリマーによる輪を取り付け、その輪の温度による形状の変化によりミセルに負荷をかけ、割るとゆうものである。</br>
-今回はこのフラワーミセルの原理を応用しリポソームとＤＮＡによってリポソームを割ることを試みる。</br>
-リポソームはDOPCで作った通常のものとDOPC、DPPC、cholesterolの三種類を混ぜ、相分離を形成しているものの二種類を用いる。</br>
-これはリポソーム表面の状態の違いによる収率の違いを調査するためである。</br>
-これらのリポソームにコレステロール修飾されているDNA一本鎖ストランドを加えて、表面にDNAを生やす。</br>
-そこに先程修飾したDNAに相補なDNA一本鎖ストランドを加える。このＤＮＡは両端が相補に結合するように設計されているためリポソーム表面でDNAのループが形成されるようになる。</br>
-次にループを形成しているDNAに相補なトリガーストランドを加える。これとループDNAハイブリタイゼーションし結合する。この際DNAが持続長以下の長さに設計してあるため、DNAはまっすぐに保とうとする。その際に生じる力でリポソームに負荷がかかり、リポソームが割れるはずである。</br></br>
-リポソーム内部の溶液中にトリガーストランドを入れておくことができるので、この系であれば連鎖反応を引き起こすことも容易であると考えられる。</br>
-</p>
-</article> -->
-    </section>
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