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</header>  
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<p class="sukima"> Design
</p>
  <section id="tabs">
<article data-title="Egg-type trigger">
<h3 id="designsubproject1">Egg-type initiator</h3></br>
<img src="http://openwetware.org/images/5/5a/Alginate-design-01.png" alt="example-tab2" border="0"></br>
<div align="center">
                    Fig1 process of Egg-type initiator</div><br>
<br>


Egg-type initiator consists of two layers.<br>
<div id="ttop">
<a href="#top" class="page_top" onfocus="this.blur();" onclick="scrollTo(0,0); return false;" title="Top"></a></div>


<h5>The first layer: “alginate gel membrane”</h5>
<section role="main">
The alginate gel membrane has a solution phase inside them, and resembles artificial salmon caviars (JINKOH-IKURA in Japanese).<br><br>
        <article>
        <h2>Design</h2>


<h5>The second layer: “temperature-sensitive liposomes”</h5>
<table id="toc" class="toc" summary="Contents"><tr><td><div id="toctitle"><h2>Contents</h2></div>
The temperature-sensitive liposomes contain PNIPAM lipids in their membrane. Temperature shift causing hydrophobicity change of the PNIPAM induces collapse of the liposomes (See “Characters of the PNIPAM molecular”). The temperature-sensitive liposomes encapsulate a chelate compound (EGTA) and DNAs.<br><br>  
<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>


The two layers realize a dual disruption system as follows (Fig1). 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.<br>
</ul>
The alginate gel membrane encapsulates many temperature-sensitive liposomes.
</li>
Please refer following URL.<br>
</ul>
http://www.sigmaaldrich.com/etc/medialib/docs/SAJ/Brochure/1/j_recipedds2.Par.0001.File.tmp/j_recipedds2.pdf<br>
</td></tr></table>
2.<br>
Warming the temperature-sensitive liposomes from room temperature to over 32 ºC causes disruption of the liposomes.<br>
3.<br>
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.<br>
<h2 id=chain>Project goal</h2>
As a result of the melting, trigger DNAs (DNA origami structure or DNA strands for the flower micelle approach (See design: chain-reactive burst)) are released from the melted alginate membrane.<br>
&nbsp;In Lipo-HANABI project, we need to develop the following two subsystems.<br><br>


<h5>An application to DNA origami formation using the dual disruption system</h5>
i) Sensing system (First stage): liposome disruption by temperature control. <br>
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 “DNA origami formation through urea dilution”).<br><br>


<h5>※Principle of this system</h5>
ii) Amplification system (Second stage): a chain-reactive disruption of the liposomes activated by the First stage. <br><br>
<h5>Alginate gels</h5>
Alginate is widely used in foods additives and drug stabilizers. Alginate becomes gel under certain concentrations of calcium, but lowering or chelating calcium melts alginate gels (alginate become sols under the condition).<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>


<h5>Characters of the PNIPAM molecular</h5>
<h4 id=5>DNA origami approach </h4>


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>
Reference<br>
http://www.sigmaaldrich.com/etc/medialib/docs/SAJ/Brochure/1/j_recipedds2.Par.0001.File.tmp/j_recipedds2.pdf<br>




<div align="center"><img src="http://openwetware.org/images/d/df/PNIPAM-function-02.png" width="600"></div></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>.  
Fig.6 Function of PNIPAM<br>
In this approach,　we use “Origami-anchor DNA” which connects DNA Origami with liposome membrane.
A schematic image how liposome containing PNIPAM disrupt at high temperature is shown.<br><br>


<h5>DNA origami formation through urea dilution</h5>
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>


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>
<div align="center">
 
<Img Src="http://openwetware.org/images/c/c5/%E8%86%9C%E3%80%80%E5%8F%8D%E7%99%BAdfhr.png">
</article>
</div>
 
<div class="caption">Fig.2 Stress on liposome membrane</div>
 
 
 
 
 
 
 
 
 
<article data-title="Chain-reactive burst">
 
<h3 id="chain">Chain-reactive burst</h3></br>
 
 
 
<br>
We designed chain-reactive burst systems as follows.Each liposome contains triggers, drugs, and has receptors of the trigger. When liposomes are destroyed, new triggers and drugs are released.To achieve liposome's burst by outside trigger, following two approaches are raised. <br>
リポソームが連鎖的に割れるようなシステムをデザインしました。リポソーム内部のtriggerは、リポソームが割れると放出され、周囲のリポソームを割ります。リポソームに外部からの負荷をかけて割ることを目的に、以下の2つのアプローチを考案しました。<br>
 
<br>
<li>i)膜を湾曲させるアプローチ<br><h5>i)A bending approach</h5></li>
<li>ii)フラワーミセルによるアプローチ</br><h5>ii)A flower micelle approach</h5></li>
<br>
<br>
At first, we considered a theory to disrupt liposomes by a trigger DNA signal. About the change of vesicles by destroyed, estimated the difference of free energies of vesicles by calculations about free energies of different kinds of size. And also discuss what kind of size that vesicles are more stable.<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>


Please see the details below link (Go to Calcultion pageリンクを張る).<br>
<Design of DNA origami><br>
このリポソームを割るときの理論的アプローチについて考察しました。ベシクルが壊れる変化について、大きさの違いによる自由エネルギーの値から、どのような大きさのときにより安定で、どのくらいのエネルギー差があるのかを計算により見積もった。詳細はCalculationのページへ。（リンクを張る）<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>


<h4>i)膜を湾曲させるアプローチ<br>i)A bending approach</h4><br>
<div align="center">
 
<Img Src="http://openwetware.org/images/4/45/Outsidefig8.png">
<Img Src="http://openwetware.org/images/d/d2/Bending-flow.png" Align="center" width="900px" ><br>
</div>
<div align="center">Fig2 process of bending membranes</div><br>
<div class="caption">Fig.3 Rectangular origami</div>
 
1.リポソーム表面にccDNAを付着させる。
Cholesterol modified DNA strands attaches surface of liposomes.<br>
2.相補的DNA鎖付きのDNAオリガミを加える。
Then, DNA origami with complementaly strand are added as triggers.<br>
3．表面にbindしたオリガミ構造体によって膜表面に負荷がかかる。(詳細は後述)
  Triggers bind surface of liposomes and get a load on membrane.<br>
4．負荷によって膜がburstする。
  By the load of trigger, liposome was destroyed.<br>
<br>
 
<h5>a)Mechanism of bending membranes</h5>
 
 
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>
<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>
<div class="caption-right">
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。
<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>
<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>
<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>
&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>
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。
&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>
<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>
&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>
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>
<div align="center">
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>
<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>
Most membrane bending proteins combine the above three mechanisms.<br>
<Img Src="http://openwetware.org/images/1/17/Flor4.png" width="70%" hight="800" ><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>
<div class="caption">Fig.8 How to disrupt a liposome</div>
<br>
      </article>
以上から、膜を不安定にさせるためには、<br>
</section>
<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>
 
<h5>b) Rigid scaffolds</h5>
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>
We also designed rectangle and triangle to make the pressure of the collision highest.<br>
<Img Src="http://openwetware.org/images/6/63/Outsidefig3.png" Align="left">
<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>
 
 
 
 
 
<h4>Ⅱフラワーミセルによるアプローチ</br>ii)Utilizing flower micelles</h4><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>
 
 
<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>
We tried to break liposomes by applying the basis of flower micelles.<br>
 
1.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>
1.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>
<Img Src="http://openwetware.org/images/a/aa/Flower2.png">
<br>
<br>
2.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。<br>
2.Next, we added complementary trigger strand to the loop strand. The trigger strand hybridizes with the loop strand.<br>
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>
And then strands keep straight, because we designed the trigger strand shorter than persistence length.<br>
<img src="http://openwetware.org/images/0/03/Flower3.png">
4.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>
4.This process gives pressure on the liposome and breaks them.<br>
<Img Src="http://openwetware.org/images/3/3b/Flower4.png">
<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>
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>
<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>
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>
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>
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>
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>
 
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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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