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            <a href="http://openwetware.org/wiki/Biomod/2013/Sendai"><h1 style="color:white;" ><b>Biomod<span>2013<br>&emsp; Team</span>Sendai</b></h1></a>

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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">1</span> <span class="toctext">Our Target(Lipo HANABI)</span></a></li> <li class="toclevel-1"><a href="#bending"> <span class="tocnumber">2</span> <span class="toctext">How to break</span></a></li> <ul> <li class="toclevel-2"><a href="#Flower"> <span class="tocnumber">2-1</span> <span class="toctext">step1 温度感受性リポソームの破壊</span></a></li> <li class="toclevel-2"><a href="#sensing"> <span class="tocnumber">2-2</span> <span class="toctext">step2 DNAによる連鎖的リポソームの破壊</span></a></li> <ul> <li class="toclevel-2"><a href="#5"> <span class="tocnumber">2-2-1</span> <span class="toctext">DNAオリガミによるアプローチ</span></a></li> <li class="toclevel-2"><a href="#6"> <span class="tocnumber">2-2-2</span> <span class="toctext">anchored DNAによるアプローチ</span></a></li> </li>

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

<h3 id=chain>Project goal</h3> To realize Lipo-HANABI, following two systems is needed. Our goal of this summer project is achieving these subgoals.<br>

i) To make a initiation system: liposomes disruption as a result of sensing thier environment<br> ii) To make a chain-reactive disruption system: this system need followng two subsystems. <br> ii-a)Liposomes dispution by attaching key DNA and anchor DNA<br> ii-b)Selective disruption by key DNA species<br> ii-c)Chain reactive disruption by a released key DNA<br>

<h4 id=Flower>First stage: Initiation by sensing environment</h4> <h5>Temperature sensitive liposomes</h5> To make temperature-sensitive liposomes, we used lipids conjugated with NIPAM polymer. NIPAM is hydrophlic at room temperatures, but switches to hydrophobic over 32 dC. Hydrohpobic NIPAM shrink to avoid water environment. This structure changes of NIPAM make a stress on surface tension of liposomes, and consequently disrupt liposomes.<br> <h4 id=sensing>Second stage: Amplification by chain-reactive burst of liposomes</h4> Second liposomes have two DNAs. One is "anchored DNA" (DNA with cholesterol) on the outside surface of liposomes. Another is "key DNA" inside the liposome. Liposome disruption is induced by attachment of key DNA with anchor DNA as follwoing mechanisms.

<h4 id=sensing>Liposome disruption induced by attachment of key DNA with anchor DNA </h4> <h5 id=5>DNA origami approach</h5> このアプローチは「膜タンパクでリポを割る」論文を参考にして設計したものである（リンク）<br> This approach refers a paper about<a Href="http://www.ncbi.nlm.nih.gov/pubmed/19780639"> Membrane-bending proteins</a> In this approach, a lot of DNA origamis are adsorbed on surface of liposomes using “Origami anchor DNA”, which is 10 nt DNA modified with cholesterol molecule at 3’. As a result, liposome surface gets bending stress. Finally, liposomes burst.

Hybridize When DNA origamis are on surface of liposomes, the curvature of liposomes changes by electric repulsion. We conducted a calculation（リンク）about this phenomenon. Also, electric repulsion between hybridized DNA origamis destabilized surface of liposomes. Thus, they burst.

The above reference paper, "Membrane-bending proteins", says the efficient structure design for destabilizing membranes meets the following conditions :

<ur> <li>Having rigid scaffolds</li> <li>Having large surface areas to maximize the effect of the scaffold on the membrane</li> <li>Producing a large pressure by collisions</li> </ur> <br> DNA origami is known as a designable rigid structure. Therefore, we use DNA origami in order to make rigid scaffolds. Moreover, surface area of the DNA origami is larger. Thus, in order to meet these conditions, we designed rectangle DNA origami. <br> <Img Src="http://openwetware.org/images/4/49/%E3%83%AA%E3%83%9D%E3%81%A1%E3%82%83%E3%82%93%EF%BC%91%EF%BC%92.png"> <div align="center">Fig.3 Rectangle origami</div><br>

＜Design of DNA origami＞ <br> We expect the rectangle DNA origami to work as one scaffold in itself. Following is the design of our rectangular DNA origami.<br> <Img Src="http://openwetware.org/images/4/45/Outsidefig8.png"> <div align="center">Fig.4 Rectangular origami</div> <br> <div class="caption-right">

<Img Src="http://openwetware.org/images/a/a7/Lipo5.png" ><span>Fig.5 DNA origami designed by caDNAno</span>

</div> We used caDNAno2 for our DNA origami design.<br> 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. <br> Besides, to destabilize the membrane by inserting this origami, we designed 141 staples at the center of the origami to hybridize with anchors (These anchors give our origami amphipathicity), and enabled it to insert into the membrane. <br> To sum up, the anchor not only connects DNA origami and liposomes but also inserts into the membrane and destabilizes it. <br> <Img Src="http://openwetware.org/images/3/37/%E3%83%AA%E3%83%9D%E3%81%A1%E3%82%83%E3%82%93%EF%BC%93.png"> <div align="center">Fig.6 Unstable liposome</div> <br> <h5 id=6>Flower DNAによるアプローチ</h5> このアプローチは「高分子フラワーミセル」の論文をDNAに応用したものである。

このアプローチでは１０ntとそれと一部が相補になっている５０nt3’コレ付きDNAがハイブリしたものをFlower アンカーDNAと呼ぶ。Flower アンカーDNAの一本鎖になっている４０ntの部分に鍵DNAが相補になっており、ハイブリダイゼーションによって持続長が長くなったフラワーDNAはリポソーム膜面に「引っ張り（引き裂き）」ストレスを与え、リポソームを壊すのである。 <Img Src="http://openwetware.org/images/8/8b/Flower-new.png" Align="center" ><br> <Img Src="http://openwetware.org/images/6/69/%E3%83%AA%E3%83%9D%E3%81%A1%E3%82%83%E3%82%93%EF%BC%92.png" Align="center" width="900px" hight="800" ><br> <div align="center">Fig.10 How to straighten loop</div>

<div > <Img Src="http://openwetware.org/images/e/ed/%E3%83%AA%E3%83%9D%E3%81%A1%E3%82%83%E3%82%93%E2%91%A5.png" style="width:425px;"><br> <span>Fig.8 Flower micelle method</span></div>

We tried to collapse liposomes by applying the mechanism of flower micelles.<br> フラワーミセルをリポソームに応用するためには、<br> ・多くのコレ付きDNAを表面に埋め込むこと<br> ・鍵DNAのハイブリによってDNAの性質が変化すること<br> といった要素が重要になってくる。<br><br> 以下の図のように持続長の変化によって変形するDNAを設計した

<img src="http://openwetware.org/images/0/03/Flower3.png"><br> <div align="center">Fig.7 Process of flower micelle approach</div><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> <br> 私たちはこのソフトでFlower DNAの二本とKey DNA の3種類のDNAを設計しました。

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@@ Line 72: / Line 72: @@
 </td></tr></table>
-<h3 id=chain>2 stage of Lipo-HANABI</h3>
+<h3 id=chain>Project goal</h3>
-Lipo-HANABI is driven by the following two stage mechanism. <br><br>
+To realize Lipo-HANABI, following two systems is needed. Our goal of this summer project is achieving these subgoals.<br>
-st stage (initiation by the triggering signal): A temperature-sensitive liposome as an initiator. We chose temperature as an environmental initiator because of controllability. Increasing temperature is the trigger to collapse initiator liposome and releases key DNA that is designed for collapsing 2nd stage liposomes<br><br>
+i) To make a initiation system: liposomes disruption as a result of sensing thier environment<br>
+ii) To make a chain-reactive disruption system: this system need followng two subsystems. <br>
+ii-a)Liposomes dispution by attaching key DNA and anchor DNA<br>
+ii-b)Selective disruption by key DNA species<br>
+ii-c)Chain reactive disruption by a released key DNA<br>
-nd stage: ２段階目ではリポソームの連鎖反応が起きる。In 2nd stage, a chain reactive liposomal destruction happens. The 2nd stage liposomes contain payload molecules and also the key DNA. ひとつのリポソームが割れると中に入っていたかぎDNAで次々にリポソームが割れるOnce a liposome collapse, neighbor liposomes collapse one after another.<br>
+<h4 id=Flower>First stage: Initiation by sensing environment</h4>
-As a consequence, collapse of the liposomes goes on in a chain-reactive way. <br><br>
+<h5>Temperature sensitive liposomes</h5>
+To make temperature-sensitive liposomes, we used lipids conjugated with NIPAM polymer. NIPAM is hydrophlic at room temperatures, but switches to hydrophobic over 32 dC. Hydrohpobic NIPAM shrink to avoid water environment. This structure changes of NIPAM make a stress on surface tension of liposomes, and consequently disrupt liposomes.<br>
-An advantage of using the two-stage strategy is that we can change the 1st stage to deal with various types of trigger signal, without changing the 2nd stage. <br><br>
+<h4 id=sensing>Second stage: Amplification by chain-reactive burst of liposomes</h4>
+Second liposomes have two DNAs. One is "anchored DNA" (DNA with cholesterol) on the outside surface of liposomes. Another is "key DNA" inside the liposome. Liposome disruption is induced by attachment of key DNA with anchor DNA as follwoing mechanisms.
-<h3 id=bending>２段階それぞれにおけるリポソーム破壊の方法の説明</h3>
-<h4 id=Flower>１段階目　温度感受性リポソームによる反応の開始</h4>
-１段階目で反応の着火剤として使うリポソームは温度感受性リポソームと言って、リポソームにNIPAMというポリマーを修飾したものである。
-st stage liposome is temperature-sensitive liposome; conjugated by polymer NIPAM. <br>
-NIPAMとは３２度を境に親水から疎水に切り替わる性質をもつポリマーだ<br>
-NIPAM switches to hydrophobic on the border of 32 Celsius.
-３２度より高くなると縮んで疎水性になりリポ表面が不安定化する<br>
-NIPAM shrinks and make the surface of liposome unstable more than 32 Celsius.
-その結果リポが割れる<br>
-As a consequence, liposome collapses.
-<h4 id=sensing>２段階目 DNAによる連鎖的リポソームの破壊 </h4>
-２ndステージのリポソームは外側の表面にアンカーDNA（コレステロール付きのDNA）が生え、内部には鍵DNAとペイロードがある。1stステージのイニシエーターから放出された鍵DNAと２ndステージのリポソームのアンカーDNAが結合することでリポソームは割れる。その時に放出される次の鍵DNAが別のアンカーDNAを持つリポソームに結合し、破壊する。この繰り返しにより連鎖的に割る。
-（アンカーDNAと鍵DNAはリポソーム破壊を誘発するため、）アンカーDNAが内部に存在する場合、内部の鍵DNAにより破壊されてしまう。これを避けるためには、アンカーDNAは外側だけに存在している必要がある。私たちのプロジェクトではリポソーム作製後、コレステロール修飾したアンカーDNAをリポソームに振り掛けることでこれを達成する。
-The 2nd stage liposomes have anchored DNA (DNA with cholesterol) on the outside surface and also have key DNA and payload in the interior. When key DNA released from the 1st stage initiators and anchored DNA on the 2nd stage liposomes
+<h4 id=sensing>Liposome disruption induced by attachment of key DNA with anchor DNA </h4>
+<h5 id=5>DNA origami approach</h5>
+このアプローチは「膜タンパクでリポを割る」論文を参考にして設計したものである（リンク）<br>
+This approach refers a paper about<a Href="http://www.ncbi.nlm.nih.gov/pubmed/19780639"> Membrane-bending proteins</a>
+In this approach, a lot of DNA origamis are adsorbed on surface of liposomes using “Origami anchor DNA”, which is 10 nt DNA modified with cholesterol molecule at 3’. As a result, liposome surface gets bending stress. Finally, liposomes burst.
+Hybridize When DNA origamis are on surface of liposomes, the curvature of liposomes changes by electric repulsion. We conducted a calculation（リンク）about this phenomenon. Also, electric repulsion between hybridized DNA origamis destabilized surface of liposomes. Thus, they burst.
-<br><br>
+The above reference paper, "Membrane-bending proteins", says the efficient structure design for destabilizing membranes meets the following conditions :
-（図を入れる）
-To achieve liposomal burst by outside triggers, we propose the following two approaches.<br>
-リポソームを割るには、活性化エネルギーを越えための触媒が必要である(See 計算)
-我々のプロジェクトでは、この触媒を鍵DNAで行う。<br>鍵DNAとして（１）DNAオリガミを使う方法と（２）フラワーDNAを使う方法の２つを考案した<br>
-２つのアプローチで使用するアンカーDNAは違うもので、それぞれオリガミアンカーDNA、フラワーアンカーDNAと呼ぶ。
-<h5 id=5>DNAオリガミアプローチ</h5>
-このアプローチは「膜タンパクでリポを割る」論文を参考にして設計したものである（リンク）<br>
-This approach refers a paper about<a Href="http://www.ncbi.nlm.nih.gov/pubmed/19780639"> Membrane-bending proteins</a>
-このアプローチではオリガミアンカーDNA（１０nt3’コレ修飾DNA）を通してリポソーム表面に大量のDNAオリガミを吸着させ、リポソーム膜面に「曲げ」ストレスを与えることにより、リポソームを壊す。<br><br>
-Hybridize
-リポソーム表面にオリガミがハイブリすることでリポソーム表面がまっすぐになろうとする。これについては計算を行った（リンク）また、ハイブリしたDNAオリガミ同士の反発でさらにリポソーム表面が不安定化しリポソームが割れる。
-前述の膜タンパクの論文からthe efficient design for destabilizing membranes is the structures that :<br>
 <ur>
-<li>have rigid scaffolds</li>
+<li>Having rigid scaffolds</li>
-<li>have large surface areas to maximize the effect of the scaffold on the membrane</li>
+<li>Having large surface areas to maximize the effect of the scaffold on the membrane</li>
-<li>produce a large pressure by collisions</li>
+<li>Producing a large pressure by collisions</li>
 </ur>
 <br>
+DNA origami is known as a designable rigid structure. Therefore, we use DNA origami in order to make rigid scaffolds. Moreover, surface area of the DNA origami is larger. Thus, in order to meet these conditions, we designed rectangle DNA origami.
-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>
-そこで
-We designed rectangle origami to make the pressure of the collision highest.<br>
 <Img Src="http://openwetware.org/images/4/49/%E3%83%AA%E3%83%9D%E3%81%A1%E3%82%83%E3%82%93%EF%BC%91%EF%BC%92.png">
 <div align="center">Fig.3 Rectangle origami</div><br>
-＜オリガミの設計＞
-長方形のオリガミはexpected to work as one scaffold in itself<br>
-The design of our rectangular DNA origami is as below.<br>
+＜Design of DNA origami＞ <br>
+We expect the rectangle DNA origami to work as one scaffold in itself. Following is the design of our rectangular DNA origami.<br>
 <Img Src="http://openwetware.org/images/4/45/Outsidefig8.png">
 <div align="center">Fig.4 Rectangular origami</div>
@@ Line 145: / Line 118: @@
   <Img Src="http://openwetware.org/images/a/a7/Lipo5.png" ><span>Fig.5 DNA origami designed by caDNAno</span>
 </div>
-We used <A Href="http://cadnano.org/">caDNAno2</A> for our DNA origami design.<br>
+We used caDNAno2 for our DNA origami design.<br>
-The DNA origami has a rectangle shape of 67.6nm (26 helixes) by 127 nm (374 bases).<br>
+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.<br>
+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>
-Besides, to destabilize the membrane by inserting this origami, we designed 141 staples at the center of the origami to hybridize with anchors (These anchors give our origami amphipathicity), and enabled it to insert into the membrane.
+Besides, to destabilize the membrane by inserting this origami, we designed 141 staples at the center of the origami to hybridize with anchors (These anchors give our origami amphipathicity), and enabled it to insert into the membrane. <br>
-<br>
+To sum up, the anchor not only connects DNA origami and liposomes but also inserts into the membrane and destabilizes it. <br>
-<div class="c-both"></div>
-To sum up, the anchor not only connects DNA origami and liposomes but also inserts into the membrane and destabilizes it.<br>
 <Img Src="http://openwetware.org/images/3/37/%E3%83%AA%E3%83%9D%E3%81%A1%E3%82%83%E3%82%93%EF%BC%93.png">
 <div align="center">Fig.6 Unstable liposome</div>