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

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<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=""> 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=""> <div align="center">Fig.1 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=""> <div>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Fig.2 Rectangular origami</div> <br> <div class="caption-right">

<Img Src="" ><span>Fig.3 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=""> <div>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Fig.4 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="" Align="center" ><br> <Img Src="" Align="center" width="900px" hight="800" ><br> <div align="center">Fig.5 How to straighten loop</div>

<div > <Img Src="" style="width:425px;"><br> <span>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Fig.6 Flower micelle method</span></div> <br>

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=""><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="">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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