Biomod/2012/TeamSendaiA/Project

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<!—二段組み本文 -->
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<div id="Header">
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<h1><img src="http://openwetware.org/images/c/c2/Title-kai.png"></h1>
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<h1 align="center";><!--Team Sendai--><img src="http://openwetware.org/images/c/c2/Title-kai.png" ></h1>
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<p>Tohoku University</p>
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</div>
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<!—トップメニュー -->
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<h1>Abstract</h1>
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<div align="right">
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<ul id="Topmenu">
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<li><a href=" http://openwetware.org/wiki/Biomod/2012/Tohoku/Team_Sendai">Top</a></li>
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<li><a href=" http://openwetware.org/wiki/Biomod/2012/TeamSendaiA/Project">Project</a></li>
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<li><a href=" http://openwetware.org/wiki/Biomod/2012/TeamSendaiA/Simulation">Simulation</a></li>
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<li><a href=" http://openwetware.org/wiki/Biomod/2012/TeamSendaiA/Results_%26_Discussion">Experiment</a></li>
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<li><a href=" http://openwetware.org/wiki/BIOMOD/2012/Team_Sendai/Movie">Diary</a></li>
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<li><a href=" http://openwetware.org/wiki/Biomod/2012/TeamSendaiA/Member ">Team</a></li>
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</ul>
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</div>
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<!--目次 -->
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<div id="mokuji">
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<h2>Contents</h2>
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<ol>
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<li><a href="#Motivation">Motivation</a></li>
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<li><a href="#ProjectPlan">Project plan</a></li>
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<li><a href="#Selector">Selector</a></li>
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<li><a href="#Gate">Gate</a></li>
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<li><a href="#Membrane">Membrane</a></li>
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<li><a href="#Future">Future</a></li>
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</ol>
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<!-- コンテンツ -->
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<div id="Content">
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<!--
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<a name="Motivation"></a><h2>Motivation</h2>
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<p>
<p>
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現在チャネルの研究は進められているが、それらは単純な穴であり、選択性を持って物質交換を行うものは少ない。そこで我々は選択性をもったチャネルの開発に着手した。</br>
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All the creatures on Earth are made of cells. The exterior and the interior of the cell are compartmentalized by biomembranes.</br>
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我々が設計したチャネルはこうである。まず、穴としてDNAオリガミで作った六角柱を用意(Gate)。その内側に一本鎖DNAを数本並べておく。この一本鎖DNAをSelectorと名付けた。DNAはその相補性により、DNAはもとよりRNAやタンパク質などの様々な生体分子と相互作用を持ち、また簡単に配列を変更することが可能であるため、六角柱内に一本鎖DNAが並ぶ構造によって、汎用性を持った選択的なチャネルを作成することが可能である。Selectorの配列はチャネルの奥に行くほどターゲット分子との結合を強いように設計する。こうすることでターゲット分子は選別されながら、チャネルの出口へ向かって行くことができる。</br>
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A nanodevice that is able to actively transport only the specific oligonucleotide through the biomembrane has a great potential to deliver siRNA into the cell or to extract mRNA expressed in the cell.</br>
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また、我々はチャネルの取り付く細胞膜のモデルとしてリポソームを使う。
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Here, we decided to create a novel device with such a function. </br>
 +
We have designed <strong>a cylindrical injector/extractor device made of DNA origami</strong> (we named this device: <strong>CELL-GATE</strong>). Inside the cylinder, a cascade of single stranded DNAs is planted. Once the outer-most ssDNA binds to a target oligonucleotide, the target is passed to the inner-ones one by one because of the higher bonding energy assigned to the inner ones.</br>
 +
We also investigate how the cylinder penetrates the biomembrane by using liposome as a model membrane.</br>
</p>
</p>
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-->
 
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<a name="ProjectPlan"></a><h2>Project plan</h2>
 
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<p>
 
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Our project is divided large three parts, Selector, Gate and Liposome.</br>
 
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So we'll do each experiments abreast and finally we'll mix. </br>
 
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我々のプロジェクトは大きく三つに分けることができる。セレクター、チャネル、リポソームである。そこで我々はこの三つの実験を並行して行う。それぞれの実験がうまくいったところで組み合わせる。(D-Hertを分解した画像をこの下に欲しい)
 
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</p>
 
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<a name="Selector"></a><h2>Selector</h2>
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<div id="pop">
 +
<h2><!--Abstract-->Project Image</h2>
<p>
<p>
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Working Selector</br>
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<strong>Our PROJECT CELL-GATE is divided into three sub-projects:</strong><br>
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<div align="center">
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<a href="http://openwetware.org/wiki/Biomod/2012/TeamSendai/Idea#PORTER">PORTER</a>: Composed of single stranded DNA (ssDNA) sequences. Each ssDNA is called  Porter. Porters are designed to transfer target DNA strands into (or out from) the membrane.</br>
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<img src="http://openwetware.org/images/4/45/Suceed.gif" width="420px" height="300px"><br>
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<a href="http://openwetware.org/wiki/Biomod/2012/TeamSendai/Idea#GATE">GATE</a>: Cylindrical DNA nanostructure connecting the inside and outside of membrane like a channel. A cascade of the Porters is planted inside this cylinder. We can use this Gate as an injector or an extractor.</br>
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</div>
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<a href="http://openwetware.org/wiki/Biomod/2012/TeamSendai/Idea#MEMBRANE">MEMBRANE</a>: The exterior and the interior of the cell are compartmentalized by biomembranes. We use liposome as a model membrane.
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  Inside the gate, a cascade of three single stranded DNAs is planted. We named the DNAs Selector1, Selector2, and Selector3 from the outside of the gate. In addition, another Selector, which is called Selector4, is in the liposome.<br>
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[In the gate] Selector1and 2 have complementary sequences to a target oligonucleotide here and there and consecutive adenine sequences in other portion. We made an attempt to capture a target distant from the gate with high specificity. So we lay out a long Selector1. By catching a target and making loops, it can shrink and go in the gate.<br>
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  Selector3 is complementary to the target, but it is shorter than the target. When it binds to the target, the upper end of the target makes a toehold.<br>
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  We designed the inner Selector has higher bonding energy. So once the outer-most ssDNA binds to a target, the target is passed to the inner-ones one by one.<br>
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[In liposome]  Selector4 is perfectly complementary to the target. After the target reaches Selector3, Selector4 conveys the target into liposome.<br>
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<br>
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<table style="clear:right;width:650px;border-style:solid;border-width:2px;margin:0 auto">
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<tr>
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<th style="width:100px;">Name</th><th style="width:450px;">Sequence(5' to 3')</th><th style="width:100px;">Tm(°C)</th>
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</tr>
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<tr>
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<td style="border-style:solid;border-width:1px;">target</td><td style="border-style:solid;border-width:1px;">* - ACTAG<font color="green">TGAG</font><font color="orange">TGCAGCAGTCGTACCA</font></td><td style="border-style:solid;border-width:1px;"></td>
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</tr>
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<tr>
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<td style="border-style:solid;border-width:1px;">Selector1</td><td style="border-style:solid;border-width:1px;">AAAAAAAAAAAAAAAAA<font color="red">TGGTAC</font>AAAAAAAA<font color="red">GACTG</font>AAAAAAAA<font color="red">CTGCA</font></td><td style="border-style:solid;border-width:1px;">30.6</td>
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</tr>
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<tr>
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<td style="border-style:solid;border-width:1px;">Selector2</td><td style="border-style:solid;border-width:1px;">AAAAAAAAAAA<font color="red">TGGTAC</font>AAAA<font color="red">GCTGCA</font></td><td style="border-style:solid;border-width:1px;">36.5</td>
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</tr>
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<tr>
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<td style="border-style:solid;border-width:1px;">Selector3</td><td style="border-style:solid;border-width:1px;"><font color="red">TGGTACGACTGCTGCA</font></td><td style="border-style:solid;border-width:1px;">62.3</td>
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</tr>
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<tr>
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<td style="border-style:solid;border-width:1px;">Selector4</td><td style="border-style:solid;border-width:1px;"><font color="red">TGGTACGACTGCTGCA<font color="blue">CTCA</font></font></td><td style="border-style:solid;border-width:1px;">68.0</td>
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</tr>
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</table>
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<br>
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*Red-orange and blue-green regions are complementary DNA sequences.<br>
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*Black region is added to differ the molecular weight of each sample(for distinguishing them during electrophoresis).<br>
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<br>
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<a href=" http://openwetware.org/wiki/Biomod/2012/TeamSendaiA/Results_%26_Discussion#Selector">Experiment page</a>
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</p>
</p>
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<img src="http://openwetware.org/images/c/c5/Projectcellgate.png" alt="D-Heart" width="440px" usemap="#D-Heart">
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<map name="D-Heart">
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<area shape="rect" coords="310,8,390,45" href="http://openwetware.org/wiki/Biomod/2012/TeamSendai/Idea#PORTER" alt="Porter">
 +
<area shape="rect" coords="310,95,370,132" href="http://openwetware.org/wiki/Biomod/2012/TeamSendai/Idea#GATE" alt="Gate">
 +
<area shape="rect" coords="33,213,165,245" href="http://openwetware.org/wiki/Biomod/2012/TeamSendai/Idea#MEMBRANE" alt="Membarane">
 +
</map>
 +
Please CLICK ITEMS, GATE, PORTER, AND MEMBRANE in the picture!
 +
</div>
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<!--Gateコンテンツ-->
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<div id="pop">
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<h2>Movie</h2>
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<a name="Gate"></a><h2>Gate</h2>
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<font size="4">
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<a name="Strategy"></a><h3>Strategy</h3>
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</font>
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<p>
<p>
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Gate should be able to transport the target with selector inside gate and go through cell membrane.
 
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To transport the target with selector, we decided to make hexagonal tube as gate.
 
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The reasons we adopted hexagonal tube as gate are that surfaces of hexagonal tube are suitable for being attached selector,
 
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high strength of honeycomb structure are easy to be observed. To go through cell membrane, we placed the staple attached lipid on center of gate.</br>
 
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We expect gate is introduced liposome simultaneously with creation of liposome.
 
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In addition, we attached edge of the gate to adenine staple like a "mustache" to make easy watching by AFM and interrupt other DNA approaching.
 
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We think because of our selector 1 is enough long, only target is transported into the gate. </br>
 
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In addition to this, we made the cholesterol hexagonal tube. The reason that designed this tube is coupling into a liposome film using a characteristic of the cholesterol like a lipid. (cf.Figure 2.3 )
 
</br>
</br>
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<font size="4">
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<iframe width="420" height="315" src="http://www.youtube.com/embed/8OB8ytltxwY" frameborder="0" allowfullscreen></iframe>
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<h3>Structure image</h3>
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</font>
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<p>
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This is the hexagonal tube design by caDNAno using honeycomb structure.</br>
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<br>
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<img src="http://openwetware.org/images/9/91/Cadnano3D.gif" width="315px" height="405px" alt="Structure image"/>
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</br>
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<p>
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We made 3shape’s hexagonal tube.</br>
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1: Mere hexagonal tube</br>
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2: Hexagonal tube with the adenine at the entrance</br>
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3: The cholesterol hexagonal tube</br>
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<img src="http://openwetware.org/images/c/c4/スクリーンショット_2012-10-15_1.11.25.png" width="474px" height="351px"/ >
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<img src="http://openwetware.org/images/0/0b/スクリーンショット_2012-10-15_1.11.35.png" width="471px" height="356px" align="right"/ >
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<img src="http://openwetware.org/images/5/5e/スクリーンショット_2012-10-15_1.22.38.png" width="456px" height="351px"/ >
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</br>
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<a href=" http://openwetware.org/wiki/Biomod/2012/TeamSendaiA/Results_%26_Discussion#Gate">
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</br>
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Experiment page</a>
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</p>
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<a name="Membrane"></a><h2>Membrane</h2>
 
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<p>
 
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We use liposomes as a model of cell membrane. To insert cell-gate into the</br>
 
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liposome, we stretched out ssDNA of 10 nt from the side of the hexagonal tube. Then,</br>
 
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we extend the complementary ssDNA, and modified the cholesterol at the end of them.</br>
 
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We choose the cholesterol because cholesterol is strongly hydrophobic. We expect</br>
 
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that cholesterol penetrate into the hydrophobic portion of the liposome. We confirmed</br>
 
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the tube modifying the cholesterol by electrophoresis. We use fluorescein to confirm</br>
 
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that the tube insert into the liposome correctly.</br>
 
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(細胞膜のモデルとして、リポソームを使用する。リポソームにセルゲートを刺さるように</br>
 
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するために、六角形筒の横から10塩基のシングルストランドDNAを伸ばした。さらに、</br>
 
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相補的なシングルストランドDNAを伸ばして、それらの末端にコレステロール修飾を行</br>
 
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った。コレステロールを修飾したのは、コレステロールが強い疎水性であるからである。</br>
 
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コレステロールがリポソームの疎水性部分に入り込むことで、筒がリポソームに刺さりや</br>
 
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すくなると考えた。コレステロール修飾は電気泳動で確かめた。筒がリポソームに刺さ</br>
 
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っているかを確かめる方法として、蛍光分子を利用する。)</br>
 
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<a href=" http://openwetware.org/wiki/Biomod/2012/TeamSendaiA/Results_%26_Discussion#Membrane">Experiment page</a>
 
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<a name="Future"></a><h2>Future</h2>
 
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Single stranded DNA can bind not only to DNA, also to various biomolecules like RNA and proteins.
 
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By changing Selector DNA sequences in the gate, we’ll capture biomolecules just as we want to.<br>
 
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In the future, we expect to make the molecular robot which can transport the object to inside the cell and collect the object from the cell using Cell-gate.</br>
 
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And now we use liposome as a model of the cell.</br>
 
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We think that by using liposome not only as a model of the cell but a bag to put something into,</br>
 
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we can make organelles to collect unnecessary object and release medicine.</br>
 
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将来的にはこのセルゲートを使うことで細胞内への物質の運搬、細胞内からの物質の回収ができるロボットの作製を目指している。</br>
 
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また、実験を行うにあたって細胞のモデルとしてリポソームを現在用いているが、単にモデルとしてリポソームを用いるだけでなくこのリポソームでできた袋を蓄積場として、</br>
 
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不要な物質の回収や、優良物質の散布ができる小器官のようなものを作製し細胞内で働かせるといった応用も考えられる。</br>
 
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Current revision

Team Sendai Top

Abstract

All the creatures on Earth are made of cells. The exterior and the interior of the cell are compartmentalized by biomembranes.
A nanodevice that is able to actively transport only the specific oligonucleotide through the biomembrane has a great potential to deliver siRNA into the cell or to extract mRNA expressed in the cell.
Here, we decided to create a novel device with such a function.
We have designed a cylindrical injector/extractor device made of DNA origami (we named this device: CELL-GATE). Inside the cylinder, a cascade of single stranded DNAs is planted. Once the outer-most ssDNA binds to a target oligonucleotide, the target is passed to the inner-ones one by one because of the higher bonding energy assigned to the inner ones.
We also investigate how the cylinder penetrates the biomembrane by using liposome as a model membrane.

Project Image

Our PROJECT CELL-GATE is divided into three sub-projects:
PORTER: Composed of single stranded DNA (ssDNA) sequences. Each ssDNA is called Porter. Porters are designed to transfer target DNA strands into (or out from) the membrane.
GATE: Cylindrical DNA nanostructure connecting the inside and outside of membrane like a channel. A cascade of the Porters is planted inside this cylinder. We can use this Gate as an injector or an extractor.
MEMBRANE: The exterior and the interior of the cell are compartmentalized by biomembranes. We use liposome as a model membrane.

D-Heart Porter Gate Membarane Please CLICK ITEMS, GATE, PORTER, AND MEMBRANE in the picture!

Movie



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