Biomod/2012/TeamSendai/Design: Difference between revisions

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<li><a href="http://openwetware.org/wiki/Biomod/2012/TeamSendai/FinalTop#">Top</a></li>
<li><a href="http://openwetware.org/wiki/Biomod/2012/Tohoku/Team_Sendai">Top</a></li>
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Revision as of 01:11, 20 October 2012

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<ul id="menu"> <li><a href="http://openwetware.org/wiki/Biomod/2012/Tohoku/Team_Sendai">Top</a></li> <li> <a href="#">Project</a> <ul> <li> <a href=" http://openwetware.org/wiki/Biomod/2012/TeamSendai/Idea">Idea</a> </li> <li> <a href=" http://openwetware.org/wiki/Biomod/2012/TeamSendai/Simulation">Simulation</a> </li> <li> <a href=" http://openwetware.org/wiki/Biomod/2012/TeamSendai/Design">Design</a> </li> </ul> </li> <li> <a href="#">Experiment</a> <ul> <li> <a href=" http://openwetware.org/wiki/Biomod/2012/TeamSendai/Result">Result</a> <ul> <li> <a href=" http://openwetware.org/wiki/Biomod/2012/TeamSendai/Result#Porter">Porter</a> <li> <a href=" http://openwetware.org/wiki/Biomod/2012/TeamSendai/Result#Cylinder">Cylinder</a> </li> <li> <a href=" http://openwetware.org/wiki/Biomod/2012/TeamSendai/Result# Vesicle">Vesicle</a> </li> </ul> <li><a href=" http://openwetware.org/wiki/Biomod/2012/TeamSendai/Method">Method</a> </li> </ul> </li> <li> <a href=" http://openwetware.org/wiki/Biomod/2012/TeamSendai/Diary">Diary</a> </li> <li> <a href=" http://openwetware.org/wiki/Biomod/2012/TeamSendai/Team ">Team</a> </li> </ul>

<!-- コンテンツ --> <div id="Content"> <!-- <a name="Motivation"></a><h2>Motivation</h2> <p> 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> 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> 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。 </p> --> <a name="ProjectPlan"></a><h2>Project plan</h2> <p> Our project is divided large three parts, Selector, Gate and Liposome.</br> So we'll do each experiments abreast and finally we'll mix. </br>

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） </p>

<a name="Selector"></a><h2>Selector</h2> <p> Working Selector</br> <div align="center"> <img src="http://openwetware.org/images/4/45/Suceed.gif" width="420px" height="300px"><br> </div> 

 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>

[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> 

 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>
 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>

[In liposome] Selector4 is perfectly complementary to the target. After the target reaches Selector3, Selector4 conveys the target into liposome.<br> <br> <table style="clear:right;width:650px;border-style:solid;border-width:2px;margin:0 auto"> <tr> <th style="width:100px;">Name</th><th style="width:450px;">Sequence(5' to 3')</th><th style="width:100px;">Tm(°C)</th> </tr> <tr> <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> </tr> <tr> <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> </tr> <tr> <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> </tr> <tr> <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> </tr> <tr> <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> </tr> </table> <br>

  • Red-orange and blue-green regions are complementary DNA sequences.<br>
  • Black region is added to differ the molecular weight of each sample(for distinguishing them during electrophoresis).<br>

<br> <a href=" http://openwetware.org/wiki/Biomod/2012/TeamSendaiA/Results_%26_Discussion#Selector">Experiment page</a>

</p>


<!--Gateコンテンツ-->



<a name="Gate"></a><h2>Gate</h2> <font size="4"> <a name="Strategy"></a><h3>Strategy</h3> </font> <p> 


Gate should be able to transport the target with selector inside gate and go through cell membrane.

To transport the target with selector, we decided to make hexagonal tube as gate. The reasons we adopted hexagonal tube as gate are that surfaces of hexagonal tube are suitable for being attached selector, 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> We expect gate is introduced liposome simultaneously with creation of liposome. 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. We think because of our selector 1 is enough long, only target is transported into the gate. </br> 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> <font size="4"> <h3>Structure image</h3> </font> <p>

This is the hexagonal tube design by caDNAno using honeycomb structure.</br> <br>

<img src="http://openwetware.org/images/9/91/Cadnano3D.gif" width="315px" height="405px" alt="Structure image"/> </br> <p> We made 3shape’s hexagonal tube.</br> 1: Mere hexagonal tube</br> 2: Hexagonal tube with the adenine at the entrance</br> 3: The cholesterol hexagonal tube</br>

<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"/ >

<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"/ > <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"/ > </br>

<a href=" http://openwetware.org/wiki/Biomod/2012/TeamSendaiA/Results_%26_Discussion#Gate"> </br> Experiment page</a> </font> </p>


</br>









<a name="Membrane"></a><h2>Membrane</h2> <p> We use liposomes as a model of cell membrane. To insert cell-gate into the</br> liposome, we stretched out ssDNA of 10 nt from the side of the hexagonal tube. Then,</br> we extend the complementary ssDNA, and modified the cholesterol at the end of them.</br> We choose the cholesterol because cholesterol is strongly hydrophobic. We expect</br> that cholesterol penetrate into the hydrophobic portion of the liposome. We confirmed</br> the tube modifying the cholesterol by electrophoresis. We use fluorescein to confirm</br> that the tube insert into the liposome correctly.</br> （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> 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> 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> 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> 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> 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> 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> <a href=" http://openwetware.org/wiki/Biomod/2012/TeamSendaiA/Results_%26_Discussion#Membrane">Experiment page</a> </p>




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