Biomod/2013/Sendai/design: Difference between revisions

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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>  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>  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  the keys. To make the liposome, we used lipids conjugated with NIPAM polymer.<br>  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>  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>  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>

 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>  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>  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>  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>  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>  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>  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>  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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Biomod/2013/Sendai/design: Difference between revisions

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+            <a href="http://openwetware.org/wiki/Biomod/2013/Sendai/calcuation" class="whiteSendai">Calculation</a>
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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>
-    </header>
+</header>
-    <section id="tabs">
-      <article data-title="Design">
-            <h2>Design</h2>
-<p>
-<h3>全体のプロジェクト</h3></br>
-私たちは、制御された薬の放出系を行うために膜構造を破壊してその反応が連鎖していくシステムを作りたい。そしてこのシステムの実現のためには以下の２つが必要である。</br>
-①好きなタイミングで反応の連鎖を開始できるように、内部にリポソーム膜を破壊する役割を持つＤＮＡであるトリガーが入っているアルギン酸膜を作製し、ある条件を満たせばそのアルギン酸膜が内側から破壊されてトリガーが放出されるシステムを作る。</br>
-②反応が連鎖できるように内部にトリガーが入っており、外側にリポソームが結合すれば割れるように設計されたリポソーム膜を作製する。</br>
-私たちは「内側からアルギン酸膜を破壊するサブプロジェクト」と「外側からリポソームを破壊するサブプロジェクト」の二つのサブプロジェクトを実行してこのシステムの完成を目指しました。</br></br>
-<!--
-<a href="#designsubproject1">内側からアルギン酸膜を破壊するサブプロジェクト</a><br>
-<a href="#designsubproject2">外側からリポソームを破壊するサブプロジェクト <font size="2">リポソーム班</font> </a><br>
-<a href="#designsubproject3">外側からリポソームを破壊するサブプロジェクト <font size="2">B-Z班</font> </a>
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-        <article data-title="アルギン酸">
-<h3 id="designsubproject1">内側からアルギン酸膜を破壊するサブプロジェクト</h3></br>
-内側からアルギン酸膜を破壊するために私たちは以下のようなシステムをデザインしました。（Fig1の数字と下記の説明の説明は対応している）</br>
-①内部にアルギン酸を溶かすキレート剤（ＥＧＴＡ）と尿素とトリガー（アルギン酸膜外にあるリポソームを割ることができるもの）となるＤＮＡオリガミ/ＤＮＡストランドを入れたニッパム分子付きのリポソームを作製します。ニッパムとは３２度以下では水和していて親水性だが、３２度以上では収縮して疎水性になる。ニッパムがリポソームに修飾されると、３２度以下ではニッパム分子の水和により安定な状態になるが、３２度以上ではニッパム分子が疎水性になって不安定な状態になるので、３２度以上になった時にリポソームが割れることになる。</br>
-参考</br>
-http://www.sigmaaldrich.com/etc/medialib/docs/SAJ/Brochure/1/j_recipedds2.Par.0001.File.tmp/j_recipedds2.pdf</br>
-</br>
-②温度を約３２度に上げることで、ニッパム分子が収縮しリポソームが破壊されます。</br>
-③リポソームが破壊されたことで内部のキレート剤がアルギン酸膜を破壊し、それと同時に尿素が希釈されることでＤＮＡがハイブリタイゼーションしてトリガーとなるＤＮＡオリガミが形成されます。</br>
-④破壊されたアルギン酸膜からトリガーのＤＮＡオリガミが外に出ていきます。</br>
-このようなシステムの設計によって、好きな時に温度を３７度に上げればトリガーを放出することが可能になります。</br></br>
-<img src="http://openwetware.org/images/8/80/Design-alginate-00.png" alt="example-tab2" border="0"></br>
-Fig1 内側からアルギン酸膜を破壊し、トリガーとなるDNAオリガミを放出するシステム
-        </article>
+<div id="ttop">
+<a href="#top" class="page_top" onfocus="this.blur();" onclick="scrollTo(0,0); return false;" title="Top"></a></div>
+ <section role="main">
+        <article>
+         <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>
-        <article data-title="リポソーム">
+</ul>
+</li>
+</ul>
+</td></tr></table>
-<h3>外側からリポソームを破壊するサブプロジェクト</h3></br>
+<h2 id=chain>Project goal</h2>
-<h4 id="designsubproject2">リポソーム班</h4></br>
+&nbsp;In Lipo-HANABI project, we need to develop the following two subsystems.<br><br>
-        </article>
+i) Sensing system (First stage): liposome disruption by temperature control. <br>
-        <article data-title="B-Z">
+ii) Amplification system (Second stage): a chain-reactive disruption of the liposomes activated by the First stage. <br><br>
-<h3>外側からリポソームを破壊するサブプロジェクト</h3><br>
+<h3 id=Flower>First stage: Sensing system </h3>
-<h4 id="designsubproject3">ＢＺ班</h4><br>
+&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>
-まずリポソームを割る方法として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、割るとゆうものである。</br>
+<h4 id=5>DNA origami approach </h4>
-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>
-リポソームはDOPCで作った通常のものとDOPC、DPPC、cholesterolの三種類を混ぜ、相分離を形成しているものの二種類を用いる。</br>
-これはリポソーム表面の状態の違いによる収率の違いを調査するためである。</br>
-これらのリポソームにコレステロール修飾されているDNA一本鎖ストランドを加えて、表面にDNAを生やす。</br>
-そこに先程修飾したDNA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>
-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ハイブリタイゼーションし結合する。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。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></br>
-リポソーム内部の溶液中にトリガーストランドを入れておくことができるので、この系であれば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>
+&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.
-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>
+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>
-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>
-<font size="-2">
+<div align="center">
-コレステロール付きDNAの配列<br>
+<Img Src="http://openwetware.org/images/c/c5/%E8%86%9C%E3%80%80%E5%8F%8D%E7%99%BAdfhr.png">
-<font color="red">CCAGAAGACG</font> -コレステロール<br>
+</div>
-ループが40nt<br>
+<div class="caption">Fig.2 Stress on liposome membrane</div>
-<font color="red">CGTCTTCTGG</font>TTTTTTTTTT<font color="blue">GCGAACCACGGTTCCCAGCGTGACCTTCATGCTTAAGTTT</font><font color="red">CGTCTTCTGG</font><br>
+<br>
-ループが40ntのトリガーストランド<br>
+&nbsp;From the reference, we learned that efficient structure design for destabilizing membranes should have the following properties: <br>
-<font color="blue">AAACTTAAGCATGAAGGTCACGCTGGGAACCGTGGTTCGC</font><br>
+<ur><li>Having rigid scaffolds</li>
-ループが20nt<br>
+<li>Having large surface areas to maximize the effect of the scaffold on the membrane</li></ur>
-<font color="red">CGTCTTCTGG</font>TTTTTTTTTTTT<font color="blue">CATAACATGAGGCGCCGT</font><font color="red">CGTCTTCTGG</font><br>
-ループが20ntのトリガーストランド<br>
-<font color="blue">ACGGCGCCTCATGTTATGAA</font><br>
-ループが10nt<br>
-<font color="red">CGTCTTCTGG</font>TTTTTTTTTT<font color="blue">CTGTAACTAA</font><font color="red">CGTCTTCTGG</font><br>
-ループが10ntのトリガーストランド<br>
-<font color="blue">TTAGTTACAG</font><br>
-<img src="http://openwetware.org/images/e/ec/Kari-design-bz.jpg">
+<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>
-</font>
+<div align="center">
+<Img Src="http://openwetware.org/images/4/45/Outsidefig8.png">
+</div>
-<!-- </p> -->
+<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>
         </article>
+</section>
-    </section>
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