Difference between revisions of "Biomod/2013/Sendai/design"

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<div id="ttop">
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<a href="#top" class="page_top" onfocus="this.blur();" onclick="scrollTo(0,0); return false;" title="Top"></a></div>
  
 
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<ul>
 
<ul>
 
<li class="toclevel-2"><a href="#Flower">
 
<li class="toclevel-2"><a href="#Flower">
<span class="tocnumber"></span> <span class="toctext">1st stage:Sensing system</span></a></li>
+
<span class="tocnumber"></span> <span class="toctext">First stage:Sensing system</span></a></li>
 
<li class="toclevel-2"><a href="#sensing">
 
<li class="toclevel-2"><a href="#sensing">
<span class="tocnumber"></span> <span class="toctext">2nd stage:Amplification system</span></a></li>
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<span class="tocnumber"></span> <span class="toctext">Second stage:Amplification system</span></a></li>
 
<ul>
 
<ul>
 
<li class="toclevel-3"><a href="#5">
 
<li class="toclevel-3"><a href="#5">
 
<span class="tocnumber"></span> <span class="toctext">DNA origami approach</span></a></li>
 
<span class="tocnumber"></span> <span class="toctext">DNA origami approach</span></a></li>
 
<li class="toclevel-3"><a href="#6">
 
<li class="toclevel-3"><a href="#6">
<span class="tocnumber"></span> <span class="toctext">FlowerDNA approach</span></a></li>
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<span class="tocnumber"></span> <span class="toctext">Flower DNA approach</span></a></li>
 
</li>
 
</li>
  
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<h2 id=chain>Project goal</h2>
 
<h2 id=chain>Project goal</h2>
In Lipo-HANABI project we need to develop the following subsystems<br><br>
+
&nbsp;In Lipo-HANABI project, we need to develop the following two subsystems.<br><br>
  
i) Sensing system (1st stage): liposome disruption by temperature control. <br><br>
+
i) Sensing system (First stage): liposome disruption by temperature control. <br>
  
ii) Amplification system (2nd stage): a chain-reactive disruption of the liposomes activated by the 1st stage. <br><br>
+
ii) Amplification system (Second stage): a chain-reactive disruption of the liposomes activated by the First stage. <br><br>
  
<h3 id=Flower>1st stage: Sensing system </h3>
+
<h3 id=Flower>First stage: Sensing system </h3>
To make temperature-sensitive liposomes, we used lipids conjugated with NIPAM polymer.<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>
NIPAM is hydrophilic at room temperature, but switches to hydrophobic over 32 C. When it becomes hydrophobic, it shrinks to avoid water molecules. This structural change of NIPAM induces stress on the surface of liposomes, and consequently disrupts liposomes.<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">
 
<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 align="center">Fig.1 Temperature-sensitive liposome</div>  
+
</div>
<h3 id=sensing>2nd stage: Amplification system </h3>
+
<div class="caption">Fig.1 Temperature-sensitive liposome</div>  
We assume that 1st stage liposomes contain key molecules that initiate chain reactive burst of 2nd stage liposomes.<br>
+
<h3 id=sensing>Second stage: Amplification system </h3>
There are two different approaches to realize the 2nd stage.<br>
+
&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>
 
   A) DNA Origami approach<br>
 
   B) Flower DNA approach<br>
 
   B) Flower DNA approach<br>
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This approach is inspired by a paper about <a Href="http://www.ncbi.nlm.nih.gov/pubmed/19780639"> Membrane-bending proteins</a>
+
&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.
  
In this approach, we name DNA stuck into liposome membrane, Origami-anchor DNA. A lot of DNA origamis are adsorbed on the surface of liposomes by using Origami-anchor DNA. DNA origami is supposed as a stiff, straight board like structure 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 of liposome repel each other because of negative charges on DNA backbone. This effect may add more stress on the membrane. We did analysis on this phenomenon (link).<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>
  
<Img Src="http://openwetware.org/images/1/12/%E8%86%9C%E3%80%80%E5%8F%8D%E7%99%BAnew.png"><br>
+
<div align="center">
<div align="center">Fig.2 Stress on liposome membrane</div><br>
+
<Img Src="http://openwetware.org/images/c/c5/%E8%86%9C%E3%80%80%E5%8F%8D%E7%99%BAdfhr.png">
 
+
</div>
From the reference, we learned that efficient structure design for destabilizing membranes should have the following properties: .<br>
+
<div class="caption">Fig.2 Stress on liposome membrane</div>
•  Having rigid scaffolds<br>
 
• Having large surface areas to maximize the effect of the scaffold on the membrane<br>
 
 
 
<ur>
 
 
<br>
 
<br>
 +
&nbsp;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>
 +
&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>
  
<Design of DNA origami> <br>
+
<div align="center">
DNA origami is known as a designable rigid structure made of DNA. We use DNA origami to make rigid scaffolds. In order to meet the requirements, we designed 2D rectanglar 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">
 
<Img Src="http://openwetware.org/images/4/45/Outsidefig8.png">
<div align="center">Fig.3 Rectangular origami</div>
+
</div>
 +
<div class="caption">Fig.3 Rectangular origami</div>
 
<br>
 
<br>
 
<div class="caption-right">
 
<div class="caption-right">
  <Img Src="http://openwetware.org/images/a/a7/Lipo5.png" ><span>Fig.4 DNA origami designed by caDNAno</span>
+
  <Img Src="http://openwetware.org/images/a/a7/Lipo5.png" style="padding-left:10mm"><span>Fig.4 DNA origami designed by caDNAno</span>
 
</div>
 
</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.  
+
&nbsp;We use <a href="http://cadnano.org/">caDNAno2</a> for our DNA origami design.  
Also, we put 141 staples sticking out from bottom face of the origami. Those staples hybridize with cholesterol-modified Origami-anchor DNA, which has high affinity with lipid membrane.
+
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,
<Img Src="http://openwetware.org/images/2/2a/Outsidefig5o8o.png" width="50%" height="50%">
+
so that we could distinguish the two sides with AFM (Atomic Force Microscope) observation.  
<div align="center">Fig.5 Unstable liposome</div>
+
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>
 
<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>
 
<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>.
This approach is inspired by a paper about <a href="http://pubs.acs.org/doi/ipdf/10.1021/jp104711q">Polymer Flower-micelle</a>. <br>
+
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>
To adapt Flower-micelle to our project, we should remark the followings.<br>
+
<li>Adding another ssDNA (complementary to the above DNA) which induces a structural change by DNA hybridization</li>
• Sticking a lot of cholesterol-modified DNA into liposome surface<br>
+
<li>The induced structural change on the DNA results in disruption of the liposome</li>
• Change of DNA’s property by hybridization with Key DNA<br>
 
In this approach, we call 50nt3’DNA partly hybridized with 10ntDNA, Flower-anchor DNA. 50nt3’DNA is cholesterol-modified and the part of it hybridizes with 10ntDNA. The rest of its 40nt complements Key DNA. <br>
 
<Img Src="http://openwetware.org/images/4/45/Flower-new54.png"  ><br>
 
<div align="center">Fig6 Liposome with Flower-anchor DNA</div>
 
The hybridization makes Flower-anchor DNA longer. It gives stretching stress to liposome membrane and ends up disrupting liposomes. <br>
 
 
 
We designed the sequences of Flower-anchor DNA and Key DNA by <A Href="http://www.dna.caltech.edu/DNAdesign/">DNA design</A>, software for designing DNA sequences. <br>
 
 
<br>
 
<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>
<img src="http://openwetware.org/images/6/65/Flower3new8.png"><br>
+
<div align="center">
<div align="center">Fig.7 Process of flower micelle approach</div><br><br>
+
<Img Src="http://openwetware.org/images/3/3d/Flower-newfg.png" width="450px" height="350px" ></div><br>
<Img Src="http://openwetware.org/images/d/dd/Flower4newa.png" Align="center" width="900px" hight="800" ><br>
+
<div class="caption">Fig.6 Liposome with Flower-anchor DNA</div>
<div align="center">Fig.8 How to straighten loop</div>
 
 
 
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。
 
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>
 
 
 
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。
 
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
 
 
 
 
 
We designed the sequences of Flower-anchor DNA and Key DNA by <A Href="http://www.dna.caltech.edu/DNAdesign/">DNA design</A>, software for designing DNA sequences. <br>
 
 
<br>
 
<br>
私たちはこのソフトでFlower DNAとKey DNA のDNAを設計しました。
+
&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>
 
       </article>
 
</section>
 
</section>

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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"></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> &nbsp;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> &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>

<h4 id=5>DNA origami approach </h4>


&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.

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> &nbsp;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> &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>

<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> &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>

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