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<ul>
<ul>
<li class="toclevel-1"><a href="#chain">
<li class="toclevel-1"><a href="#chain">
<span class="tocnumber">1</span> <span class="toctext">Our Target(Lipo HANABI)</span></a></li>
<span class="tocnumber"></span> <span class="toctext">Project goal</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>
<ul>
<li class="toclevel-2"><a href="#Flower">
<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>
<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">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>
<span class="tocnumber"></span> <span class="toctext">Second stage:Amplification system</span></a></li>
<ul>
<ul>
<li class="toclevel-2"><a href="#5">
<li class="toclevel-3"><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>
<span class="tocnumber"></span> <span class="toctext">DNA origami approach</span></a></li>
<li class="toclevel-2"><a href="#6">
<li class="toclevel-3"><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>
<span class="tocnumber"></span> <span class="toctext">Flower DNA approach</span></a></li>
</li>
</li>


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<h3 id=chain>Project goal</h3>
<h2 id=chain>Project goal</h2>
To realize Lipo-HANABI, following two systems is needed. Our goal of this summer project is achieving these subgoals.<br>
&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>


i) To make a initiation system: liposomes disruption as a result of sensing thier environment<br>
<h3 id=Flower>First stage: Sensing system </h3>
ii) To make a chain-reactive disruption system: this system need followng two subsystems. <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>
ii-a)Liposomes dispution by attaching key DNA and anchor DNA<br>
&nbsp;This structural change of NIPAM induces stress on the surface of the liposome, and consequently disrupts them.<br>
ii-b)Selective disruption by key DNA species<br>
<div align="center">
ii-c)Chain reactive disruption by a released key DNA<br>
<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=Flower>First stage: Initiation by sensing environment</h4>
<h4 id=5>DNA origami approach </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="http://www.ncbi.nlm.nih.gov/pubmed/19780639"> 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.  
&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.


The above reference paper, "Membrane-bending proteins", says the efficient structure design for destabilizing membranes meets the following conditions :
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>


<ur>
<div align="center">
<li>Having rigid scaffolds</li>
<Img Src="http://openwetware.org/images/c/c5/%E8%86%9C%E3%80%80%E5%8F%8D%E7%99%BAdfhr.png">
<li>Having large surface areas to maximize the effect of the scaffold on the membrane</li>
</div>
<li>Producing a large pressure by collisions</li>
<div class="caption">Fig.2 Stress on liposome membrane</div>
</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>
<br>
<Img Src="http://openwetware.org/images/4/49/%E3%83%AA%E3%83%9D%E3%81%A1%E3%82%83%E3%82%93%EF%BC%91%EF%BC%92.png">  
&nbsp;From the reference, we learned that efficient structure design for destabilizing membranes should have the following properties: <br>
<div align="center">Fig.3 Rectangle origami</div><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">
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.4 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.5 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 used caDNAno2 for our DNA origami design.<br>
&nbsp;We use <a href="http://cadnano.org/">caDNAno2</a> for our DNA origami design.  
The DNA origami has a rectangle shape of 67.6nm (26 helixes) by 127 nm (374 bases).
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 by 161 bases at one edge of this origami, so that we could distinguish the two sides during AFM (Atomic Force Microscope) observation. <br>
We cut out a smaller rectangle of 10 helixes (161 bases) at one of the corners,
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>
so that we could distinguish the two sides with AFM (Atomic Force Microscope) observation.  
To sum up, the anchor not only connects DNA origami and liposomes but also inserts into the membrane and destabilizes it. <br>
Also, we put 141 staples sticking out from the bottom face of the origami.
<Img Src="http://openwetware.org/images/3/37/%E3%83%AA%E3%83%9D%E3%81%A1%E3%82%83%E3%82%93%EF%BC%93.png">
Those staples hybridize with cholesterol-modified Origami-anchor DNA, which has high affinity with lipid membrane.<br>
<div align="center">Fig.6 Unstable liposome</div>
<br>
<br>
<h5 id=6>Flower DNAによるアプローチ</h5>
<div align="center">
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。
<Img Src="http://openwetware.org/images/a/a0/Outsidefig5rg.png"　width="450px" height="350px">
 
</div>
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。
<div class="caption">Fig.5 Unstable liposome</div>
<Img Src="http://openwetware.org/images/8/8b/Flower-new.png" Align="center" ><br>
<br><br>
<Img Src="http://openwetware.org/images/6/69/%E3%83%AA%E3%83%9D%E3%81%A1%E3%82%83%E3%82%93%EF%BC%92.png" Align="center" width="900px" hight="800" ><br>
<h4 id=6>Flower DNA approach</h4>
<div align="center">Fig.10 How to straighten loop</div>
&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>
<div >
<ur><li>Embedding a lot of cholesterol-modified ss DNA on the liposome surface</li>
<Img Src="http://openwetware.org/images/e/ed/%E3%83%AA%E3%83%9D%E3%81%A1%E3%82%83%E3%82%93%E2%91%A5.png" style="width:425px;"><br>
<li>Adding another ssDNA (complementary to the above DNA) which induces a structural change　by DNA hybridization</li>
<span>Fig.8 Flower micelle method</span></div>
<li>The induced structural change on the DNA results in disruption of the liposome</li>
 
<br>
We tried to collapse liposomes by applying the mechanism of flower micelles.<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>
フラワーミセルをリポソームに応用するためには、<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>
・多くのコレ付きDNAを表面に埋め込むこと<br>
<div align="center">
・鍵DNAのハイブリによってDNAの性質が変化すること<br>
<Img Src="http://openwetware.org/images/3/3d/Flower-newfg.png" width="450px" height="350px" ></div><br>
といった要素が重要になってくる。<br><br>
<div class="caption">Fig.6 Liposome with Flower-anchor DNA</div>
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="http://openwetware.org/images/b/b1/Flower3gj.png"><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="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の二本とKey DNA の3種類の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>
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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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