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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>2 stage of Lipo-HANABI</h3>
<h2 id=chain>Project goal</h2>
Lipo-HANABI is driven by the following two stage mechanism. <br><br>
&nbsp;In Lipo-HANABI project, we need to develop the following two subsystems.<br><br>


1st stage (initiation by the triggering signal): A temperature-sensitive liposome as an initiator. We chose temperature as an environmental initiator because of controllability. Increasing temperature is the trigger to collapse initiator liposome and releases key DNA that is designed for collapsing 2nd stage liposomes<br><br>
i) Sensing system (First stage): liposome disruption by temperature control. <br>


2nd stage: ２段階目ではリポソームの連鎖反応が起きる。In 2nd stage, a chain reactive liposomal destruction happens. The 2nd stage liposomes contain payload molecules and also the key DNA. ひとつのリポソームが割れると中に入っていたかぎDNAで次々にリポソームが割れるOnce a liposome collapse, neighbor liposomes collapse one after another.<br>
ii) Amplification system (Second stage): a chain-reactive disruption of the liposomes activated by the First stage. <br><br>
As a consequence, collapse of the liposomes goes on in a chain-reactive way. <br><br>


An advantage of using the two-stage strategy is that we can change the 1st stage to deal with various types of trigger signal, without changing the 2nd 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>


<h3 id=bending>２段階それぞれにおけるリポソーム破壊の方法の説明</h3>
<h4 id=5>DNA origami approach </h4>
<h4 id=Flower>１段階目　温度感受性リポソームによる反応の開始</h4>
１段階目で反応の着火剤として使うリポソームは温度感受性リポソームと言って、リポソームにNIPAMというポリマーを修飾したものである。
1st stage liposome is temperature-sensitive liposome; conjugated by polymer NIPAM. <br>


NIPAMとは３２度を境に親水から疎水に切り替わる性質をもつポリマーだ<br>
NIPAM switches to hydrophobic on the border of 32 Celsius.
３２度より高くなると縮んで疎水性になりリポ表面が不安定化する<br>
NIPAM shrinks and make the surface of liposome unstable more than 32 Celsius.
その結果リポが割れる<br>
As a consequence, liposome collapses.


<h4 id=sensing>２段階目 DNAによる連鎖的リポソームの破壊 </h4>
２ndステージのリポソームは外側の表面にアンカーDNA（コレステロール付きのDNA）が生え、内部には鍵DNAとペイロードがある。1stステージのイニシエーターから放出された鍵DNAと２ndステージのリポソームのアンカーDNAが結合することでリポソームは割れる。その時に放出される次の鍵DNAが別のアンカーDNAを持つリポソームに結合し、破壊する。この繰り返しにより連鎖的に割る。
（アンカーDNAと鍵DNAはリポソーム破壊を誘発するため、）アンカーDNAが内部に存在する場合、内部の鍵DNAにより破壊されてしまう。これを避けるためには、アンカーDNAは外側だけに存在している必要がある。私たちのプロジェクトではリポソーム作製後、コレステロール修飾したアンカーDNAをリポソームに振り掛けることでこれを達成する。


The 2nd stage liposomes have anchored DNA (DNA with cholesterol) on the outside surface and also have key DNA and payload in the interior. When key DNA released from the 1st stage initiators and anchored DNA on the 2nd stage liposomes
&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">
<br><br>
</div>
 
<div class="caption">Fig.2 Stress on liposome membrane</div>
（図を入れる）
To achieve liposomal burst by outside triggers, we propose the following two approaches.<br>
 
リポソームを割るには、活性化エネルギーを越えための触媒が必要である(See 計算)
我々のプロジェクトでは、この触媒を鍵DNAで行う。<br>鍵DNAとして（１）DNAオリガミを使う方法と（２）フラワーDNAを使う方法の２つを考案した<br>
 
２つのアプローチで使用するアンカーDNAは違うもので、それぞれオリガミアンカーDNA、フラワーアンカーDNAと呼ぶ。
 
<h5 id=5>DNAオリガミアプローチ</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>
このアプローチではオリガミアンカーDNA（１０nt3’コレ修飾DNA）を通してリポソーム表面に大量のDNAオリガミを吸着させ、リポソーム膜面に「曲げ」ストレスを与えることにより、リポソームを壊す。<br><br>
Hybridize
リポソーム表面にオリガミがハイブリすることでリポソーム表面がまっすぐになろうとする。これについては計算を行った（リンク）また、ハイブリしたDNAオリガミ同士の反発でさらにリポソーム表面が不安定化しリポソームが割れる。
 
前述の膜タンパクの論文からthe efficient design for destabilizing membranes is the structures that :<br>
<ur>
<li>have rigid scaffolds</li>
<li>have large surface areas to maximize the effect of the scaffold on the membrane</li>
<li>produce a large pressure by collisions</li>
</ur>
<br>
 
To make rigid scaffolds, we took note of DNA origami, because DNA origami is a method for making rigid structures of any shape. Moreover, we adopted a 2D structure to make the surface area largest.<br>
<br>
<br>
そこで
&nbsp;From the reference, we learned that efficient structure design for destabilizing membranes should have the following properties: <br>
We designed rectangle origami to make the pressure of the collision highest.<br>
<ur><li>Having rigid scaffolds</li>
<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">  
<li>Having large surface areas to maximize the effect of the scaffold on the membrane</li></ur>
<div align="center">Fig.3 Rectangle origami</div><br>


＜オリガミの設計＞
<Design of DNA origami><br>
長方形のオリガミはexpected to work as one scaffold in itself<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>


The design of our rectangular DNA origami is as below.<br>
<div align="center">
<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 <A Href="http://cadnano.org/">caDNAno2</A> 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).<br>
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.  
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>
<br>
<div class="c-both"></div>
<div align="center">
To sum up, the anchor not only connects DNA origami and liposomes but also inserts into the membrane and destabilizes it.<br>
<Img Src="http://openwetware.org/images/a/a0/Outsidefig5rg.png"　width="450px" height="350px">
<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">
</div>
<div align="center">Fig.6 Unstable liposome</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>
<br>
<h5 id=6>Flower DNAによるアプローチ</h5>
&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>
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。
&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">
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。
<Img Src="http://openwetware.org/images/3/3d/Flower-newfg.png" width="450px" height="350px" ></div><br>
<Img Src="http://openwetware.org/images/8/8b/Flower-new.png" Align="center" ><br>
<div class="caption">Fig.6 Liposome with Flower-anchor DNA</div>
<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>
<div align="center">Fig.10 How to straighten loop</div>
 
<div >
<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>
<span>Fig.8 Flower micelle method</span></div>
 
We tried to collapse liposomes by applying the mechanism of flower micelles.<br>
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>
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/0/03/Flower3.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>
       </article>
</section>
</section>

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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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