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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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<h2 id=chain>Project goal</h2>
&nbsp;In Lipo-HANABI project, we need to develop the following two subsystems.<br><br>


<h3 id=chain>1. Our target(Lipo-HANABI) </h3>
i) Sensing system (First stage): liposome disruption by temperature control. <br>
今回のプロジェクトでは生物的な分子放出システム構築の基本的な例として次の２段階の分子放出システムの構築を目指す。
温度感受性リポソームを使って温度上昇を感知し、その内部の反応を開始するDNAを放出する。（１段階目）そして、そのDNAがもととなって、温度感受性リポソーム周辺の、内部に同じDNAをもつリポソームが連鎖的に割れていく（２段階目）という系だ。<br>
この系は一点から周囲へとリポソームの破壊が連鎖的に広がっていく系であるため、その様子が日本のHANABI（Japanese Firework）に似ていることから私たちはこの系をLipo-HANABIと呼ぶことにした。<br>


このように２段階にするメリットは、１種類目のリポソームが特定の刺激（ここでは温度上昇）に対して反応するように作ることができれば、２種類目のリポソームは同じものでも連鎖反応が起こせることである。そうすることで増幅率や、耐ノイズ性を２段階目で保証しやすい。異なる刺激に反応するシステム（例えば光やpHなど）をつくるには、１段階目だけ設計しなおせばよい。<br>
ii) Amplification system (Second stage): a chain-reactive disruption of the liposomes activated by the First stage. <br><br>
今回のプロジェクトで第一段階目の入力として、「温度」を用いる理由は、比較的条件の設定が簡便であり、局所的な加熱が可能であり、（顕微鏡下でスポットを加熱することができる）温熱療法など、生体組織でも使われており、本システムを例えばDDSとして利用する場合には入力として使えると考えたためである。<br>
 
このLipo-HANABIというシステムを実現するために私たちは温度感受性リポソーム（for１段階目）とDNAによって破壊することができるリポソーム（for２段階目）の２種類をつくり、１種類目のリポソームが外部刺激（温度上昇）により壊れると、２種類目のリポソームを壊す鍵DNAが放出される。２種類目のリポソームにはそれ自身を壊す鍵DNAが入っているため、連鎖的にリポソームが壊れ、大量の内容分子と鍵DNAが２種類目のリポソームの連鎖的な破壊により放出される。<br><br>
In our project, we aim to construct a molecular releasing system as a basic example of biological one. We designed the system as follows. <br>
(1)A temperature-sensitive liposome sense increase in temperature and release DNA to start the next reaction. <br>
(2)The first DNA chain-reactively collapse liposomes which have the same DNA sequences in its inside. <br>
In this system, the collapse of liposome prevail from one point to surround chain-reactively. So we call this system Lipo-HANABI because the shape of this system is look like Hanabi, Japanese fireworks. <br>
The merit of constructing two phases is to use the same type of liposome in phase (2) when we would like to take chain reaction. <br>
The above system confirm amplification factor and prevention of noise. When we would like to change what the system senses, we have only to make the first phase liposome.
The reason to select temperature as the first phase input is as follows. <br>
(1)We can easily make the condition. <br>
(2)We can heat locally in using microscope. <br>
(3)Heat is used in our body tissue and the system is likely to be used as input when we apply to DDS. <br>
To realize the Lipo-HANABI system, we have to make temperature-sensitive liposome and the other one to be collapsed by DNA. <br>
The first liposome is collapsed by rising temperature. It releases DNA to collapse the other ones. <br>
There are DNA to collapse the same kind of ones in the liposomes. So we can take chain-reactive collapse of liposome. <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. How to collapse liposomes</h3>
<h4 id=5>DNA origami approach </h4>


このシステムで最も重要なのは、まず特定の刺激に対してリポソームを壊し、DNAによるリポソームの破壊が連鎖的に起こることである。<br>
１段階目は温度で不安定化するリポソームを用いる。(温度感受性リポソーム) <br>
２段階目は、一段階目のリポソームに内包されていた鍵DNAで不安定化されるリポソームを用いる。この２段階目のリポソームに、鍵DNA と一緒にpayloadを加えることで分子の放出が行える。しかもそれは指数関数的に分子の濃度が増加していく放出である。<br><br>
The most important point for this project is to collapse liposome from a specific stimulus and take chain-reactive collapse of liposomes.
We made unstable liposomes in heating them. We call this liposome “the first liposomes”. (temperature-sensitive liposome)
We used unstable liposomes in when they are attached to DNA which are confined in the first-phase liposomes. We can release molecules by confining DNA and payload in the second- phase liposome. Payload means the materials to function in the specific place.
In addition, this system raise the solution of the molecules exponentially.




<h4 id=Flower>2-1）Step1 The collapse of temperature-senstive loposomes</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>.
We used temperature-sensitive liposomes in the first phase.
In this approach,　we use “Origami-anchor DNA” which connects DNA Origami with liposome membrane.
Warming the temperature-sensitive liposomes from room temperature to high temperature causes collapse of the liposomes.
We used NIPAM to collapse liposome. Nipam is a polymer and there are various kind of NIPAM. we selected PNIPAM because it can be a switch because it is hydrophobic by raising temperature.
Characters of the PNIPAM molecular are as below.<br>
NIPAM is hydrophilic at less than 32 ºC, but it become hydrophobic and shrinks at > 32 ºC. Therefore, liposomes containing a modified NIPAM (poly(NIPAM-co-AA-co-ODA) in their membranes become unstable at high temperature (temperature-sensitive liposomes). Consequently, increasing temperature collapse the liposomes.<br>
Reference(
<a href=
"http://www.sigmaaldrich.com/etc/medialib/docs/SAJ/Brochure/1/j_recipedds2.Par.0001.File.tmp/j_recipedds2.pdf">pdf</a>)
<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>


<div align="center"><img src="http://openwetware.org/images/1/1f/Sendai-napam-des-fig1-01.png" width="600"></div><br>
<div align="center">
Fig.3 A schematic image how liposome containing PNIPAM collapsed at high temperature is shown.<br><br>図、親水性→疎水性が分かるように文字を入れる<br>
<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>
<h4 id=sensing>2-2）Step2 DNAによる連鎖的リポソームの破壊</h4>
Each liposome in 2nd Step contains triggers and payload inside, and aptomor for the 鍵DNA on its surface. Aptomor DNA is the one on the surface of the liposome.  When liposomes are collapsed, new triggers and payload are released. To achieve liposomal burst by outside 鍵DNA, we propose the following two approaches. DNA origamiによるアプローチとAnchored DNAによるアプローチである<br>
 
 
<h5 id=5>2-2-1)  DNA origamiによるアプローチ</h5>
<h6>このアプローチの概要</h6>
 
 
一段目から放出される鍵分子はDNAオリガミでそのDNAオリガミにより2段目の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>
 
 
<Img Src="http://openwetware.org/images/2/20/Anchor.jpg" ><br>
 
リポソームを作製した後にコレステロール修飾したDNAをリポソームに振り掛ける。こうすることでリポソームの内側にコレステロール修飾したDNAが生えないようにすることができる。
Cholesterol-conjugated DNA strands (in the rest of this document, referred to as “Anchor”) attach to the surfaces of liposomes.<br>
 
<Img Src="http://openwetware.org/images/0/0b/Askld.png" Align="center" width="900px" hight="800" >
 
<div align="center">Fig.1 Process of bending approach</div><br>
<br>
Our DNA origamiによる approach consists of the following 3steps.<br>
1. Then, DNA origami complementary to the aptamer is added as triggers.<br>
2. Triggers bind to the surfaces of liposomes and give a load on the membrane.<br>
3. Due to the load by 鍵分子のオリガミ, liposomes are destroyed.<br>
<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>


<h6>このアプローチの原理的根拠</h6>
<div align="center">
 
 
 
トリガーで使用するDNAオリガミはリポソームを割るのに最適な形にデザインするべきだ。
To destroy liposomes, we focused on the mechanism the living things use to bend cell membranes. We consider that if we could make use of the mechanism of bending membranes (destabilizing membranes), it would lead to the collapse of membranes. The following three mechanisms have been proposed as of now (<a href="http://www.ncbi.nlm.nih.gov/pubmed/19780639">Membrane-bending proteins</a>)<br>
 
<div class="caption-left">
<Img Src="http://openwetware.org/images/a/ae/Designfig2.png" width="280px" height="400px">
<span>Fig.2 Mechanism of bending membranes</span></div>
<br>
The mechanism A is that amphipathic molecules are inserted into the cell membrane and the bending is caused. The inner hydrophobic part of the lipid bilayer has a strong adhesive power for the two leaflets. Thus, once the amphipathic molecules are inserted into one leaflet of the membrane and expand it, the other leaflet bends according to it, making its surface area smallest.<br>
<br>
The mechanism B is that the molecule attached to the membrane becomes a rigid scaffold and distort the membrane under itself, or stabilize the already bended membrane.<br>
<br>
The mechanism C is that lipid molecules are clustered in one leaflet of the membrane and the inequality of lipid quantity makes the membrane bend.<br>
<div class="c-both"></div>
 
Most membrane bending proteins combine the above three mechanisms.<br>
In addition, a theory that protein crowding causes the bending of cell membranes ( <A Href="http://www.ncbi.nlm.nih.gov/pubmed/22902598">Membrane bending by protein- protein crowding</A>) has recently been suggested. This mechanism is that the collision of membrane proteins produces lateral pressure on membranes and distorts them.<br>
<br>
 
<h6>このアプローチのDNAデザイン</h6>
 
 
Due to the above reasons, 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>
 
We also designed rectangle and triangle to make the pressure of the collision highest.<br>
<Img Src="http://openwetware.org/images/4/49/Outsidefig3-1.png">
<div align="center">Fig.3 Rectangle origami</div><br>
We suppose that rectangle and triangle structures are most effective for the following reasons. <br>
Rectangle is expected to work as one scaffold in itself; triangle (the most efficient figure that covers a sphere) structures, to gather and work as one big rigid scaffold.<br>
<br>
The design of our rectangular DNA origami is as below.<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 <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 aptamers (These aptamers 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 aptamer 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">
 
</div>
<Img Src="http://openwetware.org/images/8/8e/Outsidefi.png">
<div class="caption">Fig.5 Unstable liposome</div>
<div align="center">Fig.6 Unstable liposome</div>
<br><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>
<h5 id=6>2-2-2) Anchored DNAによるアプローチ</h5>
<li>Adding another ssDNA (complementary to the above DNA) which induces a structural change　by DNA hybridization</li>
<h6>このアプローチの概要</h6>
<li>The induced structural change on the DNA results in disruption of the liposome</li>
 
 
このアプローチでは一段階目の温度感受性リポソームから放出される鍵分子はDNAストランドでそのDNAストランドは二段階目のリポソーム表面に埋め込んであるDNAストランドに相補になっている（この埋め込んであるDNAをアンカーDNAという）。アンカーDNAに構造変化を起こさせ、リポソーム膜面に「引っ張り（引き裂き）」ストレスを与えることにより、リポソームを壊す。<br>
 
<Img Src="http://openwetware.org/images/a/a7/Design-flower-flow2.png" Align="center" ><br>
 
 
First, we mix anchors (the same strands as used in 2-2-1) DNA origamiによる approach), loop strands, and liposomes.<br>
Each of the loop strands is designed to have two complementary parts to anchors at its both ends. So when it binds to the anchors at the both ends, the middle part remains single-stranded and becomes a loop. <br>
As an anchors is cholesterol-conjugated and has high affinity for a liposome, it floats on a liposome and enables the anchors-loop strand complex attach to the liposome. In other words, a loop strand hybridizes with a liposome via two anchors.<br>
 
 
 
<Img Src="http://openwetware.org/images/f/fd/Sag2.png" Align="center" width="900px" hight="800" ><br>
<div align="center">Fig.10 How to straighten loop</div>
 
1. We add a trigger strand corresponding (complementary) to the loop strand on the liposome. The trigger strand hybridizes with the loop part, making it change to be straight.<br>
<br>
<br>
<img src="http://openwetware.org/images/0/03/Flower3.png"><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>
<div align="center">Fig.7 Process of flower micelle approach</div><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>
2. The double-stranded part keeps straight (though it was originally a loop part), because the trigger strand is designed to be shorter than its persistence length.<br>
<div align="center">
3.In this process, some stress is placed on the liposome, and it is collapsed.<br>
<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>
 
 
<h6>このアプローチの原理的根拠</h6>
 
 
 
<div <!--class="caption-right"-->>
<Img Src="http://openwetware.org/images/6/6f/Design-flowermicelle.png" style="width:425px;"><br>
<span>Fig.8 Flower micelle method</span></div>
 
 
There is a method called <a href="http://pubs.acs.org/doi/ipdf/10.1021/jp104711q">flower micelles</a>.<br>
In this method, we cover the surfaces of liposomes with many copolymer rings. The rings can be distorted by heating, place some stress on the liposomes, and collapse them.<br>
 
 
 
<h6>このアプローチのDNAデザイン</h6>
 
 
 
 
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、
・多くのコレ付きDNAを表面に埋め込むこと
・トリガーDNAのハイブリによってDNAの構造が変化すること
といった要素が重要になってくる。
 
そこで、we cover the surfaces of liposomes with many copolymer rings. The rings can be distorted by heating, place some stress on the liposomes, and collapse them.<br>
<div class="c-both"></div>
<br>
<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>
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>
<div class="caption">Fig.7 Process of flower DNA approach</div><br><br>
We arranged three kinds of loop strands. <br>
<Img Src="http://openwetware.org/images/1/17/Flor4.png"  width="70%" hight="800" ><br>
Each loop strand has a 40nt, 20nt, or 10nt loop part (shown below in black and blue), which becomes a loop after the hybridization of the whole loop strand with aptamers.<br>
<div class="caption">Fig.8 How to disrupt a liposome</div>
The blue part of a loop strand is complementary to a corresponding trigger strand (also shown in blue). So a loop strand and a trigger strand are expected to hybridize with each other, place some stress on a liposome, and collapse it. <br>
      </article>
The red part of a loop strand is complementary to an aptamer (shown in red). Cooperating with aptamers, it enables the whole loop strand to attach to the surface of a liposome. <br>
Aptamers are the same strands as those used in i)Bending approach.<br>
 
<br>
<table border cellspacing="3" bgcolor="lightyellow">
<tr bgcolor="lightyellow">
<td> The kinds of DNA strands </td>
<td> Its sequence </td>
</tr>
<tr bgcolor="moccasin">
<td> Aptamer DNA </td>
<td> CCAGAAGACG -cholesterol
</td>
</tr>
<tr bgcolor="moccasin">
<td> 40nt loop DNA </td>
<td> CGTCTTCTGGTTTTTTTTTTGCGAACCACGGTT<br>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;CCCAGCGTGACCTTCATGCTTAAGTTTCGTCTTCTGG </td>
</tr>
<tr bgcolor="moccasin">
<td> Trigger DNA for 40 nt loop DNA </td>
<td> AAACTTAAGCATGAAGGTCACGCTGGGAACCGTGGTTCGC </td>
</tr>
<tr bgcolor="moccasin">
<td> 20nt loop DNA </td>
<td> CGTCTTCTGGTTTTTTTTTTTTCATAACATGAGGCGCCGTCGTCTTCTGG </td>
</tr>
<tr bgcolor="moccasin">
<td> Trigger DNA for 20 nt loop DNA </td>
<td> ACGGCGCCTCATGTTATGAA </td>
</tr>
<tr bgcolor="moccasin">
<td> 10nt loop DNA </td>
<td> CGTCTTCTGGTTTTTTTTTTCTGTAACTAACGTCTTCTGG </td>
</tr>
<tr bgcolor="moccasin">
<td> Trigger DNA for 10 nt loop DNA </td>
<td> TTAGTTACAG </td>
</tr>
</table>
 
<br>
<br>
 
 
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{{Biomod/2013/Sendai/sandbox/template2}}
{{Biomod/2013/Sendai/sandbox/template2}}

Latest revision as of 22:54, 26 October 2013

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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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