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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">Step1 温度感受性リポソームの破壊</span></a></li>
<span class="tocnumber">1</span> <span class="toctext">First stage: Sensing system</span></a></li>
<ul>
<ul>
<li class="toclevel-2"><a href="#bending">
<li class="toclevel-2"><a href="#bending">
<span class="tocnumber">1-1</span> <span class="toctext">温度感受性リポソームを破壊する実験</span></a></li>
<span class="tocnumber">1-1</span> <span class="toctext">Disruption of temperature sensitive liposomes</span></a></li>
</ul>
</ul>
<li class="toclevel-1"><a href="#Flower">
<li class="toclevel-1"><a href="#Flower">
<span class="tocnumber">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">2</span> <span class="toctext">Second stage: Amplification system</span></a></li>
<ul>
<ul>
<li class="toclevel-2"><a href="#sensing">
<li class="toclevel-2"><a href="#sensing">
<span class="tocnumber">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">2-1</span> <span class="toctext">DNA Origami approach </span></a></li>
<ul>
<ul>
<li class="toclevel-2"><a href="#5">
<li class="toclevel-2"><a href="#5">
<span class="tocnumber">2-1-1</span> <span class="toctext">デザインしたDNAオリガミの作製</span></a></li>
<span class="tocnumber">2-1-1</span> <span class="toctext">Making DNA Origami</span></a></li>
<li class="toclevel-2"><a href="#6">
<li class="toclevel-2"><a href="#6">
<span class="tocnumber">2-1-2</span> <span class="toctext">DNAオリガミに蛍光付きDNAがハイブリしていることの確認実験</span></a></li>
<span class="tocnumber">2-1-2</span> <span class="toctext">Labeling DNA Origami with fluorescent-tagged DNA</span></a></li>


<li class="toclevel-2"><a href="#7">
<li class="toclevel-2"><a href="#7">
<span class="tocnumber">2-1-3</span> <span class="toctext">DNAオリガミによりリポソームを破壊する実験</span></a></li>
<span class="tocnumber">2-1-3</span> <span class="toctext">Disruption of liposomes by DNA Origami (microscopic analysis)</span></a></li>
<li class="toclevel-2"><a href="#13">
<span class="tocnumber">2-1-4</span> <span class="toctext">Disruption of liposomes by DNA Origami (quantitative analysis)</span></a></li>
 
<li class="toclevel-2"><a href="#8">
<li class="toclevel-2"><a href="#8">
<span class="tocnumber">2-1-4</span> <span class="toctext">DNAによる配列特異性を証明する実験</span></a></li>
<span class="tocnumber">2-1-5</span> <span class="toctext">Confirming sequence specificity of DNA</span></a></li>
</ul>
</ul>
<li class="toclevel-1"><a href="#9">
<li class="toclevel-1"><a href="#9">
<span class="tocnumber">2-2</span> <span class="toctext">フラワーミセルによるアプローチ</span></a></li>
<span class="tocnumber">2-2</span> <span class="toctext">Flower DNA approach</span></a></li>
<ul>
<ul>
<li class="toclevel-2"><a href="#10">
 
<span class="tocnumber">2-2-1</span> <span class="toctext">SPRによるループ構造の確認</span></a></li>
<li class="toclevel-2"><a href="#11">
<li class="toclevel-2"><a href="#11">
<span class="tocnumber">2-2-2</span> <span class="toctext">フラワーミセルによりリポソームを破壊する実験</span></a></li>
<span class="tocnumber">2-2-1</span> <span class="toctext">Disruption of liposomes by Flower DNA</span></a></li>
<li class="toclevel-2"><a href="#12">
<li class="toclevel-2"><a href="#12">
<span class="tocnumber">2-2-3</span> <span class="toctext">DNAによる配列特異性を証明する実験</span></a></li>
<span class="tocnumber">2-2-2</span> <span class="toctext">Confirming sequence specificity of DNA</span></a></li>




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</td></tr></table>
</td></tr></table>


<h3 id=chain>1 Step1 温度感受性リポソームの破壊</h3>
<h3 id=chain>First stage: Sensing system</h3>
<h4 id=bending>1-1温度感受性リポソームを破壊する実験</h4>
<h4 id=bending>1-1Disruption of temperature sensitive liposomes</h4>
<h6>Purpose</h6>
<h5>Purpose</h5>
私たちのプロジェクトでは外部刺激を感知するイニシエーターの一例としてニッパム修飾したリポソーム（温度感受性リポソーム）を使用する。そのため、温度上昇によりニッパム修飾のリポソームが割れること実験により確認する。<br>
In our project, we planned to use liposomes conjugated with NIPAM polymer as a chain reaction initiator. NIPAM (poly-N-isopropyl acrylamide) is a temperature sensitive molecule that has a unique critical temperature (Tc: 32°C ). <br>
When the temperature increased over than Tc, the hydrophilic polymer changes its property hydrophobic. It is expected that the change should disrupt the membrane lipid alignment. Here we confirm that the possibility of breaking liposomes with NIPAM by increasing temperature.<br>
NIPAM was purchesed from SIGMA ALDRICH.<br>
<br>


In our project, liposome collapses by temperature shift is a crucial step. Thus, we should confirm the temperature sensitivity of PNIPAM lipids-based liposome.（訳途中）<br>
<h5>Method</h5>
The liposomes were prepared by natural swelling method. Obtained sample included a mixture of unilamellar and multilamellar liposomes.<br>
Then we added NIPAM-conjugated lipids (dissolved in ultra pure water (Milli-Q)) to the liposomes solution.<br>
The liposomes were observed on the slide glass by phase-contrast microscopy. <br>
After confirming the formation of the liposomes, a petri dish with hot water (~90˚C) was put on the sample slide glass to increase the temperature.<br>
Detailed Protocol<br>
<A href="http://openwetware.org/wiki/Biomod/2013/Sendai/protocol">Protocol</A><br>


<h6>Method</h6>
<h5>Result</h5>
脂質にはEggPC、バッファーにはLパラフィン？を使用し、ボルテックス法によりリポソームを作製した。<br>
リポソームにNIPAM (in クロロホルム)を質量比？？？の割合で加えた。<br>
スライドガラスを作製し、位相差顕微鏡でリポソームがあることを確認した。<br>
リポソームが確認できたら、スライドガラスの上にお湯を乗せて温度を上げた。<br>
Protocol<br>
（対応するプロトコルへのリンク）<br>


<h6>Result</h6>
温度を上げる前のリポソームの状態は図１のようになった。<br>


<img src="http://openwetware.org/images/8/89/Snap_20131018_193341_8127.jpg"><br>
図１　温度を上げる前のニッパム付きのリポソーム<br>


スライドガラスにお湯を乗せて温度を上昇させた後のリポソームの様子は以下図２、図３のようになった。観察しているリポソームは図１のものと同じである。まず、図２のようにバックグラウンドがザラザラになって、しばらくすると図３のようにリポソームが確認できなくなった。ピントを調節してもリポソームは確認できなかった。<br>
<img src="http://openwetware.org/images/9/9e/2-1-1fig1.png" width="100%" height="100%"><br>


<img src="http://openwetware.org/images/2/21/Snap_20131018_193358_8128.jpg"><br>
<div class="caption">Fig.1 Phase contrast images of liposomes in NIPAM solution. Temperature increased from RT to enough over than Tc (left to right).</div><br>
図２　温度上昇後のニッパム付きのリポソーム<br>
<br>
<img src="http://openwetware.org/images/6/63/Snap_20131018_193431_8129.jpg"><br>
Fig.1 shows the continuous images before and after the temperature increase. The view sight was the same position. <br>
図３　温度を上昇させた後のニッパム付きのリポソームが消えた様子<br>
NIPAM polymer turned into globular states with increasing temperature. Liposomes disappeared by increasing temperature (> Tc).<br>


<h6>Discussion</h6>
図１で見えていたリポソームが、図２，３のように消えてしまったのでニッパム付きのリポソームが割れたと考えられる。しかし、リポソームによっては温度を上げた後も残っているものがいくつか確認できた。これはリポソームがマルチラメラ（脂質二重膜が複数重なっているもの）になっているものと考えられ、脂質二重膜が単一層のユニラメラよりも割れにくいからだと考えられる。上記図１，２，３で定点観察したリポソームはユニラメラであると考えられる。<br>


<h3 id=Flower>2 Step2 DNAによる連鎖的リポソームの破壊</h5>
<h4 id=sensing>2-1 DNAオリガミによるアプローチ</h4>
<h5 id=5>2-1-1 デザインしたDNAオリガミの作製</h5>
<h6>Purpose</h6>
In our project, we used DNA origami as triggers for collapsing liposomes. We designed a rectangular DNA origami with a chipped edge and tried to make it.<br>
<h6>Method</h6>
We mixed M13mp18, staples, 5xTAE Mg2+, and mQ in a microtube and annealed it for 2.5 hours.<br>
<A href="http://openwetware.org/wiki/Biomod/2013/Sendai/protocol">Protocol</A><br>
<br>
<br>


<h6>Result</h6>
We confirmed that our DNA origami was well formed by AFM (Atomic Force Microscope) (Fig.1).<br>
<Img Src="http://openwetware.org/images/d/d9/Outsideafm2.png"> <br>
Fig.1 AFM image of DNA origami (M13: 4nM, staples:20nM)<br>
<br>


<h6>Discussion</h6>
<h5>Discussion</h5>
Just like our design, rectanglar origamis with chipped edges were observed.
Thermosensitive polymer NIPAM can disrupt the coexisting liposomes by the polymers phase transition. <br>
On the other hand, some liposomes still present even at the high temperature. In this experiment, some fractions were multi-lamellar liposomes. Since globular states of NIPAM (hydrophobic) at high temperature attack the liposome membrane from the outside, it is not surprising that the multi-lamellar liposomes consist of many lipid bilayers are more difficult to disrupt. Therefore, we suppose that liposomes disrupted by temperature shift in Fig.1 were uni-lamella ones. <br>
<u>These results confirmed that triggering by heat disrupted the liposomes.</u><br>
 


<h5 id=6>2-1-2 DNAオリガミに蛍光付きDNAがハイブリしていることの確認実験</h5>
<h6>Purpose</h6>
If the origami is fluorescently labeled, it is much easier to observe the effect of DNA origami on liposomes. So we labeled our origami by hybridizing it with fluorescent tagged DNAtrands.<br><br>


<h6>Method</h6>
<h3 id=Flower>Second stage: Amplification system</h5>
Our DNA origami has many staples that can bind to fluorescent tagged DNA for labeling. We mixed fluorescent tagged DNA together with DNA origami staples in annealing solution.<br>
<h4 id=sensing>2-1 DNA Origami approach</h4>
In addition, to see if the origami binds to the fluorescent tagged DNA in shorter time, we added the fluorescent tagged DNA into control annealing solution, which contained no fluorescent tagged DNA, and left it for 40 minutes.<br>
<h5 id=5>2-1-1 Making DNA Origami</h5>
To see the origami was well labeled with fluorescent molecules, we used electrophoresis. <br>
<h5>Purpose</h5>
Electrophoresis was conducted with a 1% agarose gel, CV100V for 50 minutes.<br>
In our project, to use DNA Origami as the Key DNA to break liposomes, we design the rectangular DNA Origami with a chipped edge.
<br>
<h5>Method</h5>
Mixing M13mp18, staples, 5xTAE Mg2+, and mQ in a microtube and annealing for 2.5 hours.<br>
<A href="http://openwetware.org/wiki/Biomod/2013/Sendai/protocol">Protocol</A><br>
<A href="http://openwetware.org/wiki/Biomod/2013/Sendai/protocol">Protocol</A><br>
<h5>Result</h5>
We obtain DNA Origami same as our design. The result was confirmed by AFM (Atomic Force Microscope.)<br>
<br>
<br>
By scanning a gel before staining, we can see only the bands of DNAtructures with fluorescent molecules; scanning a gel after staining, we can see the bands of all DNAtructures. So we scanned a gel before and after staining (we scanned both a non-stained and a stained gel). <br>
<div align="center"><Img Src="http://openwetware.org/images/0/0f/%E3%82%AA%E3%83%AA%E3%82%AC%E3%83%9F%E3%81%AEfig.1.jpg"> </div><br>
First we saw the bands of our origami in a non-stained gel. Then, we compared the bands with those in a stained gel. If the bands of origami in a non-stained gel were at the same height as those in a stained gel, we can say that our origami is successfully fluorescently labeled.<br>
<div class="caption">Fig.2 AFM image of DNA Origami (M13: 4nM, staples:20nM)</div><br>
<br>
<br>


<h6>Result</h6>  
<h5>Discussion</h5>
In a non-stained gel (Fig.2), only bands in lane 3 and 4 from the left (*Ori, **Ori) can be seen. They are fluorescent labeled structures. In addition, as they gave the same result, 40 minutes is long enough for fluorescent labeling.<br>
<u>As shown in Fig. 2, DNA Origami was well-formed.</u><br>
<Img Src="http://openwetware.org/images/5/58/S_Outside-gel-3.2.png" width="300"><br>
Fig.2 Non-stained gel image: only bands in two lanes can be seen. From the left, they are DNA origami with fluorescent molecules in pre-annealing (Ori*), and DNA origami with fluorescent molecules in post-annealing (Ori**)<br>
<br>
<br>
In a stained gel (Fig.3), marker (lane 1) had the longest DNAtrand of 20kb. Comparing this and M13mp18 (lane 2) with annealed DNA origamis (lane 3,4,5), the bands of the origamis are at the higher position. Therefore, we concluded that in lane3~5, DNA origami structure made of M13 and staples were made as we had expected. <br>
<h5 id=6>2-1-2 Labeling DNA Origami with fluorescent-tagged DNA</h5>
We considered that the bands in lane3~5 are seen as if they were diffused, just because our origami has many staples binding to the fluorescent tagged DNA, and each origami attaches to different number of them, and its molecular weight varies.<br>
<h5>Purpose</h5>
<Img Src="http://openwetware.org/images/2/2d/S_Outside-gel-2.2.png" width="300"> </br>
To observe the fluorescent effect of DNA Origami on liposomes by microscope, we labeled our Origami by hybridizing with the fluorescent-tagged DNA strand.  
Fig.3 Stained gel image: from the left, marker, M13mp18, Ori*, Ori**, and DNA origami with no fluorescent molecule (Ori)<br>
<br><br>
 
<h5>Method</h5>
Our DNA Origami is composed of many staples that can bind to the same fluorescent-tagged DNA for labeling. We mixed fluorescent-tagged DNA with DNA Origami staples before annealing, and after annealing solution. Labeling of the DNA Origami was confirmed by gel-electrophoresis. Gel-electrophoresis was conducted with a 1% Agarose gel, 100V for 50 minutes.<br>
<A href="http://openwetware.org/wiki/Biomod/2013/Sendai/protocol">Protocol</A><br>
<br>
<br>
 
<h5>Result</h5>  
<h6>Discussion</h6>
Figure 3 shows florescence detection of the gel before staining (Left) and after SYBR Gold staining (Right). In a non-stained gel, only lane 3 and 4 (*Ori, **Ori) was found. The fluorescent bands of Origami in a non-stained gel were at the same height as that in a stained gel, we conclude that our Origami was successfully fluorescently labeled irrespective of the timing of adding the fluorescent DNA.<br>
Combining the results of Fig.2 and 3, the fluorescent labeled bands in lane3 and 4 in Fig.2 are at the same height as those of DNA origami in Fig.3. Thus, we concluded our origami was successfully fluorescently labeled.<br>
<div align="center">
<Img Src="http://openwetware.org/images/8/8e/Fig5and6.jpg" width="640" height="360"></div><br>
<div class="caption">Fig.3 Labeling of DNA origami by fluorescence tagged DNA. Left panel shows non-stained gels, and right panel shows the same gels after SYBR Gold stain. From the left, marker, M13mp18, DNA Origami with fluorescent molecules added in pre-annealing (Ori*), DNA Origami with fluorescent molecules added in post-annealing (Ori**), and DNA Origami with no fluorescent molecule (Ori).<br></div>
<br>
<br>
<h5>Discussion</h5>
<u>The results indicate we succeeded to label our Origami by the fluorescence DNA.</u><br>
<br>
<br>


<h5 id=7>2-1-3 Investigating the interaction of DNA origami and liposomes<h5>
<h5 id=7>2-1-3 Disrupting liposomes by DNA Origami (microscopic analysis)<h5>
<h6>Purpose</h6>
<h5>Purpose</h5>
To collapse liposome with our origami, first we investigated how our DNA origami affected liposomes.<br>
To break liposomes with our Origami, first we investigate how our DNA Origami affects liposomes.<br>
<br>
<br>
<h6>Principle</h6>
<h5>Principle</h5>
To collapse liposomes with our origami, many origamis have to hybridize with the surface of liposomes.<br>
To break liposomes with our Origami, a lot of Origami has to hybridize to the surface of the liposomes.<br>
To begin with, we added cholesterol-conjugated single-stranded DNA (in the rest of this document, referred to as Anchored DNA) into liposomes, and made them float on the surface. If the Anchored DNA have a complementary part to our origami, the origami is expected to hybridize with the surface. In this way, many origamis would hybridize with liposome via Anchored DNA.<br>
To begin with, we added cholesterol-conjugated single-stranded DNA (in the rest of this document, referred to as Origami-anchor DNA) into liposomes, and made it float on the surface. The Origami-anchor DNA has a complementary part to our Origami, so the Origami is expected to hybridize to Origami-anchor DNA on the liposomes. In this way, lots of Origami would hybridize to liposomes via Origami-anchor DNA.<br>
<br>
<br>
<h6>Method</h6>
<h5>Method</h5>
We added Anchored DNA into liposomes at the final concentration of 0.018, 0.069, 1.8, and 6.9µM. Then we observed the samples with a phase microscope. Next, adding fluorescently labeled DNA origamis into the above liposomes, we saw if some change would happen with a fluorescent microscope.<br>
To begin with, we mixed cholesterol-conjugated single-stranded DNA (in the rest of this document, referred to as Origami-anchor DNA) into liposomes, and made it float on the surface. Origami-anchor DNA has a complementary part to our Origami. We should note that the anchor DNA was added after liposome formation to avoid the anchor DNA are inserted on inner side of liposome. <br>
We added Origami-anchor DNA into liposomes at the final concentration of 0.018, 0.069, 1.8, and 6.9µM (NOTE: the concentration of DNA origami is constant, only the concentrations of the anchor DNA varied).  
Then we observed the samples with a fluorescent microscope. <br>
Next, adding the fluorescently labeled DNA Origami into the above liposomes, we observed the samples again.<br>
<A href="http://openwetware.org/wiki/Biomod/2013/Sendai/protocol">Protocol</A><br>
<A href="http://openwetware.org/wiki/Biomod/2013/Sendai/protocol">Protocol</A><br>
<br>
<br>
<h6>Result</h6>
<h5>Result</h5>
In all four conditions, liposomes were observed with a phase microscope. We confirmed the formation of multilamella liposomes (Fig.4~7).<br>
 
<br>
In all four conditions, liposomes were observed with a fluorescent microscope.  
We used the mixture of uni- and multi-lamella liposomes (Fig.4~7).<br>


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<Img Src="http://openwetware.org/images/7/72/Lipofig4.png" width="400"></br>
<div align="center"><Img Src="
Fig.4 Phase microscope image of liposomes (cholesterol-conjugated DNA: 0.018µM)<br>
http://openwetware.org/images/d/d7/2-2-1fig4.png"></div><br>
<div class="caption">Fig.4 Fluorescent microscope image of liposomes <br>(Origami-anchor DNA: 0.018µM; Left: without fluorescence, Right: with fluorescence)</div><br>
<br>
<div align="center"><Img Src="
http://openwetware.org/images/9/94/2-2-1fig5.png"></div><br>
<div class="caption">Fig.5 Fluorescent microscope image of liposomes <br>(Origami-anchor DNA: 0.069µM; Left: without fluorescence, Right: with fluorescence)</div><br>
<br>
In the presence of fluorescent molecules, when the concentration of Origami-anchor DNA was 0.018, 0.069µM, many gleaming (in green color) liposomes were observed. These results confirmed that the fluorescently labeled Origami well hybridized to the liposomal surface but that did not disrupt (Fig.4,5). <br>
<br>
<br>
<Img Src="http://openwetware.org/images/d/d0/Lipofig5.png" width="400"></br>
<div align="center"><Img Src="
Fig.5 Phase microscope image of liposomes (cholesterol-conjugated DNA: 0.069µM)<br>
http://openwetware.org/images/9/94/2-2-1fig6.png"></div><br>
<div class="caption">Fig.6 Fluorescent microscope image of liposomes <br>(Origami-anchor DNA: 1.8µM; Left: without fluorescence, Right: with fluorescence)</div><br>
<br>
<br>
<Img Src="http://openwetware.org/images/d/de/Lipofig6.png" width="400"></br>
On the other hand, when the concentration of Origami-anchor DNA was 1.8µM, few gleaming liposomes could be seen with a fluorescent microscope (Fig.6). This result indicates the possibility that liposomes have broken.<br>
Fig.6 Phase microscope image of liposomes (cholesterol-conjugated DNA: 1.8µM)<br>
<br>
<br>
<Img Src="http://openwetware.org/images/d/d7/Lipofig7.png" width="400"></br>
<div align="center"><Img Src="
Fig.7 Phase microscope image of liposomes (cholesterol-conjugated DNA: 6.9µM)<br>
http://openwetware.org/images/c/c0/2-2-1fig7.png"></div><br>
<div class="caption">Fig.7 Fluorescent microscope image of liposomes <br>(Origami-anchor DNA: 6.9µM; Left: without fluorescence, Right: with fluorescence)</div><br>
<br>
<br>
Adding fluorescently labeled DNA origamis into the above liposomes, we saw if some change would happen with a fluorescent microscope.<br>
When the concentration of Origami-anchor DNA is 6.9µM, some liposomes were gleaming and others distorted, forming networks (Fig.7).
When the concentration of Anchored DNA was 0.018, 0.069µM, many gleaming (in green color) liposomes were observed. We confirmed that the fluorescently labeled origamis well hybridized with the liposomal surface (Fig.8,9,10). <br>
<table>
<tr>
  <td>
  <Img Src="http://openwetware.org/images/6/6c/Lipofig8.png" width="400">
  </td>
  <td>
  <Img Src="http://openwetware.org/images/a/a6/Lipofig9.png" width="400">
  </td>
</tr>
</table>
Fig.8,9 fluorescent microscope image of liposomes (cholesterol-conjugated DNA: 0.018µM)<br>
<Img Src="http://openwetware.org/images/b/b4/Lipofig10.png" width="400"></br>
Fig.10 fluorescent microscope image of liposomes (cholesterol-conjugated DNA: 0.069µM)<br>
<br>
<br>
On the other hand, when the concentration of Anchored DNA was 1.8µM, few gleaming liposomes could be seen with a fluorescent microscope (Fig.11). This result indicates the possibility that liposomes were collapsed.<br>
<Img Src="http://openwetware.org/images/1/18/Lipofig11.png" width="400"></br>
Fig.11 fluorescent microscope image of liposomes (cholesterol-conjugated DNA: 1.8µM)<br>
<br>
<br>
When the concentration of Anchored DNA is 6.9µM, some liposomes were gleaming and others distorted, forming networks (Fig.12).<br>
<h5>Discussion</h5>
From these results, we put forward the following hypothesis about the interaction of DNA Origami and liposomes. When the concentration of the anchor DNA is low (0.018, 0.069µM), liposomes was still stable. When the concentration is middle (1.8µM), more DNA Origami hybridizes to the surface and loads on it. This loading made liposomes become fragile and easy to break. When the concentration is high (6.9µM), disrupted liposomes were connected with others, and consequently, form networks via Origami-anchor DNA and DNA Origami complex.
<Img Src="http://openwetware.org/images/8/88/Lipofig12.png" width="400"></br>
Fig.12 fluorescent microscope image of liposomes (cholesterol-conjugated DNA: 6.9µM)<br>
<br>
<br>
<h6>Discussion</h6>
Anyway, <u>these data strongly indicated the designed DNA origami disrupted liposomes with high concentration of the anchor DNA.</u>
From these results, we put forward the following hypothesis about the interaction of DNA origami and liposomes.<br>
When the concentration of Anchored DNA is low (0.018, 0.069µM), DNA origamis hybridize with the surface of the liposomes relatively stablely. When the concentration is middle (1.8µM), more DNA origamis hybridizes with the surface and place stress on it. Then, liposomes become fragile and easy to be collapsed. When the concentration is high (6.9µM), some liposomes exist individually, and others form networks via Anchored DNA and DNA origami complexes.<br>
<Img Src="http://openwetware.org/images/7/7c/Experimentinsidefig.png"><br>
<br>
<br>
According to this hypothesis, when the concentration of Anchored DNA is 1.8µM, DNA origami collapses liposomes. Therefore, in the following experiment, we checked if DNA origami would collapse liposomes at this concentration of Anchored DNA.<br>
<br>
<br>
<br>
<br>


<!--
<h5 id=13>2-1-4 Disrupting liposomes by DNA Origami (quantitative analysis)<h5>
国内大会後の記事。国内大会前の結果の方がよかったからけしておく
<h5>Purpose</h5>
<h5>2-1-3 DNAオリガミによりリポソームを破壊する実験</h5>
The above experiments in 2-1-3 microscopic analysis suggest that our DNA Origami disrupted liposomes. Thus, we performed more quantitative analysis.<br>
<h6>Purpose</h6>
<br>
DNAオリガミによるアプローチではDNAオリガミがリポソーム表面にハイブリしてリポソームに負荷をかけて割れる。本実験ではDNAオリガミによってリポソームが割れるかどうかを確かめる。<br>
<h5>Method</h5>
<div class="caption-right">
<img src="http://openwetware.org/images/f/f5/2-1-4liposome-size-graph%28lipo-leg-origami%29.png" style="padding-left:10mm;width: 250px;"><span> Fig.8 Threshold cutting in<br>the flow cytometer analysis<br>by EV-SS plot (Sample 3)</span>
</div>
We did the experiment using Flow cytometer (Cell Lab Quanta SC Flow Cytometer) in the same way as experiment 2-1-4. Only 7-13 μm diameter liposomes were analyzed (cut off by EV value) (Fig.8). Liposomes showing over 100 SS value (the indicator of sample complexity) were also omitted because of reliability of the data. Liposomes encapsulating GFP molecules were used in this experiment.<br>
<br>
Following 50 μL of samples(Fig.9) were examined with the Flow cytometer.
<A href="http://openwetware.org/wiki/Biomod/2013/Sendai/protocol">Protocol</A><br>
<img src="http://openwetware.org/images/9/95/2-1-4samplefigdeka.png">
<br><div class="caption">Fig.9 3 types of samples</div>
<br>


<h6>Method</h6>
<h5>Result</h5>
As figure below, we were able to observe liposomes containing GFP, by the confocal microscope.<br>
<div align="center">
<img src="http://openwetware.org/images/8/87/Lipo-scalebar-2.png"></div>
<br><div class="caption">Fig.10 Liposomes conteaining GFP(confocal microscope)</div>
<br>
<div align="center">
<img src="http://openwetware.org/images/8/85/2-1-4result.png"></div>
<br><div class="caption">Fig.11 Quantitative analysis by flow cytometer</div>


DNAオリガミに蛍光をハイブリさせたものをアニーリングにより作製する<br>
<br>
界面通過法によりリポソームをつくる<br>
The x axis of the above graph is the fluorescence intensity of only liposomes, and the y axis represents the number of liposomes.<br>
位相差顕微鏡でリポソーム2μℓを観察する（この時点ではまだ割れない）<br>
Without addition, liposomes having high fluorescence intensity (over 100 a.u.) were mainly observed. When a surfactant NP40, which must disrupt liposomes, were added, the fraction of the high fluorescence liposomes decreased. When the key DNA origami was added, the fraction of the high fluorescence liposomes were completely diminished.<br>
位相差顕微鏡で観察しているリポソーム2μℓにコレステロールレグ5μℓをピペッティングして混ぜ、観察する。（鍵DNAオリガミを入れていないのでこの時点ではまだ割れない）<br>
位相差顕微鏡で観察しているリポソーム＋コレステロールレグのサンプルに精製したDNAオリガミ4μℓを加え、顕微鏡で観察する<br>


Protocol
<h5> Discussion </h5>
（対応するプロトコルへのリンク）
<u>These results confirmed that our designed DNA origami actually disrupted liposomes. (The efficiency was higher than 2% surfactant!)</u>
<br>
<br>
<h5 id=8>2-1-5 Confirming sequence specificity of DNA flow cytometer</h5>
<h5>Purpose</h5>
To confirm the selectivity of Key DNAs to the anchor DNA, we compared the effect of the complementary Key DNA and the no-binding Key DNA.<br>
<br>
<h5>Method</h5>
Experimental conditions were the same in 2-1-4 except samples.<br>
Sample 1 (Complement). Liposomes + Origami-anchor DNA(A) + Key DNA(A)<br>
Sample 2 (no binding pair). Liposomes + Orgiami-anchor DNA(A) + Key DNA(B)<br>
<A href="http://openwetware.org/wiki/Biomod/2013/Sendai/protocol">Protocol</A><br>
<br>
<h5>Result</h5>
The results were shown in figure 12.<br>
<div align="center">
<img src="http://openwetware.org/images/6/6d/2-1-5matometagazou-deka.png" style="
    width: 90%;
"></div>
<br><div class="caption">Fig.12 Results of complementary key DNA and no binding Key DNA</div>


<h6>Result</h6>
リポソームのみの位相差顕微鏡の画像は図７のようになった。リポソームが確認できる。<br>
図７ リポソーム2μℓの位相差顕微鏡画像<br>
リポソームにコレステロールレグを加えたサンプルの位相差顕微鏡画像は図８のようになった。この時点でもリポソームが確認できた。<br>
図８ リポソーム2μℓ+コレステロールレグ5μℓの位相差顕微鏡画像<br>
リポソーム＋コレステロールレグに精製したDNAオリガミを加えた位相差顕微鏡画像は図９のようになった。DNAオリガミを加えた３分後にネットワークのような構造が確認された。このネットワーク構造はDNAオリガミを加える前は確認できなかったものである。<br>
図９ リポソーム2μℓ+コレステロールレグ5μℓ+精製したDNAオリガミ4μℓの位相差顕微鏡画像<br>
<h6>Discussion</h6>
このネットワーク構造はDNAオリガミを加えたあとに発生した。そのため、このネット―ワークのような構造はリポソームが割れた跡なのではないかと考えられる。<br>
しかし、今回は顕微鏡で観察しているリポソームにコレステロールレグやDNAオリガミのサンプルを加えたためリポソームの濃度が薄くなるので一つのリポソームを定点観察することが難しく、リポソームが割れる瞬間を観察できなかった。<br>
浸透圧で割れない程度の粘性の高いリポソームやアガロースゲルで固定してリポソームを観察すれば定点観察できると考えられる。<br>
-->
<h5 id=8>2-1-4 DNAによる配列特異性を証明する実験</h5>
<h6>Purpose</h6>
このプロジェクトにおいてリポソームをDNAで割る理由は、DNAの配列特異性を利用して割れるリポソーム間に関係性を持たせるためである。そこで配列の異なる２種類のDNAを生やしたリポソームを用意して片方だけに相補なDNAを加え、２種類のリポソームのうちDNAが相補になっている片方のリポソームだけが破壊されることを確かめた。<br>
<h6>Method</h6>
内部にGFP（緑の蛍光）を含んだリポソームAとローダミン（赤の蛍光）を含んだリポソームBの２種類のリポソームを、界面通過法により作製する。どちらのリポソームも
<!--脂質の組成は？-->
<br>
<br>
リポソームAには5'-CCAGAAGACG-chol-3'の配列を持つコレステロール付きのDNAをリポソームBには配列5'-TCCACTAACG-chol-3'をもつコレステロール付きのDNAを振り掛けた。<br>
In the sample1, Origami-anchor DNA and Key DNA are complementary each other. In the sample B, the Key DNA has a different sequence that does not hybridize with anchor DNA. The X axis in the figures shows fluorescent intensity. Y axis indicate the number of count. High fluorescence (>100) means liposome with GFP, low fluorescence means that GFP inside liposomes were leaked. The non-binding key DNA does not affect liposome with anchor DNA. On the other hands, the complement key DNA disrupt liposomes.<br>
1分間遠心分離器にかけて、リポソームにくっつかなったコレステロール付きDNAとリポソームを分離する。<br>
<br>
リポソームAとリポソームBが入っているリポソームを1μℓずつ混合し、位相差顕微鏡で観察した。<br>
<h5>Discussion</h5>
精製したDNAオリガミ
<u>These results demonstrated the selectivity of the Key DNA sequence, and strongly supported our designed DNA actually disrupted liposomes via hybridization of the Key DNA and the anchor (keyhole) DNAs.</u><br>
<!--蛍光ついている？？-->
<br>
を4μℓをリポソームA,Bの混合サンプルに加え、位相差顕微鏡で観察した。<br>
Protocol<br>
（対応するプロトコルへのリンク）<br>


<h6>Result</h6>


<h6>Discussion</h6>
<h4 id=9>2-2 Flower DNA approach</h4>
 
<!-------------SPRコメントアウトここから-------------------->
<h4 id=9>2-2 フラワーミセルによるアプローチ</h4>
<!--
<h5 id=10> 2-2-1 SPRによるループ構造の確認</h5>
<h5 id=10> 2-2-1 Confirming the formation of the loop structure by SPR</h5>
<h6>Purpose</h6>
<h5>Purpose</h5>
To collapse liposomes by flower micelle method, we aim to attach many loop strands to the surface of liposomes. <br>
To break liposomes by flower DNA method, we aim to attach many loop strands to the surface of liposomes. <br>
To achieve this, we adopt the same hybridization method via Anchored DNA as used in i)Bending approach into liposomes: the Anchored DNA has a complementary part to our loop strand and the loop strand is expected to hybridize with liposomes.<br>
To achieve this, we adopt the same hybridization method via Anchored DNA as used in i)Bending approach into liposomes: the Anchored DNA has a complementary part to our loop strand and the loop strand is expected to hybridize to liposomes.<br>
We checked the hybridization of liposomes and Anchored DNA, and that of Anchored DNA and our loop strands. <br>
We checked the hybridization of liposomes and Anchored DNA, and that of Anchored DNA and our loop strands. <br>
<br>
<br>


<h6>Principle</h6>
<h5>Principle</h5>
As our loop strand is too small to observe with an AFM or a fluorescent microscope, we used an apparatus called SPR.<br>
As our loop strand is too small to observe with an AFM or a fluorescent microscope, we used an apparatus called SPR.<br>
SPR is a Surface Plasmon Resonance assay that estimates the weight of molecules attached to membrane surface, by the change of the reflection of the laser beam.<br>
SPR is a Surface Plasmon Resonance assay that estimates the weight of molecules attached to membrane surface, by the change of the reflection of the laser beam.<br>
Line 300: Line 301:
<br>
<br>


<h6>Method</h6>
<h5>Method</h5>
<ur><li>1. Inject 45µl DOPC (100mM) into SPR</li>
<ur><li>1. Inject 45µl DOPC (100mM) into SPR</li>
<li>2. Inject 5µl NAOH to SPR in order to stabilize the point</li>
<li>2. Inject 5µl NAOH to SPR in order to stabilize the point</li>
<li>3. Inject 10µl Anchored DNA (0.1µM) to SPR</li>
<li>3. Inject 10µl Anchored DNA (0.1µM) to SPR</li>
<li>4. Inject 10µl loop DNA of 40 bp (0.1µM) to SPR</li>
<li>4. Inject 10µl loop DNA of 40 bp (0.1µM) to SPR</li>
<li>5. Inject 10 µl trigger DNA of 40 bp (0.1µM) to SPR</li>
<li>5. Inject 10 µl Key DNA of 40 bp (0.1µM) to SPR</li>
<br>
<br>
<br>
<A href="http://openwetware.org/wiki/Biomod/2013/Sendai/protocol">Protocol</A><br>


<h6>Result</h6>
<h5>Result</h5>
The result was shown in Fig.15 below.<br>
The result was shown in Fig.15 below.<br>
 
 
<Img Src="http://openwetware.org/images/f/fd/Flowerex2.png"></br>
<Img Src="http://openwetware.org/images/f/fd/Flowerex2.png"></br>
Fig.15 The transition of SPR value<br>
Fig.13 The transition of SPR value<br>
<br>
<br>
As the first injection of Anchored DNA caused no change of SPR value, we injected Anchored DNA for two times. <br>
As the first injection of Anchored DNA caused no change of SPR value, we injected Anchored DNA for two times. <br>
Fig 15 shows that SPR value increased after injecting Anchored DNA and loop DNA. Moreover, we should note that after injecting trigger DNAome changes of SPR value were observed.<br>
Fig 13 shows that SPR value increased after injecting Anchored DNA and loop DNA. Moreover, we should note that after injecting Key DNA some changes of SPR value were observed.<br>
<br>
<br>


<h6>Discussion</h6>
<h5>Discussion</h5>
Fig.15 shows the behavior of materials on the surface of liposomes. The increase of SPR value after injecting Anchored DNA indicates that Anchored DNA successfully combined with liposomes.
Fig.13 shows the behavior of materials on the surface of liposomes. The increase of SPR value after injecting Anchored DNA indicates that Anchored DNA successfully combined with liposomes.
Similarly, it is considered that loop DNA combined with Anchored DNA. <br>
Similarly, it is considered that loop DNA combined with Anchored DNA. <br>
Thus, we confirmed the formation of the loop structures on liposomes.<br>
Thus, <u>we confirmed the formation of the loop structures on liposomes.</i><br>
<br>
<br>
-->
<!---------------SPRコメントアウトここまで------------------>


<h5 id=11>2-2-2フラワーミセルによりリポソームを破壊する実験</h5>
<h6>Purpose</h6>
フラワーミセルアプローチでは鍵DNAストランドがリポソーム表面に生えているアンカーDNAにハイブリしてリポソームが割れる必要がある。それを確かめるために。


<h6>Method</h6>
DNAオリガミに蛍光をハイブリさせたものをアニーリングにより作製する
膜染色（テキサスレッド）したリポソームをつくる
リポソームのみを蛍光顕微鏡で観察する。
リポソームにDNAオリガミを加えてその後の様子を観察する
（対応するプロトコルへのリンク）


<h6>Result</h6>
<h5 id=11>2-2-1 Liposome disruption by flower DNA approach</h5>
<h5>Purpose</h5>
We evaluated whether flower DNA approach works to disrupt liposomes.<br>
<br>
<h5>Method</h5>
We made phase-separated liposomes (DOPC: DPPC: cholesterol= 1: 1: 1) with encapsulating texas-Red (conjugated with dextran) dye inside by the water-in-oil emulsion method. Because of the difference of solid-liquid temperature between critical DOPC and DPPC, these two lipids were phase separated at room temperature. Cholesterol, which is also used in the anchor DNA, presents in the both phases. We expected that the anchor DNA in the solid phase (DPPC domain) provide strong stress on liposomes by hybridizing with the Key DNA. <br>
<br>
First, the Flower-anchor DNA (stained with SYBR Gold) was added into the liposomes.
Next, we added the Key DNA that stretches the Flower anchored DNA into the mixture. The liposomes were observed in a chamber on a slide glass with a fluorescent microscope.<br>
<A href="http://openwetware.org/wiki/Biomod/2013/Sendai/protocol">Protocol</A><br>


<h6>Discussion</h6>


<h5 id=12>2-2-3 DNAによる配列特異性を証明する実験</h5>
<h5>Result</h5>
<h6>Purpose</h6>
<div align="center">
<img src="http://openwetware.org/images/2/21/S-%E3%83%95%E3%83%A9%E3%83%AF%E3%83%BC%E3%80%80%E6%9C%80%E5%88%9D%E3%81%AE%E7%94%BB%E5%83%8F.jpg"></div>
<div class="caption">Fig.13 Results of complementary key DNA and no binding Key DNA</div><br>


<h6>Method</h6>
We observed shrinking of liposomes (detected by red filter) by adding the Key DNA (Figure 13). Shifting to green filter showed that the flower-anchor DNA (dyed with SYBR Gold) presented around shrunk liposomes. We should note that such aggregation of the flower anchor DNA was not observed before the addition of the key DNA.<br>
（対応するプロトコルへのリンク）
<div align="center"><img src="http://openwetware.org/images/5/5e/2-2-1fig7%E3%81%82%E3%81%A3%E3%81%97%E3%82%85%E3%81%8F%E3%81%B0%E3%82%93.png"></div><br>
 
<div class="caption">Fig.14 The contact surface between Key DNA solution and liposomes</div>
<h6>Result</h6>
Next, we made solution mixing chamber. In this chamber, we can observe the contact surface between Key DNA solution and liposomes solution. The right side of the boundary is Key DNA solution and the left side is liposome solution. Network structures (Green, which indicate the anchor DNA) were observed on the boundary of solutions. This result strongly supported the key DNA is the factor to form the network structure. <div align="center"><br><img src="http://openwetware.org/images/0/03/S-tashiro1.jpg">
 
<img src="http://openwetware.org/images/2/29/S-tashiro2.jpg"></div><br>
<h6>Discussion</h6>
<div class="caption">Fig.15 Zoom up of Fig.14</div><br>
When zoom up in the networks, liposomes that lost Texas-Red dextran were observed. <br>
<br>


<h5>Discussion</h5>
In this experiment, three different changes were observed by adding the Key DNA: i) shrinking, ii) network structure formation, and iii) leak of fluorescence. <u>These results suggest that we achieved disruption of liposomes by our flower DNA approach. </u><br>
<br>
<h5 id=12> 2-2-2 Confirming sequence specificity of DNA</h5>
<h5>Purpose</h5>
We demonstrate the selectivity of our Key DNA: the Key DNA only affects the corresponding Flower-anchor DNA and liposomes.<br>
<br>
<h5>Method</h5>
Two types of phase separated liposomes were prepared by droplet transfer methods. One type is liposomes with GFP inside (Green liposome); the other type is liposomes with Texas-Red dextran inside (Red liposome).<br>
The anchor DNA for Green liposome is named “A-flower-anchor DNA”, and the anchor DNA for Red liposome is named “B-flower-anchor DNA”. Each flower-anchor DNA can bind only the complementary Key DNA. This time, only Key DNA for Red liposomes (complementary to B-flower-anchor DNA) is added.<br>
After adding B-Key DNA, the number of each color liposomes is counted to confirm the selectivity.<br>
As a control, only buffer is added instead of B-Key DNA.<br>
<A href="http://openwetware.org/wiki/Biomod/2013/Sendai/protocol">Protocol</A><br>
<br>
<h5>Result</h5>
Fig.16 shows fluorescent microscope image of liposomes added B-Key DNA (left) and control buffer(right).
Green rectangles represent Green liposomes; Red, Red liposomes.<br>
In the left, only Green liposomes (marked with green rectangles) and no Red liposomes can be seen.
In the right, almost the same number of Green and Red liposomes are seen.<br>
<div align="center"><img src="http://openwetware.org/images/9/94/2-2-2fig%E3%81%95%E3%81%84%E3%81%94tyannpiyonn.png"></div>
<div class="caption">Fig.16　fluorescent microscope image of liposomes (left: with B-Key DNA, right: with control buffer)</div><br>
<table border cellspacing="3" bgcolor="lightyellow">
<tr bgcolor="moccasin">
<td> Additives </td>
<td> B-key DNA </td>
<td> Buffer </td>
</tr>
<tr bgcolor="moccasin">
<td> Green：Red </td>
<td>17:2 (n = 19)</td>
<td>16:17 (n= 33)</td>
</tr>
</table>
<div class="captiontable">Table1  Ratio of Green and Red liposomes</div><br>
Table1 shows the Ratio of Green and Red liposomes.<br>
When the control buffer is added, the number of Green and Red liposomes are almost the same. On the other hand, when B-Key DNA is added, much less number of red liposomes is seen compared to the number of Green liposomes. Other images are shown in <A href="http://openwetware.org/wiki/Biomod/2013/Sendai/protocol">Protocol</A>.<br>
<br>
<h5>Discussion</h5>
Comparing the left and right images, the ratio of Red to Green liposomes decreases due to the addition of B-Key DNA. This means that B-Key DNA only breaks Red liposomes and it has little effect on Green liposomes.<br>
<u>The selectivity of Key DNA has been successfully demonstrated. </u></h6>


         </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>Experiment</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">1</span> <span class="toctext">First stage: Sensing system</span></a></li> <ul> <li class="toclevel-2"><a href="#bending"> <span class="tocnumber">1-1</span> <span class="toctext">Disruption of temperature sensitive liposomes</span></a></li> </ul> <li class="toclevel-1"><a href="#Flower"> <span class="tocnumber">2</span> <span class="toctext">Second stage: Amplification system</span></a></li> <ul> <li class="toclevel-2"><a href="#sensing"> <span class="tocnumber">2-1</span> <span class="toctext">DNA Origami approach </span></a></li> <ul> <li class="toclevel-2"><a href="#5"> <span class="tocnumber">2-1-1</span> <span class="toctext">Making DNA Origami</span></a></li> <li class="toclevel-2"><a href="#6"> <span class="tocnumber">2-1-2</span> <span class="toctext">Labeling DNA Origami with fluorescent-tagged DNA</span></a></li>

<li class="toclevel-2"><a href="#7"> <span class="tocnumber">2-1-3</span> <span class="toctext">Disruption of liposomes by DNA Origami (microscopic analysis)</span></a></li> <li class="toclevel-2"><a href="#13"> <span class="tocnumber">2-1-4</span> <span class="toctext">Disruption of liposomes by DNA Origami (quantitative analysis)</span></a></li>

<li class="toclevel-2"><a href="#8"> <span class="tocnumber">2-1-5</span> <span class="toctext">Confirming sequence specificity of DNA</span></a></li> </ul> <li class="toclevel-1"><a href="#9"> <span class="tocnumber">2-2</span> <span class="toctext">Flower DNA approach</span></a></li> <ul>

<li class="toclevel-2"><a href="#11"> <span class="tocnumber">2-2-1</span> <span class="toctext">Disruption of liposomes by Flower DNA</span></a></li> <li class="toclevel-2"><a href="#12"> <span class="tocnumber">2-2-2</span> <span class="toctext">Confirming sequence specificity of DNA</span></a></li>


</li>


</ul> </li> </ul> </td></tr></table>

<h3 id=chain>First stage: Sensing system</h3> <h4 id=bending>1-1Disruption of temperature sensitive liposomes</h4> <h5>Purpose</h5> In our project, we planned to use liposomes conjugated with NIPAM polymer as a chain reaction initiator. NIPAM (poly-N-isopropyl acrylamide) is a temperature sensitive molecule that has a unique critical temperature (Tc: 32°C ). <br> When the temperature increased over than Tc, the hydrophilic polymer changes its property hydrophobic. It is expected that the change should disrupt the membrane lipid alignment. Here we confirm that the possibility of breaking liposomes with NIPAM by increasing temperature.<br> NIPAM was purchesed from SIGMA ALDRICH.<br> <br>

<h5>Method</h5> The liposomes were prepared by natural swelling method. Obtained sample included a mixture of unilamellar and multilamellar liposomes.<br> Then we added NIPAM-conjugated lipids (dissolved in ultra pure water (Milli-Q)) to the liposomes solution.<br> The liposomes were observed on the slide glass by phase-contrast microscopy. <br> After confirming the formation of the liposomes, a petri dish with hot water (~90˚C) was put on the sample slide glass to increase the temperature.<br> Detailed Protocol<br> <A href="http://openwetware.org/wiki/Biomod/2013/Sendai/protocol">Protocol</A><br>

<h5>Result</h5>


<img src="http://openwetware.org/images/9/9e/2-1-1fig1.png" width="100%" height="100%"><br>

<div class="caption">Fig.1 Phase contrast images of liposomes in NIPAM solution. Temperature increased from RT to enough over than Tc (left to right).</div><br> <br> Fig.1 shows the continuous images before and after the temperature increase. The view sight was the same position. <br> NIPAM polymer turned into globular states with increasing temperature. Liposomes disappeared by increasing temperature (> Tc).<br>


<br>


<h5>Discussion</h5> Thermosensitive polymer NIPAM can disrupt the coexisting liposomes by the polymers phase transition. <br> On the other hand, some liposomes still present even at the high temperature. In this experiment, some fractions were multi-lamellar liposomes. Since globular states of NIPAM (hydrophobic) at high temperature attack the liposome membrane from the outside, it is not surprising that the multi-lamellar liposomes consist of many lipid bilayers are more difficult to disrupt. Therefore, we suppose that liposomes disrupted by temperature shift in Fig.1 were uni-lamella ones. <br> <u>These results confirmed that triggering by heat disrupted the liposomes.</u><br>


<h3 id=Flower>Second stage: Amplification system</h5> <h4 id=sensing>2-1 DNA Origami approach</h4> <h5 id=5>2-1-1 Making DNA Origami</h5> <h5>Purpose</h5> In our project, to use DNA Origami as the Key DNA to break liposomes, we design the rectangular DNA Origami with a chipped edge. <br>

<h5>Method</h5> Mixing M13mp18, staples, 5xTAE Mg2+, and mQ in a microtube and annealing for 2.5 hours.<br> <A href="http://openwetware.org/wiki/Biomod/2013/Sendai/protocol">Protocol</A><br>


<h5>Result</h5> We obtain DNA Origami same as our design. The result was confirmed by AFM (Atomic Force Microscope.)<br> <br> <div align="center"><Img Src="http://openwetware.org/images/0/0f/%E3%82%AA%E3%83%AA%E3%82%AC%E3%83%9F%E3%81%AEfig.1.jpg"> </div><br> <div class="caption">Fig.2 AFM image of DNA Origami (M13: 4nM, staples:20nM)</div><br> <br>

<h5>Discussion</h5> <u>As shown in Fig. 2, DNA Origami was well-formed.</u><br> <br> <h5 id=6>2-1-2 Labeling DNA Origami with fluorescent-tagged DNA</h5> <h5>Purpose</h5> To observe the fluorescent effect of DNA Origami on liposomes by microscope, we labeled our Origami by hybridizing with the fluorescent-tagged DNA strand. <br><br>

<h5>Method</h5> Our DNA Origami is composed of many staples that can bind to the same fluorescent-tagged DNA for labeling. We mixed fluorescent-tagged DNA with DNA Origami staples before annealing, and after annealing solution. Labeling of the DNA Origami was confirmed by gel-electrophoresis. Gel-electrophoresis was conducted with a 1% Agarose gel, 100V for 50 minutes.<br> <A href="http://openwetware.org/wiki/Biomod/2013/Sendai/protocol">Protocol</A><br> <br> <h5>Result</h5> Figure 3 shows florescence detection of the gel before staining (Left) and after SYBR Gold staining (Right). In a non-stained gel, only lane 3 and 4 (*Ori, **Ori) was found. The fluorescent bands of Origami in a non-stained gel were at the same height as that in a stained gel, we conclude that our Origami was successfully fluorescently labeled irrespective of the timing of adding the fluorescent DNA.<br> <div align="center"> <Img Src="http://openwetware.org/images/8/8e/Fig5and6.jpg" width="640" height="360"></div><br> <div class="caption">Fig.3 Labeling of DNA origami by fluorescence tagged DNA. Left panel shows non-stained gels, and right panel shows the same gels after SYBR Gold stain. From the left, marker, M13mp18, DNA Origami with fluorescent molecules added in pre-annealing (Ori*), DNA Origami with fluorescent molecules added in post-annealing (Ori**), and DNA Origami with no fluorescent molecule (Ori).<br></div> <br> <br> <h5>Discussion</h5> <u>The results indicate we succeeded to label our Origami by the fluorescence DNA.</u><br> <br>

<h5 id=7>2-1-3 Disrupting liposomes by DNA Origami (microscopic analysis)<h5> <h5>Purpose</h5> To break liposomes with our Origami, first we investigate how our DNA Origami affects liposomes.<br> <br> <h5>Principle</h5> To break liposomes with our Origami, a lot of Origami has to hybridize to the surface of the liposomes.<br> To begin with, we added cholesterol-conjugated single-stranded DNA (in the rest of this document, referred to as Origami-anchor DNA) into liposomes, and made it float on the surface. The Origami-anchor DNA has a complementary part to our Origami, so the Origami is expected to hybridize to Origami-anchor DNA on the liposomes. In this way, lots of Origami would hybridize to liposomes via Origami-anchor DNA.<br> <br> <h5>Method</h5> To begin with, we mixed cholesterol-conjugated single-stranded DNA (in the rest of this document, referred to as Origami-anchor DNA) into liposomes, and made it float on the surface. Origami-anchor DNA has a complementary part to our Origami. We should note that the anchor DNA was added after liposome formation to avoid the anchor DNA are inserted on inner side of liposome. <br> We added Origami-anchor DNA into liposomes at the final concentration of 0.018, 0.069, 1.8, and 6.9µM (NOTE: the concentration of DNA origami is constant, only the concentrations of the anchor DNA varied). Then we observed the samples with a fluorescent microscope. <br> Next, adding the fluorescently labeled DNA Origami into the above liposomes, we observed the samples again.<br> <A href="http://openwetware.org/wiki/Biomod/2013/Sendai/protocol">Protocol</A><br> <br> <h5>Result</h5>

In all four conditions, liposomes were observed with a fluorescent microscope. We used the mixture of uni- and multi-lamella liposomes (Fig.4~7).<br>

<!--ここは表を使ってコンパクトに-->

<div align="center"><Img Src=" http://openwetware.org/images/d/d7/2-2-1fig4.png"></div><br> <div class="caption">Fig.4 Fluorescent microscope image of liposomes <br>(Origami-anchor DNA: 0.018µM; Left: without fluorescence, Right: with fluorescence)</div><br> <br> 

<div align="center"><Img Src="

http://openwetware.org/images/9/94/2-2-1fig5.png"></div><br> <div class="caption">Fig.5 Fluorescent microscope image of liposomes <br>(Origami-anchor DNA: 0.069µM; Left: without fluorescence, Right: with fluorescence)</div><br> <br> In the presence of fluorescent molecules, when the concentration of Origami-anchor DNA was 0.018, 0.069µM, many gleaming (in green color) liposomes were observed. These results confirmed that the fluorescently labeled Origami well hybridized to the liposomal surface but that did not disrupt (Fig.4,5). <br> <br> <div align="center"><Img Src=" http://openwetware.org/images/9/94/2-2-1fig6.png"></div><br> <div class="caption">Fig.6 Fluorescent microscope image of liposomes <br>(Origami-anchor DNA: 1.8µM; Left: without fluorescence, Right: with fluorescence)</div><br> <br> On the other hand, when the concentration of Origami-anchor DNA was 1.8µM, few gleaming liposomes could be seen with a fluorescent microscope (Fig.6). This result indicates the possibility that liposomes have broken.<br> <br> <div align="center"><Img Src=" http://openwetware.org/images/c/c0/2-2-1fig7.png"></div><br> <div class="caption">Fig.7 Fluorescent microscope image of liposomes <br>(Origami-anchor DNA: 6.9µM; Left: without fluorescence, Right: with fluorescence)</div><br> <br> When the concentration of Origami-anchor DNA is 6.9µM, some liposomes were gleaming and others distorted, forming networks (Fig.7). <br> <br> <h5>Discussion</h5> From these results, we put forward the following hypothesis about the interaction of DNA Origami and liposomes. When the concentration of the anchor DNA is low (0.018, 0.069µM), liposomes was still stable. When the concentration is middle (1.8µM), more DNA Origami hybridizes to the surface and loads on it. This loading made liposomes become fragile and easy to break. When the concentration is high (6.9µM), disrupted liposomes were connected with others, and consequently, form networks via Origami-anchor DNA and DNA Origami complex. <br> Anyway, <u>these data strongly indicated the designed DNA origami disrupted liposomes with high concentration of the anchor DNA.</u> <br> <br> <br>

<h5 id=13>2-1-4 Disrupting liposomes by DNA Origami (quantitative analysis)<h5> <h5>Purpose</h5> The above experiments in 2-1-3 microscopic analysis suggest that our DNA Origami disrupted liposomes. Thus, we performed more quantitative analysis.<br> <br> <h5>Method</h5> <div class="caption-right"> 

<img src="http://openwetware.org/images/f/f5/2-1-4liposome-size-graph%28lipo-leg-origami%29.png" style="padding-left:10mm;width: 250px;"><span> Fig.8 Threshold cutting in<br>the flow cytometer analysis<br>by EV-SS plot (Sample 3)</span>

</div> We did the experiment using Flow cytometer (Cell Lab Quanta SC Flow Cytometer) in the same way as experiment 2-1-4. Only 7-13 μm diameter liposomes were analyzed (cut off by EV value) (Fig.8). Liposomes showing over 100 SS value (the indicator of sample complexity) were also omitted because of reliability of the data. Liposomes encapsulating GFP molecules were used in this experiment.<br> <br> Following 50 μL of samples(Fig.9) were examined with the Flow cytometer. <A href="http://openwetware.org/wiki/Biomod/2013/Sendai/protocol">Protocol</A><br> 

<img src="http://openwetware.org/images/9/95/2-1-4samplefigdeka.png">

<br><div class="caption">Fig.9 3 types of samples</div> <br>

<h5>Result</h5> As figure below, we were able to observe liposomes containing GFP, by the confocal microscope.<br> <div align="center"> <img src="http://openwetware.org/images/8/87/Lipo-scalebar-2.png"></div> <br><div class="caption">Fig.10 Liposomes conteaining GFP(confocal microscope)</div> <br> <div align="center"> <img src="http://openwetware.org/images/8/85/2-1-4result.png"></div> <br><div class="caption">Fig.11 Quantitative analysis by flow cytometer</div>

<br> The x axis of the above graph is the fluorescence intensity of only liposomes, and the y axis represents the number of liposomes.<br> Without addition, liposomes having high fluorescence intensity (over 100 a.u.) were mainly observed. When a surfactant NP40, which must disrupt liposomes, were added, the fraction of the high fluorescence liposomes decreased. When the key DNA origami was added, the fraction of the high fluorescence liposomes were completely diminished.<br>

<h5> Discussion </h5> <u>These results confirmed that our designed DNA origami actually disrupted liposomes. (The efficiency was higher than 2% surfactant!)</u> <br> <br> <h5 id=8>2-1-5 Confirming sequence specificity of DNA flow cytometer</h5> <h5>Purpose</h5> To confirm the selectivity of Key DNAs to the anchor DNA, we compared the effect of the complementary Key DNA and the no-binding Key DNA.<br> <br> <h5>Method</h5> Experimental conditions were the same in 2-1-4 except samples.<br> Sample 1 (Complement). Liposomes + Origami-anchor DNA(A) + Key DNA(A)<br> Sample 2 (no binding pair). Liposomes + Orgiami-anchor DNA(A) + Key DNA(B)<br> <A href="http://openwetware.org/wiki/Biomod/2013/Sendai/protocol">Protocol</A><br> <br> <h5>Result</h5> The results were shown in figure 12.<br> <div align="center"> <img src="http://openwetware.org/images/6/6d/2-1-5matometagazou-deka.png" style=" 

   width: 90%;

"></div> <br><div class="caption">Fig.12 Results of complementary key DNA and no binding Key DNA</div>

<br> In the sample1, Origami-anchor DNA and Key DNA are complementary each other. In the sample B, the Key DNA has a different sequence that does not hybridize with anchor DNA. The X axis in the figures shows fluorescent intensity. Y axis indicate the number of count. High fluorescence (>100) means liposome with GFP, low fluorescence means that GFP inside liposomes were leaked. The non-binding key DNA does not affect liposome with anchor DNA. On the other hands, the complement key DNA disrupt liposomes.<br> <br> <h5>Discussion</h5> <u>These results demonstrated the selectivity of the Key DNA sequence, and strongly supported our designed DNA actually disrupted liposomes via hybridization of the Key DNA and the anchor (keyhole) DNAs.</u><br> <br>


<h4 id=9>2-2 Flower DNA approach</h4> <!-------------SPRコメントアウトここから--------------------> <!-- <h5 id=10> 2-2-1 Confirming the formation of the loop structure by SPR</h5> <h5>Purpose</h5> To break liposomes by flower DNA method, we aim to attach many loop strands to the surface of liposomes. <br> To achieve this, we adopt the same hybridization method via Anchored DNA as used in i)Bending approach into liposomes: the Anchored DNA has a complementary part to our loop strand and the loop strand is expected to hybridize to liposomes.<br> We checked the hybridization of liposomes and Anchored DNA, and that of Anchored DNA and our loop strands. <br> <br>

<h5>Principle</h5> As our loop strand is too small to observe with an AFM or a fluorescent microscope, we used an apparatus called SPR.<br> SPR is a Surface Plasmon Resonance assay that estimates the weight of molecules attached to membrane surface, by the change of the reflection of the laser beam.<br> If Anchored DNA attaches to a liposome, and then loop strand attaches to it, SPR value increases after each step.<br> We measured SPR value after each step of adding DOPC into liposomes, and loop DNA into it.<br> <br>

<h5>Method</h5> <ur><li>1. Inject 45µl DOPC (100mM) into SPR</li> <li>2. Inject 5µl NAOH to SPR in order to stabilize the point</li> <li>3. Inject 10µl Anchored DNA (0.1µM) to SPR</li> <li>4. Inject 10µl loop DNA of 40 bp (0.1µM) to SPR</li> <li>5. Inject 10 µl Key DNA of 40 bp (0.1µM) to SPR</li> <br> <A href="http://openwetware.org/wiki/Biomod/2013/Sendai/protocol">Protocol</A><br>

<h5>Result</h5> The result was shown in Fig.15 below.<br>

<Img Src="http://openwetware.org/images/f/fd/Flowerex2.png"></br> Fig.13 The transition of SPR value<br> <br> As the first injection of Anchored DNA caused no change of SPR value, we injected Anchored DNA for two times. <br> Fig 13 shows that SPR value increased after injecting Anchored DNA and loop DNA. Moreover, we should note that after injecting Key DNA some changes of SPR value were observed.<br> <br>

<h5>Discussion</h5> Fig.13 shows the behavior of materials on the surface of liposomes. The increase of SPR value after injecting Anchored DNA indicates that Anchored DNA successfully combined with liposomes. Similarly, it is considered that loop DNA combined with Anchored DNA. <br> Thus, <u>we confirmed the formation of the loop structures on liposomes.</i><br> <br> --> <!---------------SPRコメントアウトここまで------------------>


<h5 id=11>2-2-1 Liposome disruption by flower DNA approach</h5> <h5>Purpose</h5> We evaluated whether flower DNA approach works to disrupt liposomes.<br> <br> <h5>Method</h5> We made phase-separated liposomes (DOPC: DPPC: cholesterol= 1: 1: 1) with encapsulating texas-Red (conjugated with dextran) dye inside by the water-in-oil emulsion method. Because of the difference of solid-liquid temperature between critical DOPC and DPPC, these two lipids were phase separated at room temperature. Cholesterol, which is also used in the anchor DNA, presents in the both phases. We expected that the anchor DNA in the solid phase (DPPC domain) provide strong stress on liposomes by hybridizing with the Key DNA. <br> <br> First, the Flower-anchor DNA (stained with SYBR Gold) was added into the liposomes. Next, we added the Key DNA that stretches the Flower anchored DNA into the mixture. The liposomes were observed in a chamber on a slide glass with a fluorescent microscope.<br> <A href="http://openwetware.org/wiki/Biomod/2013/Sendai/protocol">Protocol</A><br>


<h5>Result</h5> <div align="center"> <img src="http://openwetware.org/images/2/21/S-%E3%83%95%E3%83%A9%E3%83%AF%E3%83%BC%E3%80%80%E6%9C%80%E5%88%9D%E3%81%AE%E7%94%BB%E5%83%8F.jpg"></div> <div class="caption">Fig.13 Results of complementary key DNA and no binding Key DNA</div><br>

We observed shrinking of liposomes (detected by red filter) by adding the Key DNA (Figure 13). Shifting to green filter showed that the flower-anchor DNA (dyed with SYBR Gold) presented around shrunk liposomes. We should note that such aggregation of the flower anchor DNA was not observed before the addition of the key DNA.<br> <div align="center"><img src="http://openwetware.org/images/5/5e/2-2-1fig7%E3%81%82%E3%81%A3%E3%81%97%E3%82%85%E3%81%8F%E3%81%B0%E3%82%93.png"></div><br> <div class="caption">Fig.14 The contact surface between Key DNA solution and liposomes</div> Next, we made solution mixing chamber. In this chamber, we can observe the contact surface between Key DNA solution and liposomes solution. The right side of the boundary is Key DNA solution and the left side is liposome solution. Network structures (Green, which indicate the anchor DNA) were observed on the boundary of solutions. This result strongly supported the key DNA is the factor to form the network structure. <div align="center"><br><img src="http://openwetware.org/images/0/03/S-tashiro1.jpg"> <img src="http://openwetware.org/images/2/29/S-tashiro2.jpg"></div><br> <div class="caption">Fig.15 Zoom up of Fig.14</div><br> When zoom up in the networks, liposomes that lost Texas-Red dextran were observed. <br> <br>

<h5>Discussion</h5> In this experiment, three different changes were observed by adding the Key DNA: i) shrinking, ii) network structure formation, and iii) leak of fluorescence. <u>These results suggest that we achieved disruption of liposomes by our flower DNA approach. </u><br> <br> <h5 id=12> 2-2-2 Confirming sequence specificity of DNA</h5> <h5>Purpose</h5> We demonstrate the selectivity of our Key DNA: the Key DNA only affects the corresponding Flower-anchor DNA and liposomes.<br> <br> <h5>Method</h5> Two types of phase separated liposomes were prepared by droplet transfer methods. One type is liposomes with GFP inside (Green liposome); the other type is liposomes with Texas-Red dextran inside (Red liposome).<br> The anchor DNA for Green liposome is named “A-flower-anchor DNA”, and the anchor DNA for Red liposome is named “B-flower-anchor DNA”. Each flower-anchor DNA can bind only the complementary Key DNA. This time, only Key DNA for Red liposomes (complementary to B-flower-anchor DNA) is added.<br> After adding B-Key DNA, the number of each color liposomes is counted to confirm the selectivity.<br> As a control, only buffer is added instead of B-Key DNA.<br> <A href="http://openwetware.org/wiki/Biomod/2013/Sendai/protocol">Protocol</A><br> <br> <h5>Result</h5> Fig.16 shows fluorescent microscope image of liposomes added B-Key DNA (left) and control buffer(right). Green rectangles represent Green liposomes; Red, Red liposomes.<br> In the left, only Green liposomes (marked with green rectangles) and no Red liposomes can be seen. In the right, almost the same number of Green and Red liposomes are seen.<br> <div align="center"><img src="http://openwetware.org/images/9/94/2-2-2fig%E3%81%95%E3%81%84%E3%81%94tyannpiyonn.png"></div> <div class="caption">Fig.16　fluorescent microscope image of liposomes (left: with B-Key DNA, right: with control buffer)</div><br> <table border cellspacing="3" bgcolor="lightyellow"> <tr bgcolor="moccasin"> <td> Additives </td> <td> B-key DNA </td> <td> Buffer </td> </tr> <tr bgcolor="moccasin"> <td> Green：Red </td> <td>17:2 (n = 19)</td> <td>16:17 (n= 33)</td> </tr> </table> <div class="captiontable">Table1 Ratio of Green and Red liposomes</div><br> Table1 shows the Ratio of Green and Red liposomes.<br> When the control buffer is added, the number of Green and Red liposomes are almost the same. On the other hand, when B-Key DNA is added, much less number of red liposomes is seen compared to the number of Green liposomes. Other images are shown in <A href="http://openwetware.org/wiki/Biomod/2013/Sendai/protocol">Protocol</A>.<br> <br> <h5>Discussion</h5> Comparing the left and right images, the ratio of Red to Green liposomes decreases due to the addition of B-Key DNA. This means that B-Key DNA only breaks Red liposomes and it has little effect on Green liposomes.<br> <u>The selectivity of Key DNA has been successfully demonstrated. </u></h6> 

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