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<p class="sukima">Experiment
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  <section role="main">
  <article data-title="Experiment">
        <article>
            <h2>Experiment</h2>
<h2>Experiment</h2>
           
<p>
<h3>About</h3></br>


<a href="#experimentsubproject1">内側からアルギン酸膜を破壊するサブプロジェクト</a><br>
<table id="toc" class="toc" summary="Contents"><tr><td><div id="toctitle"><h2>Contents</h2></div>
<a href="#experimentsubproject2">外側からリポソームを破壊するサブプロジェクト <font size="2">リポソーム班</font> </a><br>
<ul>
<a href="#experimentsubproject3">外側からリポソームを破壊するサブプロジェクト <font size="2">B-Z班</font> </a><br>
<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>


    </article>
<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>
      <article data-title="Egg-type initiator">
<li class="toclevel-1"><a href="#9">
<h3>Step1 Egg-type initiator</h3>
<span class="tocnumber">2-2</span> <span class="toctext">Flower DNA approach</span></a></li>
 
<ul>
<h4>Experiment list</h4>
1-1) Encapsulating liposomes in alginate hydro gel beads<br>
1-2 ) Preparing of alginate gel membrane that have inner solution phase<br>
2) Constructing temperature-sensitive liposome by PNIPAM lipids<br>
3) Measurement of critical concentration of EGTA and time to melt alginate gels<br>
4) Isothermal DNA origami formation by dilution of denature reagent<br><br>
 


<h4>1-1)Encapsulating liposomes in alginate hydro gel beads</h4>
<li class="toclevel-2"><a href="#11">
<h5>Purpose</h5>
<span class="tocnumber">2-2-1</span> <span class="toctext">Disruption of liposomes by Flower DNA</span></a></li>
We need to encapsulate the temperature-sensitive liposomes in alginate gel membrane. At first, we should test whether alginate gel can encapsulate liposomes with fluorescence.<br>
<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>


<h5>Method</h5>
We made liposome by droplet-transfer method. We used 50 μℓ of 70 mM glucose solution as outer buffer and 70μℓ oil (1 mM eggPC in mineral oil) were put on the glucose solution. BSA-GFP mixture dissolved in 70 mM sucrose was used as inner solution that was dispersed in 40µℓ the oil. The dispersed droplet were put on the outer soultion, and centrifuged it for 70 seconds. After that, we obtained the liposome with fluorescence from the bottom of the tube. Then, we mixed the liposome with 1.5% sodium alginate solution. The mixture was put on a capillary and dropped it in 0.4M CaCl2 solution by centrifuging by using the device (Maeda K et al. Advanced material 2012) described in Fig.1.<br>


</li>


<img src="http://openwetware.org/images/f/f1/S_fig1.png" width="400">
Fig.1 Experimental device to form alginate gel beads</br></br>


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


 
<h3 id=chain>First stage: Sensing system</h3>
<h5>Result</h5>
<h4 id=bending>1-1Disruption of temperature sensitive liposomes</h4>
Because of GFP in the liposomes, the liposomes show fluorescence. We observed the alginate hydrogel beads by cofocus laser microscope. (Fig. 2). Liposome with fluorescence was found in the alginate hydrogel beads.<br>
 
 
 
<table>
<tr>
<td>
<img src="http://openwetware.org/images/b/bc/S_fig2.png" width="400">
</td>
<td>
<img src="http://openwetware.org/images/a/a8/Exp-1-1-02.png" width="400">
</td>
</tr>
</table>
Fig.2 Cofocal laser microscope image of alginate gel beads with liposome</br></br>
 
<h5>Discussion</h5>
From Fig.2,　liposome was very small. It was thought that this is because H2O molecule in the liposome leak under the influence of osmotic pressure outside. This problem may be improved by adjusting the density of sodium alginate solution and solution in the liposome.</br>
 
 
</br>
<h4>1-2 ) Preparing of alginate gel membrane that have inner solution phase</h4>
<h5>Purpose</h5>
<h5>Purpose</h5>
The egg-type initiator should have inner solution phase. Thus, we should develop a method to make alginate hydrogel membrane containing buffer.  
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>
<br>
<h5>Principle</h5>
We made double capillary shown in Fig. 3. Gravity by a centrifuge rotor enclosed content fluid (that is the inner solution) and sodium alginate. At the front edge of granularities, content fluid wrapped in sodium alginate. These granularities fall in drops to the solution of sodium alginate and only surface turn into gel.<br>


<h5>Method</h5>
<h5>Method</h5>
We developed a double capillary (Fig.2), made from outer thick one and inner fine one. To confirm solution in the gel beads, we used 1.5% sodium alginate solution as outer solution, and fluorescent solution (FITC) with 0.4M CaCl2 was used as inner one. Finally, the capillary containing solution was centrifuged for a few minutes. The solution from the capillary were dropped in 0.4M CaCl2<br>
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>
<img src="http://openwetware.org/images/e/ef/S_fig3.png" width="500" ></br>
The liposomes were observed on the slide glass by phase-contrast microscopy. <br>
Fig.3 Structure of double capillary</br></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>
<h5>Result</h5>
We observed alginate gel by confocal laser microscope. The inside of the gel showed fluorescence.
<br>


<table>
<tr>
  <td>
  <img src="http://openwetware.org/images/8/85/S_fig4.png" width="400">
  </td>
  <td>
  <img src="http://openwetware.org/images/d/d6/S_fig5.png" width="400">
  </td> 
</tr>
<tr>
  <td>
  Fig.4 Phase contrast microscope image of alginate hydrogel membrane
  </td>
  <td>
  Fig.5 Fluorescent microscope image of alginate hydrogel membrane
  </td>
</tr>
</table>




<h5>Discussion</h5>
<img src="http://openwetware.org/images/9/9e/2-1-1fig1.png" width="100%" height="100%"><br>
Alginate hydrogel did not become sphere, and formed tube-like gel (like frog spawn). We consider to overcoming the problem can by changing centrifugal speed.<br>


<h4>2) Function confirmation of PNIPAM</h4>
<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>
<h5>Purpose</h5>
<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.<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>


<h5>Principle</h5>
NIPAM is hydrophilic at less than 32 ºC, but it become hydrophobic and shrinks when it temperature becomes higher than 32 ºC. Therefore, the liposome that modified NIPAM becomes unstable and is broken at the time of high temperature than 32 ºC.<br>
Reference<br>
http://www.sigmaaldrich.com/etc/medialib/docs/SAJ/Brochure/1/j_recipedds2.Par.0001.File.tmp/j_recipedds2.pdf<br><br>


 
<br>
<img src="http://openwetware.org/images/6/6c/Pnipam-lipo.png" width="600"></br>
Fig.6 Collapse of liposomes containing PNIPAM
 
<h5>Method</h5>
Liposomes were prepared by the incubation method. 0.15 mM Poly(NIPAM-co-AA-co-ODA) and 0.5 mM DOPC dissolved in CHCl3. 100 μL of the lipids in glass tube was dried under Ar gas condition for an hour. The dried lipids were dissolved in the observation buffer (1xTAE with 12.5 mM MgCl2). The liposomes solution was divided into two tubes, and performed (1) and (2), respectively. <br>
(1) Setting the ultrasonic water bath at 20 ºC, and the lipids were sonicated for 15 minutes. <br>
(2) Setting the ultrasonic water bath at 40 ºC, and the lipids were sonicated for 15 minutes. <br><br>
 
We took 5μL of each mixture and dilute them by 195 μL of observation buffer. The samples were observed by microscope.<br>
 
 
 
 
<img src="http://openwetware.org/images/c/c7/PNIPAM-structure.png" width="400"><br>
Fig.7 Phospholipid decorated with PNIPAM<br><br>
 
<!--
We made phospholipid film by drying stock liquid (10mM DOPC) with Ar gas and vacuum desiccator. Then, added 500μl liquid faraffin to phospholipid film and dissolve the film to oil by supersonic dish washers in 60 for 60 min. We made inner buffer (sucrose 150mM, glucose 350mM, EGTA 100mM), added it to the phospholipid dissolves to oil, centrifuge that, and got emulsion liquid.???<br>
参考</br>
Thermoresponsive Nanostructures by Self-Assembly of a Poly(N-isopropylacrylamide)−Lipid Conjugate
Daniel N. T. Hay ,† Paul G. Rickert ,‡ Sönke Seifert ,§ and Millicent A. Firestone *†
J. Am. Chem. Soc., 2004, 126 (8), pp 2290–229　Publication Date (Web): February 3, 2004<br><br>
-->
 
<h5>Result</h5>
Liposomes were observed in the samples sonicated at 20 ºC, and liposome were not observed in the samples sonicated at 40 ºC.<br>
<table>
<tr>
  <td>
  <img src="http://openwetware.org/images/8/81/S_fig8.png" width="400">
  </td>
  <td>
  <img src="http://openwetware.org/images/3/33/S_fig9.png" width="400">
  </td> 
</tr>
<tr>
  <td>
  Fig.8 Phase contrast microscope image of liposome(20℃)
  </td>
  <td>
  Fig.9 Phase contrast microscope image of liposome(40℃)
  </td> 
</tr>
</table>




<h5>Discussion</h5>
<h5>Discussion</h5>
We observed liposomes on the same condition except temperatures. We should try to observe time-lapse image of liposomes by heating to over 32 ºC.<br>
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>






<h4>3) Measurement of critical concentration of EGTA and time to melt alginate gels</h4>
<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>
<h5>Purpose</h5>
To know critical concentration of EGTA to melt alginate gel is important to create our system. In addition, to know time for melting is essential when isothermal annealing of DNA origami was proceeded in the egg-type initiator.<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>
<h5>Method</h5>
We used large alginate hydrogel beads which is 2.6mm in diameter (volume 10µl) for observing easily.<br>
Mixing M13mp18, staples, 5xTAE Mg2+, and mQ in a microtube and annealing for 2.5 hours.<br>
First, we made alginate hydro gel beads. we took 10 µl 1.5% sodium alginate solution with micropipette and dropped it in 0.4MCaCl2 solution.<br>
<A href="http://openwetware.org/wiki/Biomod/2013/Sendai/protocol">Protocol</A><br>
Second, we made 10mM,50mM,100mM,EGTA solution. We put alginate hydro gel beads in each EGTA solution(600µl) and measured the time when alginate hydro gel beads completely collapse
<br>
 
<h5>Result</h5>
The result is the following table<br><br>
<table cellspacing="5" bgcolor="lightyellow">
<tr bgcolor="moccasin">
  <td>
  Concentration of EGTA
  </td>
  <td>
  10mM
  </td>
  <td>
  50mM
  </td>
  <td>
  100mM
  </td>
</tr>
<tr bgcolor="moccasin">
  <td>
  Time (min)
  </td>
  <td>
  -
  </td>
  <td>
  44
  </td>
  <td>
  28
  </td>
</tr>
</table>
Table.1 Concentration of EGTA and melting time<br><br>
<h5>Discussion</h5>
 
 
<h4>4) Isothermal DNA origami formation by dilution of denature reagent</h4>
<h5>Purpose</h5>
In some case, it is necessary to form trigger DNA origami in egg-type initiator. In alginate gel membrane, urea can be gradually dilute. Thus, we tested isothermal DNA origami formation by dilution of denature reagent.<br>
 
<h5>Principle</h5>
Polarity of water molecular becomes weak in the presence of urea. So urea interrupts the hydrogen bond of DNA base. For that, the melting point of DNA decreases. This enables hybridization at low temperature by decreasing the concentration of urea gradually. In this assay, we used a filter membrane system for dialysis as an alternative of alginate membrane. Urea passes the filter into outside buffer but DNA remains in the filter. Thus urea is gradually removed. The gradually decrease of urea works as an alternative of temperature shift which usually used in DNA origami formation.<br>
 
<h5>Method</h5>
We added M13mp18 and staples at the rate of 1:20 in TAE buffer with urea (6M) and Mg2+ (12.5mM). Then, we set a filter membrane system (Millipore, Amicon 3k) to floater and float it on TAE buffer with Mg2+(12.5mM). The environmental buffer was stirrer for 4 hours. Then, we observed sample remained in the filter by AFM.<br>
 
 
<img src="http://openwetware.org/images/4/4c/S_fig10.png" width="400"><br>
Fig.10 Method of urea diluting annealing<br>




<h5>Result</h5>
<h5>Result</h5>
We observed structures as we designed by AFM imaging. The result is Fig.5 as below. The scale of DNA origami is similar to our design. (for details of DNA origami design click here).<br>
We obtain DNA Origami same as our design. The result was confirmed by AFM (Atomic Force Microscope.)<br>
 
 
 
<!--And in electrophoresis, by comparing the lane of M13 and the lane of DNA origami annealed by urea diluting, the band of later lane is higher than that of former.-->
 
 
<table>
<tr>
<td>
<img src="http://openwetware.org/images/8/89/S_fig11.png" width="600">
</td>
<td>
<img src="http://openwetware.org/images/5/55/Exp-1-1-03.png" width="200">
</td>
</tr>
</table>
 
Fig.11 AFM image of DNA origami made by urea diluting annealing<br><br>
 
<h5>Discussion</h5>
We observed DNA origami we designed. However, we also observed a lot of sheet structures like fragments. We suppose that some staples did not hybridize with M13 DNA caused these fragments. Rapidly dilution of urea may cause the low yield of objective DNA origami, because stirring make urea removing faster. Nevertheless, it is necessary to measure the yield of DNA origami under low speed dilution. We should note that, in the egg-type initiator, the speed of diluting urea would be later than the speed of this experiment, and thus fast dialysis is not essential to form DNA origami in our system.<br>
 
 
<!--
 
<h4>5温度を上げればアルギン酸膜が破壊されることの確認</h4></br>
 
2と3の実験が成功したのでこれらを組み合わせて、温度を上げて内部にキレート剤であるリポソームが割れれば、アルギン酸膜が割れるかどうかを調べるために以下のような実験を行った。  </br>
まず、アルギン酸ゲルビーズを作製して位相差顕微鏡で30μℓ当たりのアルギン酸ゲルビーズの数を数えた。</br>
次に、割れた後に系全体のＥＧＴＡの濃度が実験3で調べた最適な濃度になるような量のＥＧＴＡを入れたリポソームを作製した。これをアルギン酸ゲルビーズの入っている溶液の中に入れて温 度を約３２度に上げた。その後、位相差顕微鏡で30μℓ当たりのアルギン酸ゲルビーズの数を数えた。（結果を表で示す）</br>
ＥＧＴＡ付きリポソームをいれて温度を上げた後の方がアルギン酸膜の数が減っていたので、温度を上げることでニッパム付きのリポソームが破壊されて、キレート剤であるＥＧＴＡが放出されてアルギン酸膜が破壊されたと考えられる。</br></br>
 
<h4>6アルギン酸膜内で尿素アニーリングができていることの確認</h4></br>
 
1と4の実験が成功したのでこれらを組み合わせて、アルギン酸膜内で尿素アニーリングができるかどうかを調べるために以下のような実験を行った。</br>
二重ノズルの外管に1.5%アルギン酸ナトリウム溶液を、内管に尿素とＤＮＡオリガミの材料を入れてアルギン酸膜を作製した。時間をおいてキレート剤を加えて、アルギン酸膜を破壊した。溶液をとってＡＦＭで確認した。（ＡＦＭの画像）</br>
設計したとおりのＤＮＡオリガミが観察されたのでアルギン酸膜内で尿素アニーリングができたと考えられる。</br></br>
 
 
<h4>7全体のシステムの機能確認</h4></br>
 
5と6の実験が成功したのでこれらを組み合わせて、我々が目指しているシステム全体が機能しているかどうかを調べた。</br>
まず、内部に尿素と、ＤＮＡオリガミの材料と、キレート剤であるＥＧＴＡを入れたニッパム付きリポソームを作製する。次に、二重ノズルを使ってアルギン酸膜を作製する。位相差顕微鏡でアルギン酸膜の数を数える。温度を約３２度に上げる。もう一度アルギン酸膜の数を位相差顕微鏡で数えた。また、同じ溶液をＡＦＭで観察した。</br>
温度を３２度に上げる前と後で、アルギン酸膜の数は減少してその溶液からＤＮＡオリガミがＡＦＭで観察できたので、私たちの目指しているシステムが機能していると考えられる。</br>
 
-->
 
 
 
      </article>
 
     
      <article data-title="Chain-reactive burst">
 
<h3>Step 2 Chain-reactive burst</h3>
Once the trigger DNA, which begins the interaction, is released, the next is the chain-reactive burst. If a liposome containing new triggers and active ingredients is disrupted, the released triggers come to collapse the surrounding liposomes one after another.<br>
We tackled the problem of destroying liposomes by the following two approaches.
<br>
<br>
<ur><li>i)Bending approach</li>
<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>
<li>ii)Flower micelle approach</li></ur>
<div class="caption">Fig.2 AFM image of DNA Origami (M13: 4nM, staples:20nM)</div><br>
<br>
<h4>i)Bending approach</h4>
<h4>Experiment list</h4>
The experiment necessary for realization of Bending approach is following.<br>
1)Making DNA origami<br>
1-1)Making DNA origami<br>
1-2)Labeling DNA origami<br>
2)Collapsing liposomes<br>
2-1)Making liposomes<br>
2-2)Investigating the interaction of DNA origami and liposomes<br>
2-3)Counting liposomes<br>
<br>
<br>
<h4>1)Making DNA origami</h4>
<h4>1-1)Making DNA origami<h4>
<h5>Purpose</h5>
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>
<br>
<h5>Principle</h5>
DNA origami is a method applied to making nano-structures of various shapes. DNA origami consists of two kinds of strands: scaffold and staples. Scaffold is a long round single-stranded DNA, and staples are short linear single-stranded DNAs.<br>
<br>
<h5>Method</h5>
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>
<h5>Result</h5>
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>
<br>
<h5>Discussion</h5>
<h5>Discussion</h5>
Just like our design, rectanglar origamis with chipped edges were observed.<br>
<u>As shown in Fig. 2, DNA Origami was well-formed.</u><br>
<br>
<br>
<br>
<h5 id=6>2-1-2 Labeling DNA Origami with fluorescent-tagged DNA</h5>
<h4>1-2)Labeling DNA origami<h4>
<h5>Purpose</h5>
<h5>Purpose</h5>
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 DNA strands.<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.  
<br>
<br><br>
 
<h5>Method</h5>
<h5>Method</h5>
Our DNA origami has many staples that can bind to fluorescent tagged DNAs for labeling. We mixed fluorescent tagged DNAs together with DNA origami staples in annealing solution.<br>
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>
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>
To see the origami was well labeled with fluorescent molecules, we used electrophoresis. <br>
Electrophoresis was conducted with a 1% agarose gel, CV100V for 50 minutes.<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>
By scanning a gel before staining, we can see only the bands of DNA structures with fluorescent molecules; scanning a gel after staining, we can see the bands of all DNA structures. So we scanned a gel before and after staining (we scanned both a non-stained and a stained gel). <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>
<br>
<br>
<h5>Result</h5>  
<h5>Result</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>
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>
<Img Src="http://openwetware.org/images/5/58/S_Outside-gel-3.2.png" width="300"><br>
<div align="center">
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>
<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>
In a stained gel (Fig.3), maker (lane 1) had the longest DNA strand 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>
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 DNAs, and each origami attaches to different number of them, and its molecular weight varies.<br>
<Img Src="http://openwetware.org/images/2/2d/S_Outside-gel-2.2.png" width="300"> </br>
Fig.3 Stained gel image: from the left, maker, M13mp18, Ori*, Ori**, and DNA origami with no fluorescent molecule (Ori)<br>
<br>
<br>
<h5>Discussion</h5>
<h5>Discussion</h5>
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>
<u>The results indicate we succeeded to label our Origami by the fluorescence DNA.</u><br>
<br>
<br>
<h4>2)Collapsing liposomes</h4>
 
<h4>2-1) Making liposomes</h4>
<h5 id=7>2-1-3 Disrupting liposomes by DNA Origami (microscopic analysis)<h5>
<h5>Purpose</h5>
We make liposomes that are to be collapsed by DNA origami.<br>
<br>
<h5>Principle</h5>
Phospholipids, which compose liposomes, are amphipathic molecules. They have hydrophilic and hydrophobic groups, and when they touch water, they make micelles: some hydrophilic groups take water inside. At the same time, other hydrophilic groups touch the water outside. So they form the innermost and outermost part of a micelle. On the other hand, the hydrophobic groups form the intermediate part of a micelle. <br>
In this way, spherical liposomes are made.<br>
<br>
<h5>Method</h5>
To make liposomes, first we mixed lipid (DOPC) and solvent (CHCl3) in a microtube, and desiccate it with Argon gas. Then, adding some buffer (1xTAE Mg2+), we heated it in warm water for a few hours.<br>
<A href="http://openwetware.org/wiki/Biomod/2013/Sendai/protocol">Protocol</A><br>
<br>
The result and discussion are integrated in the next passage of (2-2) Investigating the interaction of DNA origami and liposomes.<br>
<br>
<br>
<h4>2-2) Investigating the interaction of DNA origami and liposomes<h4>
<h5>Purpose</h5>
<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>
<h5>Principle</h5>
<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 DNAs (in the rest of this document, referred to as aptamer) into liposomes, and made them float on the surface. If the aptamer 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 aptamers.<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>
<h5>Method</h5>
<h5>Method</h5>
We added aptamers 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>
<h5>Result</h5>
<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>
 
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>
<br>
<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/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>
<br>
<Img Src="http://openwetware.org/images/d/d0/Lipofig5.png" width="400"></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>
Fig.5 Phase microscope image of liposomes (cholesterol-conjugated DNA: 0.069µM)<br>
<br>
<br>
<Img Src="http://openwetware.org/images/d/de/Lipofig6.png" width="400"></br>
<div align="center"><Img Src="
Fig.6 Phase microscope image of liposomes (cholesterol-conjugated DNA: 1.8µM)<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/d7/Lipofig7.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.7 Phase microscope image of liposomes (cholesterol-conjugated DNA: 6.9µM)<br>
<br>
<br>
Adding fluorescently labeled DNA origamis into the above liposomes, we saw if some change would happen with a fluorescent microscope.<br>
<div align="center"><Img Src="
When the concentration of aptamer 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>
http://openwetware.org/images/c/c0/2-2-1fig7.png"></div><br>
<table>
<div class="caption">Fig.7 Fluorescent microscope image of liposomes <br>(Origami-anchor DNA: 6.9µM; Left: without fluorescence, Right: with fluorescence)</div><br>
<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 aptamer 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>
When the concentration of Origami-anchor DNA is 6.9µM, some liposomes were gleaming and others distorted, forming networks (Fig.7).
<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 aptamer is 6.9µM, some liposomes were gleaming and others distorted, forming networks (Fig.12).<br>
<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>
<h5>Discussion</h5>
<h5>Discussion</h5>
From these results, we put forward the following hypothesis about the interaction of DNA origami and liposomes.<br>
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.
When the concentration of aptamer 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 aptamer 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 aptamer is 1.8µM, DNA origami collapses liposomes. Therefore, in the following experiment, we checked if DNA origami would collapse liposomes at this concentration of aptamer.<br>
Anyway, <u>these data strongly indicated the designed DNA origami disrupted liposomes with high concentration of the anchor DNA.</u>
<br>
<br>
<br>
<br>
<h4>2-3)Counting liposomes</h4>
<br>
 
<h5 id=13>2-1-4 Disrupting liposomes by DNA Origami (quantitative analysis)<h5>
<h5>Purpose</h5>
<h5>Purpose</h5>
To see if DNA origami collapses liposomes, we counted the number of liposomes before and after adding DNA origami. <br>
The above experiments in 2-1-3 microscopic analysis suggest that our DNA Origami disrupted liposomes. Thus, we performed more quantitative analysis.<br>
<br>
<br>
<h5>Method</h5>
<h5>Method</h5>
For the sake of observation convenience, we mixed TR-DHPE (red fluorescent dye) with lipid (DOPC) and solvate (CHCl3), and made liposomes. The liposomal surfaces were dyed by TR-DHPE.<br>
<div class="caption-right">
Then we added aptamers at the final concentration of 1.8µM, and counted the number of liposomes with a fluorescent microscope.<br>
<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>
After counting, we put DNA origami and counted the number of liposomes again.<br>
</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>
<A href="http://openwetware.org/wiki/Biomod/2013/Sendai/protocol">Protocol</A><br>
<Img Src="http://openwetware.org/images/4/41/Counting-liposome.png"></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>
<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>
<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>
<br>
<h4>ii)Flower micelle approach</h4>
The x axis of the above graph is the fluorescence intensity of only liposomes, and the y axis represents the number of liposomes.<br>
<h4>Experiment list</h4>
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>
The experiment necessary for realization of Flower micelle approach is following.<br>
 
1) Making liposome <br>
<h5> Discussion </h5>
2) Confirming the hybridization of trigger and loop DNA <br>
<u>These results confirmed that our designed DNA origami actually disrupted liposomes. (The efficiency was higher than 2% surfactant!)</u>
3) Confirming the formation of loop structure by SPR<br>
4) Collapsing liposome<br>
<br>
<br>
<br>
<br>
<h4>1)Making liposome</h4>
<h5 id=8>2-1-5 Confirming sequence specificity of DNA flow cytometer</h5>
<h5>Purpose</h5>
<h5>Purpose</h5>
We make liposomes that are to be collapsed by flower micelle method.<br>
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>Principal</h5>
We made normal liposomes made of DOPC and phase-separatied liposomes made of  DOPC, DPPC and cholesterol.<br>
Phase-separated liposomes are liposomes made by several kinds of lipids. On the surface of phase-separated liposomes several kinds of lipids separate and the liposomes are formed by some layers.<br>
As the surface lipids of the phase-separated liposomes are not so changeable as the normal liposomes, It is considered that power produced by the hybridization of the loop and trigger strands reaches the liposome more effectively.<br>
So the phase separation liposome was used for experiments this time.<br>
<br>
<br>
<h5>Method</h5>
<h5>Method</h5>
<ur><li>1. Making DOPC, DPPC, and Cholesterol lipid Lipid<br>
Experimental conditions were the same in 2-1-4 except samples.<br>
1-1 Put 7.8 mg DOPC, 7.3mg DPPC , and 3.8mg Cholesterol into each microtube, and add 1ml CHCl3.<br>
Sample 1 (Complement). Liposomes + Origami-anchor DNA(A) + Key DNA(A)<br>
1-2 Put it in a ultrasonic bath of 60 degrees Celsius for one hour.<br>
Sample 2 (no binding pair). Liposomes + Orgiami-anchor DNA(A) + Key DNA(B)<br>
1-3 10mM DOPC, DPPC, Cholesterol lipid is made.<br></li>
<A href="http://openwetware.org/wiki/Biomod/2013/Sendai/protocol">Protocol</A><br>
          <br>
            <li>2. Making phase-separated liposomes<br>
            2-1 Mix DOPC,DPPC, and Cholesterol at the ratio of 1:1:1 to make phase-separated liposomes. In this experiment, mix 4µl DOPC (10mM), 4µl DPPC (10mM),4µl Cholesterol (10mM) and 88μl buffer well.<br>
            2-2 Add 12µl Texas red (10μM) <br>
            2-3 Dry the sample using Argon gas<br>
            2-4 Hydrated the dried sample with by 100ml 1xTAE<br>
            2-5 Put the sample in hot water for three hours. Then leave it at low temperature for one hour to let the surface lipid separate.</il></ur><br>
<br>
<br>
<h5>Result</h5>
<h5>Result</h5>
As is shown in Fig.13, phase-separated liposomes were observed by a fluorescent microscope. They are basically multi-lamella liposomes.<br>
The results were shown in figure 12.<br>  
We confirmed the formation of phase-separated liposomes with a fluorescent microscope.<br>
<div align="center">
<img src="http://openwetware.org/images/6/6d/2-1-5matometagazou-deka.png" style="
<Img Src="http://openwetware.org/images/a/a2/S_flowerex1.png"></br>
    width: 90%;
Fig.13 Fluorescent microscope image of phase-separated liposomes<br>
"></div>
<br>
<br><div class="caption">Fig.12 Results of complementary key DNA and no binding Key DNA</div>
<h5>Discussion</h5>
 
Using the above-mentioned method, we successfully made phase-separated liposomes.　However, they are multi-lamella ones and should be refined to be uni-lamella ones, by methods such as electroformation or droplet-transfer method.<br>
<br>
<br>
<br>
<h4>2) Confirming the hybridization of trigger and loop DNA</h4>
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>
<h5>Purpose</h5>
We checked whether trigger DNA hybridizes with loop DNA at normal temperature by electrophoresis. <br>
<br>
<h5>Method</h5>
<ur><li>1. Prepare three microtubes and put three kinds of trigger DNAs (10, 20, 40bases; 5µl, 100nM) into each tube.</li>
<li>2. Add three kinds of loop DNAs (10, 20, 40bases; 5µl, 100nM) into corresponding tube (tube that contains trigger DNA of corresponding number of nucleotides) and leave them at normal temperature for approximately one hour.</li>
<li>3. Add 6x loading buffer with the quantity of 20% of the samples.</li>
<li>4. Make an acrylic amide gel.</li>
<li>5. Load samples (including maker) into 10 lanes.</li>
The electrophoresis was conducted with CV 100V for one hour.<br>
<br>
<h5>Result</h5>
The result was shown in Fig.14.<br>
<Img Src="http://openwetware.org/images/3/37/Flowerex3.png"></br>
Fig.14 Stained gel image<br>
<br>
The lane of 20 base loop and trigger shows a strong band at different height from the band of only 20 base loop and trigger. As for the samples of 40 base, the result was the same. <br>
On the other hand, the lane of 10 base loop and trigger shows a band at the same height as the band of only 10 base loop. No band was seen in the lane of only 10 base trigger.<br>
<br>
<br>
<h5>Discussion</h5>
<h5>Discussion</h5>
The fact that the band of 20 base loop and trigger was at the different height from the band of only 20 base loop or trigger indicates that 20 base loop and trigger DNA hybridized and made a double strand. As the samples of 40 base showed the same result, we concluded that 20 and 40base loops and triggers hybridize at normal temperature.<br>
<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>
However, as for the samples of 10 bases, there was no difference between the two band height. Therefore, 10 base loop and trigger had not hybridized. <br>
It is estimated that no band was seen in the lane of only 10 base trigger because of some kind of mistakes. Therefore we do not take this into consideration.<br>
From the above, we find that the 20 and 40nt trigger hybridizes with a loop at normal temperature.<br>
<br>
<br>
<br>
 
<h4>3) Confirming the formation of loop structure by SPR</h4>
 
<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>
<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 aptamers as used in i)Bending approach into liposomes: the aptamer 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 aptamers, and that of aptamers 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>
<h5>Principle</h5>
<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>
If aptamer attaches to a liposome, and then loop strand attaches to it, SPR value increases after each step.<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 DNAs into it.<br>
We measured SPR value after each step of adding DOPC into liposomes, and loop DNA into it.<br>
<br>
<br>
<h5>Method</h5>
<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 aptamer (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>
<h5>Result</h5>
<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 aptamers caused no change of SPR value, we injected aptamers 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 aptamers and loop DNAs. Moreover, we should note that after injecting trigger DNA, some 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>
<h5>Discussion</h5>
<h5>Discussion</h5>
Fig.15 shows the behavior of materials on the surface of liposomes. The increase of SPR value after injecting aptamers indicates that aptamers 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 DNAs combined with aptamers. <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>
-->
<!---------------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>
<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>
<br>
<h4>4) Collapsing liposome</h4>
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>
<h5>Purpose</h5>
It was tested if liposomes would be collapsed by adding trigger DNA.<br>
We demonstrate the selectivity of our Key DNA: the Key DNA only affects the corresponding Flower-anchor DNA and liposomes.<br>
<br>
<h5>Principle</h5>
Whether liposomes are collapsed or not can be decided by counting the number of liposomes before and after the trigger addition. As a control, we added the same amount of buffer instead of trigger. Liposomes are observed by a fluorescent microscope.<br>
<br>
<br>
<h5>Method</h5>
<h5>Method</h5>
<ur><li>1. Make liposomes with loop DNAs<br>
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>
1-1 Mix 2µl liposome (0.2mM) with 2µl aptamer (10µM) at normal temperature<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>
1-2 Add 2µl loop DNA (20µM)</li><br>
After adding B-Key DNA, the number of each color liposomes is counted to confirm the selectivity.<br>
<li>2. Collapse the liposomes with the loop DNAs<br>
As a control, only buffer is added instead of B-Key DNA.<br>
2-1      Add 2µl trigger DNA (20µM) </li></ur><br>
<A href="http://openwetware.org/wiki/Biomod/2013/Sendai/protocol">Protocol</A><br>
<br>
<br>
<h5>Result</h5>
<h5>Result</h5>
Fig.16 is the result of the sample added trigger DNAs; Fig.17, the sample of control experiment.<br>
Fig.16 shows fluorescent microscope image of liposomes added B-Key DNA (left) and control buffer(right).
<table>
Green rectangles represent Green liposomes; Red, Red liposomes.<br>
<tr>
In the left, only Green liposomes (marked with green rectangles) and no Red liposomes can be seen.
  <td>
In the right, almost the same number of Green and Red liposomes are seen.<br>
  <Img Src="http://openwetware.org/images/5/56/Flower5.png" width="400">
<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>
  </td>
<div class="caption">Fig.16　fluorescent microscope image of liposomes (left: with B-Key DNA, right: with control buffer)</div><br>
  <td>
<table border cellspacing="3" bgcolor="lightyellow">
  <Img Src="http://openwetware.org/images/7/77/Flower%EF%BC%94.png" width="400">
<tr bgcolor="moccasin">
  </td>
<td> Additives </td>
</tr>
<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>
</table>
Fig.16,17 Fluorescent microscope image of liposomes  
<div class="captiontable">Table1  Ratio of Green and Red liposomes</div><br>
<br>(Fig.16: sample added trigger DNAs, Fig.17: control)<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>
As it was difficult to count the number of liposomes in both cases, we did not count them.<br>
<br>
<br>
<h5>Discussion</h5>
<h5>Discussion</h5>
As we were not able to see a clear numerical change, we did not see whether liposomes were collapsed by this experiment.<br>
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>
Two ideas why liposomes were not collapsed are come up:<br>
<u>The selectivity of Key DNA has been successfully demonstrated. </u></h6>
1. The lipid ratio for making liposomes was not appropriate. We should investigate the most appropriate and effective ratio for collapsing liposomes.<br>
2. Liposomes in this experiment were multi-lamella ones: Multi-lamella liposomes have some leaflets piled up. It is considered that more power is needed to collapse them. We would try other methods except the hydration method in future to make uni-lamella liposomes (which is relatively easy to collapse).<br>
Solving the above- mentioned problems, liposomes would be destroyed.<br>


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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>

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<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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