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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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<p class="sukima">Experiment </p>

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<article data-title="Experiment">
           <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> <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> <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>

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     <article data-title="Egg-type initiator">

<h3>Step1 Egg-type initiator</h3>

<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> <h5>Purpose</h5> 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>

<h5>Method</h5> <div class="caption-right"><img src="http://openwetware.org/images/f/f1/S_fig1.png" width="400"><span>Fig.1 Experimental device to form alginate gel beads</span> </div> 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 CaCl<sub>2</sub> solution by centrifuging by using the device (Maeda K et al. Advanced material 2012) described in Fig.1.<br> <div class="c-both"></div> <br>

<h5>Result</h5> 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 H<sub>2</sub>O 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> The egg-type initiator should have inner solution phase. Thus, we should develop a method to make alginate hydrogel membrane containing buffer. <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> 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 CaCl<sub>2</sub> 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 CaCl<sub>2</sub><br>

Fig.3 Structure of double capillary</br></br>

<h5>Result</h5> We observed alginate gel by confocal laser microscope. The inside of the gel showed fluorescence. <br>

<table>

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

<h5>Discussion</h5> 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> <h5>Purpose</h5> 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>

<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> (<a href="http://www.sigmaaldrich.com/etc/medialib/docs/SAJ/Brochure/1/j_recipedds2.Par.0001.File.tmp/j_recipedds2.pdf">pdf</a>)<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>

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<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>

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  <img src="http://openwetware.org/images/8/81/S_fig8.png" width="400">
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  Fig.8 Phase contrast microscope image of liposome(20℃)
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  Fig.9 Phase contrast microscope image of liposome(40℃)
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<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>

<h4>3) Measurement of critical concentration of EGTA and time to melt alginate gels</h4> <h5>Purpose</h5> To create our system, it is important to know the critical concentration of EGTA for melting alginate gel. In addition, it is essential to know the time necessary for melting when isothermal annealing of DNA origami was proceeded in the egg-type initiator.<br>

<h5>Method</h5> We made large alginate hydrogel beads of 2.6mm in diameter (volume of 10µl) for the sake of observation convenience.<br> First, we made alginate hydrogel beads: we took 10 µl 1.5% sodium alginate solution with a micropipette and dropped it in 0.4M CaCl<sub>2</sub> solution.<br> Second, we prepared 10mM, 50mM, and 100mM EGTA solution. We put alginate hydro gel beads in each EGTA solution(600µl), and measured the time necessary for melting alginate hydro gel beads completely.

<br>

<h5>Result</h5> The result is shown in the following table1.<br><br> <table cellspacing="3" 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> From the above result, the melting time of alginate hydrogel was too short at 50 mM and 100 mM EGTA. <br> However, when the concentration of EGTA was 10mM, the alginate hydrogel never melt. So 10 mM EGTA was too thin to melt alginate hydrogels.<br> We need to measure the melting time between 10 mM and 50 mM EGTA in detail.<br>

<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 Mg<sup>2+</sup> (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> 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>

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>

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<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>

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     <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> <ur><li>i)Bending approach</li> <li>ii)Flower micelle approach</li></ur> <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)AFM observation<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)AFM observation<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. By annealing scaffold and designed staples, we can easily get DNA origami of our own design.<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> <h5>Discussion</h5> Just like our design, rectanglar origamis with chipped edges were observed.<br> <br> <br> <h4>1-2)Labeling DNA origami<h4> <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> <br> <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> 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> <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> <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> <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> In a stained gel (Fig.3), marker (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, marker, M13mp18, Ori*, Ori**, and DNA origami with no fluorescent molecule (Ori)<br> <br> <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> <br> <h4>2)Collapsing liposomes</h4> <h4>2-1) Making liposomes</h4> <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> To collapse liposome with our origami, first we investigated how our DNA origami affected liposomes.<br> <br> <h5>Principle</h5> To collapse liposomes with our origami, many origamis have to hybridize with the surface of 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> <br> <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> <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 phase microscope. We confirmed the formation of multilamella liposomes (Fig.4~7).<br> <br> <Img Src="http://openwetware.org/images/7/72/Lipofig4.png" width="400"></br> Fig.4 Phase microscope image of liposomes (cholesterol-conjugated DNA: 0.018µM)<br> <br> <Img Src="http://openwetware.org/images/d/d0/Lipofig5.png" width="400"></br>

Fig.5 Phase microscope image of liposomes (cholesterol-conjugated DNA: 0.069µM)<br>

Fig.6 Phase microscope image of liposomes (cholesterol-conjugated DNA: 1.8µM)<br>

Fig.7 Phase microscope image of liposomes (cholesterol-conjugated DNA: 6.9µM)<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 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> <table>

<tr>
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  <Img Src="http://openwetware.org/images/6/6c/Lipofig8.png" width="400">
 </td>
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  <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> 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> <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> When the concentration of aptamer is 6.9µM, some liposomes were gleaming and others distorted, forming networks (Fig.12).<br>

Fig.12 fluorescent microscope image of liposomes (cholesterol-conjugated DNA: 6.9µM)<br>

<br> <h5>Discussion</h5> From these results, we put forward the following hypothesis about the interaction of DNA origami and liposomes.<br> 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> 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> <br> <br> <h4>2-3)Counting liposomes</h4> <h5>Purpose</h5> To see if DNA origami collapses liposomes, we counted the number of liposomes before and after adding DNA origami. <br> <br> <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> Then we added aptamers at the final concentration of 1.8µM, and counted the number of liposomes with a fluorescent microscope.<br> After counting, we put DNA origami and counted the number of liposomes again.<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> <br> <br> <br> <h4>ii)Flower micelle approach</h4> <h4>Experiment list</h4> The experiment necessary for realization of Flower micelle approach is following.<br> 1) Making liposome <br> 2) Confirming the hybridization of trigger and loop DNA <br> 3) Confirming the formation of loop structure by SPR<br> 4) Collapsing liposome<br> <br> <br> <h4>1)Making liposome</h4> <h5>Purpose</h5> We make liposomes that are to be collapsed by flower micelle method.<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> <h5>Method</h5> <ur><li>1. Making DOPC, DPPC, and Cholesterol lipid Lipid<br> 1-1 Put 7.8 mg DOPC, 7.3mg DPPC , and 3.8mg Cholesterol into each microtube, and add 1ml CHCl3.<br> 1-2 Put it in a ultrasonic bath of 60 degrees Celsius for one hour.<br> 1-3 10mM DOPC, DPPC, Cholesterol lipid is made.<br></li>

          <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> <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> We confirmed the formation of phase-separated liposomes with a fluorescent microscope.<br>

<Img Src="http://openwetware.org/images/f/f2/Flower6.png"></br> Fig.13 Fluorescent microscope image of phase-separated liposomes<br> <br> <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> <h4>2) Confirming the hybridization of trigger and loop DNA</h4> <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 marker) 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> <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> 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> <h4>3) Confirming the formation of loop structure by SPR</h4> <h5>Purpose</h5> To collapse liposomes by flower micelle 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> We checked the hybridization of liposomes and aptamers, and that of aptamers 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 aptamer 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> <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 aptamer (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> <br> <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.15 The transition of SPR value<br> <br> As the first injection of aptamers caused no change of SPR value, we injected aptamers 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> <br> <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. Similarly, it is considered that loop DNAs combined with aptamers. <br> Thus, we confirmed the formation of the loop structures on liposomes.<br> <br> <br> <h4>4) Collapsing liposome</h4> <h5>Purpose</h5> It was tested if liposomes would be collapsed by adding trigger DNA.<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> <h5>Method</h5> <ur><li>1. Make liposomes with loop DNAs<br> 1-1 Mix 2µl liposome (0.2mM) with 2µl aptamer (10µM) at normal temperature<br> 1-2 Add 2µl loop DNA (20µM)</li><br> <li>2. Collapse the liposomes with the loop DNAs<br> 2-1 Add 2µl trigger DNA (20µM) </li></ur><br> <br> <h5>Result</h5> Fig.16 is the result of the sample added trigger DNAs; Fig.17, the sample of control experiment.<br> <table>

<tr>
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  <Img Src="http://openwetware.org/images/5/56/Flower5.png" width="400">
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 <td>
  <Img Src="http://openwetware.org/images/7/77/Flower%EF%BC%94.png" width="400">
 </td>
</tr>

</table> Fig.16,17 Fluorescent microscope image of liposomes <br>(Fig.16: sample added trigger DNAs, Fig.17: control)<br>

As it was difficult to count the number of liposomes in both cases, we did not count them.<br> <br> <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> Two ideas why liposomes were not collapsed are come up:<br> 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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 J. Am. Chem. Soc., 2004, 126 (8), pp 2290–229　Publication Date (Web): February 3, 2004<br><br>
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+<!--
 <h5>Result</h5>