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            <a href="http://openwetware.org/wiki/Biomod/2013/Sendai"><h1 style="color:white;" ><b>Biomod<span>2013<br>&emsp; Team</span>Sendai</b></h1></a>

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<h2>Experiment</h2>

<table id="toc" class="toc" summary="Contents"><tr><td><div id="toctitle"><h2>Contents</h2></div> <ul> <li class="toclevel-1"><a href="#chain"> <span class="tocnumber">1</span> <span class="toctext">First stage: Sensing system</span></a></li> <ul> <li class="toclevel-2"><a href="#bending"> <span class="tocnumber">1-1</span> <span class="toctext">Disruption of temperature sensitive liposomes</span></a></li> </ul> <li class="toclevel-1"><a href="#Flower"> <span class="tocnumber">2</span> <span class="toctext">Second stage: Amplification system</span></a></li> <ul> <li class="toclevel-2"><a href="#sensing"> <span class="tocnumber">2-1</span> <span class="toctext">DNA Origami approach </span></a></li> <ul> <li class="toclevel-2"><a href="#5"> <span class="tocnumber">2-1-1</span> <span class="toctext">Making DNA Origami</span></a></li> <li class="toclevel-2"><a href="#6"> <span class="tocnumber">2-1-2</span> <span class="toctext">Labeling DNA Origami with fluorescent-tagged DNA</span></a></li>

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

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

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

</li>

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

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

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

<h5>Result</h5>

<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>

<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>  <!-- <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> -->

<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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+--><br><br>
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+           <a href="http://openwetware.org/wiki/Biomod/2013/Sendai" class="whiteSendai">Top</a>
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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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      </header>
-<p class="sukima">Experiment
+<div id="ttop">
-</p>
+<a href="#top" class="page_top" onfocus="this.blur();" onclick="scrollTo(0,0); return false;" title="Top"></a></div>
-    <section id="tabs">
-<!--
+  <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="アルギン酸">
+<li class="toclevel-1"><a href="#9">
-<h3 id="experimentsubproject1">内側からアルギン酸膜を破壊するサブプロジェクト</h3></br>
+<span class="tocnumber">2-2</span> <span class="toctext">Flower DNA approach</span></a></li>
+<ul>
-<h4>プロジェクトの流れ</h4></br>
+<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>
-<!--<img src="http://openwetware.org/images/9/9b/Alginate-experiment-flow-00.png"></br>-->
+</li>
-<img src="http://openwetware.org/images/b/b0/Projectflow01.png"></br>
-<h4>1-1リポソームの作製とそれをバッファーに入れてアルギン酸ゲルビーズ内に入れる実験</br>1-1 Making liposome in alginic acid gel beads</h4></br>
+</ul>
+</li>
+</ul>
+</td></tr></table>
-アルギン酸膜内部のリポソームが割れて、リポソームの中に入っていたキレート剤がアルギン酸膜を破壊するというシステムを実現するためにはアルギン酸ゲルビーズ内にリポソームを入れる必要がある。そのために、まずリポソームを作製し、それをアルギン酸ナトリウム水溶液に入れて、アルギン酸ゲルビーズを作製する実験を行った。</br>
+<h3 id=chain>First stage: Sensing system</h3>
-リン脂質一重膜で覆われたドロップレットが油と水の界面に形成されたリン脂質一重膜を通過させることでリン脂質二重膜ベシクルを作製した。</br>
+<h4 id=bending>1-1Disruption of temperature sensitive liposomes</h4>
-グルコース100μℓにoil70μℓを加えてouterバッファーを作製した。次に、oil40μℓにBSG（？）（蛍光物質）を1μℓ加えて、ピペッティングとタッピングで白く濁るまで混ぜて、innerバッファーを作製した。そのinnerバッファーをouterバッファーの上に注いで70秒遠心にかけて、リポソームを作製した。</br>
+<h5>Purpose</h5>
-遠心した後、チューブの底にたまったベシクルを取り出し、1.5% アルギン酸ナトリウムに加え、それをキャピラリー中に入れ、400mM 塩化カルシウム 140μL中に滴下してFig1のような装置で２～３分遠心した。</br>
+In our project, we planned to use liposomes conjugated with NIPAM polymer as a chain reaction initiator. NIPAM (poly-N-isopropyl acrylamide) is a temperature sensitive molecule that has a unique critical temperature (Tc: 32°C ). <br>
-We need to put liposome in alginic acid gel beads for chelating agent which in the liposom destroy alginic acid membrane after liposome is broken. at first We make liposome and put it in a sodium alginate water solution and make alginic acid gel beads with the solution
+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>
-We make liposome by passing droplet which covered one fold of phosphatide films in one fold of phosphatide film which was formed in the interface of two things not mixing well like oil and water</br>
+NIPAM was purchesed from SIGMA ALDRICH.<br>
-We make outer buffer by adding oil 70μℓ to glucose 100μℓ.Then, we make inner buffer by adding oil 40µℓ to BSG(fluorescent substance) 1µℓ,and mixed it until it is muddy white by pipetting and tapping. We pour the inner buffer on outer buffer and centrifuged it for 70 seconds.After that, take out the liposome which collected at the bottom of the tube.</br>
+<br>
-Then, we add the liposome to a sodium alginate water solution(1.5%),and we put it in a capillary and drop it in a calcium chloride water solution(400mM ) by centrifuging it in a device such as Fig1 for 2-3 minutes</br>
-<img src="http://openwetware.org/images/7/73/%E3%82%A2%E3%83%AB%E3%82%AE%E3%83%B3%E9%85%B8%E4%BA%8C%E9%87%8D%E3%83%8E%E3%82%BA%E3%83%AB%EF%BC%91.png" width="200" ></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>
-     Fig1 アルギン酸ゲルビーズの作製</br>
+<h5>Result</h5>
-Fig1 Experimental device of alginic acid gel beads</br></br>
-リポソーム内に蛍光タンパク質が入っているためリポソームが光る。作製したアルギン酸ゲルビーズを共焦点レーザー顕微鏡で観察すると、アルギン酸ゲルビーズ内に光る円状のものが確認されたので、アルギン酸ゲルビーズの中にリポソームが入っていることが確認された。</br></br>
-Because fluorescence protein is in the liposome, liposome shines.</br>
-I observed the alginic acid gel beads by cofocus laser microscope. (Fig 2) because the globe which glittered in alginic acid gel beads is confirmed, it show that liposome was in the alginic acid gel beads.</br>
- <img src="http://openwetware.org/images/c/cc/Image0032.jpg"></br></br>
+<img src="http://openwetware.org/images/9/9e/2-1-1fig1.png" width="100%" height="100%"><br>
-     Fig2 アルギン酸ナトリウム水溶液に蛍光入りリポソームを加えて作ったアルギン酸ゲルビーズの共焦点レーザー顕微鏡画像</br>Fig2    Cofocus laser microscope image of alginic acid gel beads with liposome</br></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>
-<h4>1-2内部にバッファーの入ったアルギン酸膜の作製</br>1-2 preparing of algin acid membrane containig buffer </h4></br>
+<br>
-アルギン酸ゲルビーズの中は粘度が高く、ＤＮＡの衝突回数が減るためアニーリングができない。そこで、内部にバッファーを含んでいて、外側がアルギン酸ゲルになっているアルギン酸膜を作製する必要がある。このアルギン酸膜を作製するために、二重ノズルを作製して以下の実験を行った。</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>
-２重の毛細管に遠心機で高重力をかけることによりあらかじめ封入された内容液と外溶液（アルギン酸ナトリウム溶液）がノズル先端から内容液がアルギン酸ナトリウムに包まれた状態で粒状に放出される。これを塩化カルシウム溶液に滴下することで表面のみがゲル化して、膜状のゲルビーズが完成する。</br>
-Viscosity of beads made from algin acid is so thick that DNA can't be annealed because appulse numbers is decreased. So, we have to make algin acid membrane containing buffer. To make algin acid membrane containing buffer, we made double nosepiece and did following experiments.
-Basis</br>
-When we provided enhanced gravity to double capillary by a centrifuge rotor, enclosed content fluid and extra solution (sodium alginate) effuse from front edge of nosepiece in the state 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 and we got membranal gel beads.</br>
-Fig3のように外側の太いキャピラリーと内側の細いキャピラリーを作成する。</br>
+<h3 id=Flower>Second stage: Amplification system</h5>
-キャピラリーは外径1mmのものを熱加工した。</br>
+<h4 id=sensing>2-1 DNA Origami approach</h4>
-</br>
+<h5 id=5>2-1-1 Making DNA Origami</h5>
-外管には1.5%アルギン酸ナトリウム溶液を、内管には蛍光物質+mQをいれた</br>
+<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>
-２重キャピラリーを用いて2～3分遠心にかけ0.4M塩化カルシウム水溶液に滴下する。</br>
-Substance</br>
+<h5>Result</h5>
-We use double capillary, 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, fluorescent stuff and mQ as inner one. Finally, we put capillary containing solution into   centrifuge rotor for a few minutes and fall in drops to 0.4M calcium chloride.</br>
+We obtain DNA Origami same as our design. The result was confirmed by AFM (Atomic Force Microscope.)<br>
-<img src="http://openwetware.org/images/6/68/%E3%82%A2%E3%83%AB%E3%82%AE%E3%83%B3%E9%85%B8%E4%BA%8C%E9%87%8D%E3%83%8E%E3%82%BA%E3%83%AB%EF%BC%92.png" width="400" height="200" ></br>
+<br>
-     Fig3 二重ノズルの構造</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>
-完成したアルギン酸ゲルを共焦点レーザー顕微鏡で観察すると、下図のようにアルギン酸ゲルの内部に蛍光が確認できた。このことから内部にバッファーを含むアルギン酸膜が作製できたことが分かった。</br>
+<h5>Discussion</h5>
-We observed complete algin acid gel by confocal laser microscope and we could confirm fliorescence in the algin acid gel. Therefore, we conform argin acid membrane containing buffer.</br>
+<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>
-<h4>2ニッパムの効果でリポソームが割れることの確認実験</br>2Function confirmation of PNIPAM</h4></br>
+<!--ここは表を使ってコンパクトに-->
-このプロジェクトでは温度を約３２度に上げた時に確実にニッパムの効果でリポソームが割れる必要があるため、温度を約３２度に上げればニッパム付きリポソームが割れることを確認した。</br>
+<div align="center"><Img Src="
+http://openwetware.org/images/d/d7/2-2-1fig4.png"></div><br>
-ニッパム分子は３２度以下では水和していて親水性だが、３２度以上では収縮して疎水性になる。ニッパムがリポソームに修飾されると、３２度以下ではニッパム分子の水和により安定な状態になるが、３２度以上ではニッパム分子が疎水性になって不安定な状態になるので、３２度以上になった時にリポソームが割れることになる。</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>
-参考</br>
+  <div align="center"><Img Src="
-http://www.sigmaaldrich.com/etc/medialib/docs/SAJ/Brochure/1/j_recipedds2.Par.0001.File.tmp/j_recipedds2.pdf</br></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>
-PNIPAM付き脂質を使ってＧＵＶによりニッパム修飾されたリポソームを作製した。</br>
-PNIPAM付き脂質とはFig4のような、リン脂質にPNIPAMが結合したものである。</br>
-In this project, it is necessary that liposome destroys surely when we increase temperature to 32℃.So, we confirmed whether liposome with PNIPAM destroys when the temperature become 32℃.</br>
-PNIPAM molecular is hydrophilic by hydrating when temperature is under 32℃ .However, it becomes hydrophobic when temperature is more than 32℃ by shrinking. By decollating liposome with PNIPAM, liposome is stable under 32℃ but becomes erratic state at more than 32℃ As a result, liposome with PNIPAM destroys when the temperature is more than 32℃.</br>
-Reference</br>
-http://www.sigmaaldrich.com/etc/medialib/docs/SAJ/Brochure/1/j_recipedds2.Par.0001.File.tmp/j_recipedds2.pdf</br></br>
-We created liposome from lipid with PNIPAM(Fig4) by GUV.</br>
-<!--<img src="http://openwetware.org/images/e/e8/NIPAMgousei.png"></br>-->
-<img src="http://openwetware.org/images/f/fc/PNIPAM-phospholipid.png">
-     Fig4 PNIPAM付き脂質</br></br>
-※W/Oエマルション法を用いたGUV(Giant Unilamellar Vesicle)</br>How to make liposome by GUV(Giant Unilamellar Vesicle) using W/O emulsion technique</br>
-mM DOPCのストック溶液をArガスで乾燥させた脂質フィルムをミクロチューブ内に作り真空デシケータ内でさらに乾燥させた。リン脂質フィルムに流動パラフィン 500μLを加える。超音波洗浄機を用いて60℃で60分間リン脂質をオイルに溶かした。インナー溶液を150mM スクロース、350mM グルコース、100mM EGTAとする。オイルに溶かしたリン脂質にインナー溶液 50μLを加え、遠心分離し、エマルション溶液を作製した。アウター溶液を600mM グルコースとする。アウター溶液 100μLにエマルション溶液 70μLを加える。</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>
-リポソームを作製したら、位相差顕微鏡でリポソームの数を数えた。そして、温度を約３２度に上げて再び位相差顕微鏡でリポソームの数を数えた。（結果を表で示す）</br>
-その結果、温度を上げた後の方がリポソームの数が減っていたのでニッパム分子の効果でリポソームが割れたと考えられる。</br></br>
-We counted the number of liposome by phase contrast microscope. And we increased temperature to approximately 32 ℃ and counted the number of liposome again.</br>
-As a result, the number of liposome decreased when we raised temperature, so we  thought that liposome was broken by the effect of PNIPAM molecules.</br>
-<table>
- <tr>
-  <td>
-   <img src="http://openwetware.org/images/b/ba/Snap_20130829_125105_7050.jpg" width="400">
-  </td>
-  <td>
-   <img src="http://openwetware.org/images/1/1d/Snap_20130829_141541_7053.jpg" width="400">
-  </td>
- </tr>
-</table>
-<h4>3アルギン酸ゲルビーズを溶かすのに必要なＥＧＴＡの濃度と時間の測定</br>3 measurement of density of EGTA and time necessary to dissolve alginc acid gel</h4></br>
-アルギン酸膜内のリポソームの中にはアルギン酸ゲルを溶かすのに十分なＥＧＴＡ（キレート剤の一つ）が入っている必要がある。また、ニッパムの効果でリポソームが割れた時に尿素アニーリングとアルギン酸膜の破壊が同時に起こるが、前者にかかる時間よりも後者にかかる時間の方が短くなければならない。そのため、アルギン酸ゲルビーズを溶かすのに必要なＥＧＴＡの濃度と時間を測定した。</br>
-アルギン酸ゲルビーズを作製し、50mM,100mM,500mMのＥＧＴＡを加えたものとＥＧＴＡを加えないものを用意する。このＥＧＴＡの濃度が違う４種類のアルギン酸ゲルビーズが時間とともにどれくらい減るのかを測定する。それぞれ０分後、５分後、１０分後…のサンプルを取り出してアルギン酸ゲルビーズの数を数える。同じ実験を何回か行った。（測定結果、平均値などをまとめた表を書く。）</br></br>
-以上の結果からアルギン酸ゲルビーズをとかすのに必要なＥＧＴＡ濃度は？mMで、それにかかる時間は約？分だと分かった。</br></br>
-It is necessary for the liposome in the alginic acid membrane to hold enough EGTA (one of the chelating agents) to dissolve alginic acid gel. In addition, when liposome is broken by an effect of NIPAM, Urea annealing and the destruction of the alginic acid film happen at the same time. But Time for Urea diluting Annealing must be shorter than Time for destruction of the alginic acid membrane. Therefore We measure density and time of the EGTA necessary to dissolve alginic acid gel beads.</br>
-We make alginic acid gel beads and add 50mM,100mM,500mM EGTA to the solution. In addition, we prepare the thing which does not put EGTA in solution(control experiment)</br>
-We measure how many alginic acid gel beads decrease as time passes about the density of four kinds of EGTA.We take out the sample 0 minutes later, five minutes, ten minutes later・・・and count the number of alginic acid gel beads.We did the same experiment several times. </br>
-From these results, the density of EGTA necessary to dissolve alginic acid gel beads is ?mM. and it took about ? minutes until alginic acid gel beads melted</br>
-<h4>4尿素アニーリングによるＤＮＡオリガミの作製とその作製にかかる時間の測定</h4></br>
-尿素アニーリングによりトリガーとなるＤＮＡオリガミがきちんと形成される必要がある。また前述したとおり、このシステム（アルギン酸班のシステム）の実現には尿素アニーリングにかかる時間がアルギン酸膜が破壊される時間よりも短くなくてはならないため、尿素アニーリングにかかる時間を測定した。</br>
-尿素アニーリングの原理は以下のとおりである。</br>
-尿素があることで水分子の極性が小さくなる。DNAのハイブリダイゼーションは水素結合によって行われるので、尿素により水素結合の力が小さくなる。これにより、通常より低い温度でも融解が可能になる。これを応用したものが尿素アニーリングであり、徐々に尿素の濃度を下げることでDNAをハイブリダイゼーションさせることができ、通常の融解温度より低い温度でのアニーリング可能となる。</br></br>
-（↓プロトコルが変わりました）</br>
-尿素(8M)とDNAオリガミの材料（M13mp18と118本とステイプルが100μℓの12.5mMの酢酸マグネシウム入りの10×TAEの中に入ったもの）を入れたフィルター(アミコン(3.5kD)商品の詳細を書く）をフローターにさしてビーカーに水を入れたものの上に浮かべる。ビーカーにスターラーバーを入れて攪拌機の上に置いて、撹拌しながら一晩置いた。このようにすることで尿素がフィルターから抜けて、ＤＮＡはフィルターの中に留まるため、尿素が希釈されて尿素希釈アニーリングが行われる。フィルター内に残ったサンプルを電気泳動やＡＦＭにより観察した。</br></br>
-ＡＦＭで設計したサイズ・形の構造体が確認され、電気泳動でＭ１３のレーンと尿素アニーリングしたＤＮＡオリガミを泳動したレーンのバンドを比較すると後者の方が前者よりも高い位置にバンドが確認されたため、オリガミの構造体ができていると思われる</br></br>
-It is necessary for trigger DNA origami to be formed by Urea diluting Annealing. In addition, Time for Urea diluting Annealing must be shorter than Time for destruction of the alginic acid membrane to realize this system (system of the alginic acid group) .
-Therefore we measured the time that Urea diluting Annealing takes.</br>
-The principles of are Urea diluting Annealing is as follows. Polarity of H2O 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. So, we can do annealing by diluting urea.</br>
-We added M13mp18 and staples at the rate of 1:20 in TAE buffer with urea (6M) and Mg2+ (12.5mM).Then, we set the filter to floater and float it on TAE buffer with Mg2+(12.5mM).The devise was mixed by stirrer for 4 hours. By doing that, urea passes the filter and escape to outside buffer but DNA remains in filter, so we can do urea diluting annealing. Then, we observed sample remained in the filter by AFM and electrophoresis.</br>
-We observed structures as we designed by AFM imaging. </br>
-The result is Fig5 as below. The scale of DNA origami is similar to our design. (for details of DNA origami design click here).
-<!--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.-->
-  So, we thought we could create DNA origami by urea diluting annealing.</br>
-<img src="http://openwetware.org/images/3/30/Urea-duilting-anealing-AFMimage-00.png" width="600"></br>
-     Fig5 尿素アニーリングにより作製したDNAオリガミのAFM画像（サンプル）</br></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>
-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>
-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>
-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="Outside">
-<h3 id="experimentsubproject2">外側からリポソームを破壊するサブプロジェクト</h3>
-<p>反応を開始するトリガーDNAが放出され、反応が開始されると、次にすべきは反応の連鎖である。有効成分と、新たなトリガーを含んだリポソームを破壊すれば、放出された構造物が、連鎖反応的に周囲のリポソームを破壊する。<br>
-Once the trigger DNA, which begins the interaction, is released, the next is the chain reaction. If a liposome containing new triggers and active ingredients is broken, the released triggers come to break the surrounding liposomes one after another.<br>
-We tackled the problem of breaking liposomes by the following two approaches.<br>
-<ur>
-<li>Ⅰ膜を湾曲させるアプローチ ⅠApproach by bending membranes</li>
-<li>Ⅱフラワーミセルによるアプローチ ⅡApproach by flower micelles</li></ur>
-</br></p>
-<h4>Ⅰ膜を湾曲させるアプローチ ⅠApproach by bending membranes</h4>
-<h5>(1)リポソームを破壊するトリガーであるDNAオリガミの作成 (1)Making DNA origami that acts as a trigger for breaking liposome</h5>
-DNA origamiは、あらかじめ決まった構造体を作る際に用いられる構造である。DNA origamiは足場配列（scaffold strand）という一本鎖の長鎖DNAとステイプル配列（staple strand）という短い一本鎖DNAで構成される。<br>
-私たちのプロジェクトでは、リポソームを破壊するトリガーとしてDNA origamiを利用する。AFMによりDNAオリガミの作成を、電気泳動によりその蛍光標識(蛍光基つきDNAが、DNAオリガミに確かに接着していること)を確かめた。<br>
-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>
-In our project, we used DNA origami as triggers for breaking liposomes. <br>
-We confirmed it is well formed by AFM (Atomic Force Microscopy) and that it is also fluorescently labeled (fluorescent molecules are successfully attached to the origamis) by electrophoresis.<br>
 <br>
-<h6>①アニーリング Annealing</h6>
+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>
-マイクロチューブにM13mp18, ステイプル, 5xTAE Mg2+, mQを混合、2時間半アニーリングを行った。<br>
-We mixed M13mp18, staples, 5xTAE Mg2+, and mQ in a microtube and annealed it for 2.5 hours.<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>
-<h6>②AFM observation</h6><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>
-DNAオリガミが設計図通りにできたことを確かめるため、AFM観察を行った(Fig.1)。設計図通り、端の欠けた長方形のDNAオリガミが確認できた。<br>
-To see the origami formation, we observed the sample by AFM (Fig.1). Just like our design, rectangle origamis chipped in its edges was observed.<br>
-<Img Src="http://openwetware.org/images/d/d9/Outsideafm2.png"> <br>
-Fig.1 AFM image of DNA origami (M13: 4nM, staples:20nM)<br>
 <br>
-<h6>③電気泳動による蛍光標識の確認 Confirmation of fluorescently labeled origamis by electrophoresis</h6>
+<div align="center"><Img Src="
-DNAオリガミをリポソームに作用させるとき、そのDNAオリガミが蛍光標識されていると、確認が容易である。DNAオリガミがうまく蛍光標識されているかを確かめるため、電気泳動を行った。<br>
+http://openwetware.org/images/c/c0/2-2-1fig7.png"></div><br>
-Seeing the effect of DNA origami on liposomes, if the origami is fluorescently labeled, it is much easier to observe. To see the origami is well labeled with fluorescent molecules, we used electrophoresis.<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>
-このDNAオリガミは、標識のための蛍光基付きDNAが付くことのできるステイプルをもっている。同上の条件で、蛍光基付きDNAを混合し、2時間半アニーリングを行った。
+When the concentration of Origami-anchor DNA is 6.9µM, some liposomes were gleaming and others distorted, forming networks (Fig.7).
-また、蛍光基付きDNAがDNAオリガミに接着するのに、より早い時間でもすむかを調べるため、蛍光修飾なしDNAオリガミ溶液を10μlとり、蛍光基付きDNA0.6µlを加え、40分間放置した(これを、蛍光後付け修飾DNAオリガミとした)。<br>
-Our DNA origami has many staples that can bind to fluorescent tagged DNAs for labeling. We added fluorescent tagged DNAs in the pre-annealing buffer (the same buffer as in ① Annealing experiment) and annealed them.<br>
-In addition, to see if the origami binds to the fluorescent tagged DNA in shorter time, we added the fluorescent tagged DNA into the post-annealing buffer that hadn’t contain the fluorescent tagged DNA yet, and left them for 40 minutes.<br>
 <br>
- 染色前にゲルスキャンを行うと、蛍光基のついたストランドのみが観察されるので、蛍光修飾された構造体の位置を確認することができる。次に、染色後、ゲルスキャンを行うと、全てのDNA構造体の位置が確認できる。これらを合わせて、まず、染色後にDNAオリガミが正しく作られたことを確かめ、さらに、それが染色前にみられた蛍光修飾された構造体の位置とほぼ等しいと示すことで、DNAオリガミがうまく蛍光修飾されたことを確かめた。<br>
-Scanning a gel before its staining, we can see only the bands of DNA structures with fluorescent molecules; scanning a gel after its staining, we can see the bands of all DNA structures. So we scanned a gel before and after staining (we scanned non-stained gel and stained gel). Combining these two, first we saw the band of our origami with non-stained gel. Then, we compared the band with that in stained gel. If the band of origami in non-stained gel is in the same height as that in stained gel, we can say our origami is successfully fluorescently labeled.<br>
 <br>
- 電気泳動は、１%アガロースゲルを用い、CV100Vで50分行った。<br>
+<h5>Discussion</h5>
-Electrophoresis was conducted with 1% agarose gel, CV100V for 50 minutes.<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.
-<Img Src="http://openwetware.org/images/c/c6/Outside-gel-2.png" width="300"> </br>
- Fig.2 染色後のゲル画像。左から：ラダー、M13、蛍光付きDNAオリガミ(Ori*)、蛍光後付けDNAオリガミ(Ori**)、蛍光なしDNAオリガミ(Ori)<br clear="left">
 <br>
-染色後のゲル画像(Fig.2)の、ラダー(レーン1)の最大のDNA鎖は20kbである。これとレーン2のM13とを、アニーリングしたDNAオリガミ(レーン3，4，5)たちと比べると、DNAオリガミたちのほうが、上部にバンドが見られる。よって、レーン3~5において、M13とステイプルが反応した構造体が出来たことがわかった。なお、バンドが拡散してしまったので、バンドの高さは、バンドの真ん中で比較した。<br>
+Anyway, <u>these data strongly indicated the designed DNA origami disrupted liposomes with high concentration of the anchor DNA.</u>
 <br>
-<Img Src="http://openwetware.org/images/0/01/Outside-gel-3.png" width="300" align="left">
- Fig.3 染色前のゲル画像。左から、蛍光付きDNAオリガミ(Ori*)と、蛍光後付けDNAオリガミ(Ori**)のみが観察できる。<br clear="left">
 <br>
- 次に、染色前のゲル画像(Fig.3)をみると、レーン3,4にのみ、蛍光修飾された構造体が確認できた。これらは、染色後のゲル画像(Fig.2)でみられたDNAオリガミと等しい位置にある。よって、DNAオリガミが無事蛍光修飾されたと言える。<br>
 <br>
-<h5>(2)DNAオリガミでリポソームを割る実験 Breaking liposome with DNA origami</h5>
+<h5 id=13>2-1-4 Disrupting liposomes by DNA Origami (quantitative analysis)<h5>
-<h6>①リポソームの作成 Making liposome</h6>
+<h5>Purpose</h5>
-DNAオリガミで割るためのリポソームを作成した。<br>
+The above experiments in 2-1-3 microscopic analysis suggest that our DNA Origami disrupted liposomes. Thus, we performed more quantitative analysis.<br>
-リポソームを作るリン脂質は、両親媒性であり(親水基hydrophilic group・疎水基hydrophobic groupを持つ)、水と接触すると、親水基が内部に水を取り込み内側を、疎水基が外側を向くように整列する。こうして球状のリポソームが作成できる。<br>
-We made liposome that was to be broken by DNA origami.
-Phospholipid, which composes liposome, is amphipathic molecule. It has hydrophilic and hydrophobic groups. When it touches water, the hydrophilic groups take water inside. At the same time, they touch the water outside. So they form the innermost and outermost part. On the other hand, the hydrophobic groups forms the intermediate part. In this way, round liposomes are made.<br>
 <br>
-脂質(DOPC)、脂質を溶かす溶媒(CHCl3)をミクロチューブにいれ、Arガスで乾かした。次に、観察用Bufferを加えたのち、湯煎してリポソームを作成した。<br>
+<h5>Method</h5>
-To make liposome, first we mixed lipid (DOPC) and solvent (CHCl3) in microtube, and desiccate it with Argon gas. Then, adding buffer (1xTAE Mg2+), heat it in warm water for a few hours.<br>
+<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>
-<h6>②Investigating the interaction of DNA origami and liposome</h6>
+</div>
-次に、作成したリポソームとDNAオリガミとの相互作用を調べた。<br>
+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>
-リポソームを、DNAオリガミをトリガーとして割るには、DNAオリガミをリポソーム表面に多数ハイブリさせる必要がある。そこで、リポソームにコレステロール修飾DNAを添加し、表面に一本鎖DNAが現れるようにする。これを、DNAオリガミの一部と相補的な配列を持つ一本鎖DNAとすれば、DNAオリガミがリポソーム表面に多数ハイブリする。 <br>
-Next, we investigated how our DNA origami affects liposome.
-To break liposome with triggers of DNA origami, many origamis have to hybridize with the surface of liposome. To begin with, we added cholesterol-conjugated single-stranded DNAs into liposome, and make them float on the surface. If the single-stranded DNAs have some complementary parts to our origami, the origami comes to hybridize with the surface.<br>
 <br>
-作製したリポソームにコレステロール修飾したDNAをつけるため、このリポソームにcholesterol修飾DNAを0.018, 0.069, 1.8, 6.9µMになるように加え、位相差顕微鏡でリポソームを確認した。<br>
+Following 50 μL of samples(Fig.9) were examined with the Flow cytometer.
-To float cholesterol-conjugated DNAs on the surface of liposome, we added cholesterol-conjugated DNAs into liposome at the final concentration of 0.018, 0.069, 1.8, and 6.9µM. Then we observed the sample with a phase microscope.<br>
+<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>
-<Img Src="http://openwetware.org/images/d/da/1%E7%95%AA.jpg" width="400"></br>
+<h5> Discussion </h5>
+<u>These results confirmed that our designed DNA origami actually disrupted liposomes. (The efficiency was higher than 2% surfactant!)</u>
-Fig.4 Phase microscope image of liposome (cholesterol-conjugated DNA: 0.018µM)<br>
 <br>
-<Img Src="http://openwetware.org/images/0/0e/2%E7%95%AA.jpg" width="400"></br>
-Fig.5 Phase microscope image of liposome (cholesterol-conjugated DNA: 0.069µM)<br>
 <br>
+<h5 id=8>2-1-5 Confirming sequence specificity of DNA flow cytometer</h5>
-<Img Src="http://openwetware.org/images/5/5a/3%E7%95%AA.jpg" width="400"></br>
+<h5>Purpose</h5>
-Fig.6 Phase microscope image of liposome (cholesterol-conjugated DNA: 1.8µM)<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>Method</h5>
-<Img Src="http://openwetware.org/images/3/31/4%E7%95%AA%EF%BC%92.jpg" width="400"></br>
+Experimental conditions were the same in 2-1-4 except samples.<br>
-Fig.7 Phase microscope image of liposome (cholesterol-conjugated DNA: 6.9µM)<br>
+Sample 1 (Complement). Liposomes + Origami-anchor DNA(A) + Key DNA(A)<br>
-つの条件全てにおいて、位相差顕微鏡で、リポソームが観察され、マルチラメラリポソームの作成が確認できた(Fig.4~7)。<br>
+Sample 2 (no binding pair). Liposomes + Orgiami-anchor DNA(A) + Key DNA(B)<br>
-In all four conditions, liposomes are observed with a phase microscope. We confirmed the formation of multilamella liposomes (Fig.4~7).<br>
+<A href="http://openwetware.org/wiki/Biomod/2013/Sendai/protocol">Protocol</A><br>
 <br>
-次に、DNAオリガミを加え、蛍光顕微鏡で観察した。<br>
+<h5>Result</h5>
-cholesterol修飾DNA濃度が0.018, 0.069µMの時、多数のリポソームの膜が緑色に光っており、リポソーム膜に蛍光標識したDNAオリガミがハイブリしていることが確認できた(Fig.8,9,10)。<br>
+The results were shown in figure 12.<br>
-Next, adding fluorescently labeled DNA origamis into the above liposomes, we saw any change would happen with a fluorescent microscopy.<br>
+<div align="center">
-When the concentration of cholesterol-conjugated DNA was 0.018, 0.069µM, many gleaming (in green color) liposomes are observed. We confirmed that the fluorescently labeled origamis well hybridized with the liposome surface (Fig.8,9,10).<br>
+<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>
-<table>
- <tr>
-  <td>
-   <Img Src="http://openwetware.org/images/f/f3/1.jpg" width="400">
-  </td>
-  <td>
-   <Img Src="http://openwetware.org/images/6/69/1_%283%29.jpg" width="400">
-  </td>
- </tr>
-</table>
-Fig.8,9 fluorescent microscopy image of liposome (cholesterol-conjugated DNA: 0.018µM)<br>
 <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>
-<Img Src="http://openwetware.org/images/1/15/2.jpg" width="400"></br>
-Fig.10 fluorescent microscopy image of liposome (cholesterol-conjugated DNA: 0.069µM)<br>
 <br>
-一方で、cholesterol修飾DNA濃度が1.8µMの時は、蛍光顕微鏡では光るリポソームがほとんど確認できなかった(Fig.11)。この結果は、リポソームが割れた可能性を示唆している。<br>
+<h5>Discussion</h5>
-On the other hand,when the concentration of cholesterol-conjugated DNAs is 1.8µM, few gleaming liposome could be seen with fluorescent microscopy (Fig.11). This result indicates the possibility that liposomes were broken.<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>
-<Img Src="http://openwetware.org/images/7/79/3.jpg" width="400"></br>
-Fig.11 fluorescent microscopy image of liposome (cholesterol-conjugated DNA: 1.8µM)<br>
 <br>
- cholesterol修飾DNA濃度が6.9µMの時、膜が緑色に光っているリポソーム(Fig.12,13)のほか、<br>
-脂質が変形し、ネットワーク状に広がっているのが観察された(Fig.13~15)。<br>
-When the concentration of cholesterol-conjugated DNAs is 6.9µM, some liposome are gleaming (Fig.12,13). Others are distorted, and forming a network-like structure (Fig.13~15).<br>
+<h4 id=9>2-2 Flower DNA approach</h4>
+<!-------------SPRコメントアウトここから-------------------->
 <!--
-<Img Src="http://openwetware.org/images/9/9a/4_%282%29.jpg">
+<h5 id=10> 2-2-1 Confirming the formation of the loop structure by SPR</h5>
-<Img Src="http://openwetware.org/images/1/15/4_%2810%29.jpg">
+<h5>Purpose</h5>
-<Img Src="http://openwetware.org/images/4/45/4_%2814%29.jpg">
+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>
-<Img Src="http://openwetware.org/images/f/f4/4_%287%29.jpg" width="400"></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>
-Fig.12~15 fluorescent microscopy image of liposome (cholesterol-conjugated DNA: 6.9µM)<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>
-以上の結果から、私たちはリポソームとDNAオリガミの相互作用について、以下の仮説を考えた。<br>
+As the first injection of Anchored DNA caused no change of SPR value, we injected Anchored DNA for two times. <br>
-From these results, we put forward the following hypothesis about the interaction of DNA origami and liposome.<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>
-cholesterol修飾DNAが低濃度(0.018, 0.069µM)の時、リポソームにはDNAオリガミが安定してハイブリしている。中濃度(1.8µM)の時、リポソームにより多くのcholesterol修飾DNAを介してDNAオリガミが突き刺さるので、個々のリポソームに負荷がかかり、リポソームは割れやすくなる。cholesterol修飾DNAが十分多い(6.9µM)時、一部のリポソームは互いに独立して存在しているが、一部のリポソームは、cholesterol修飾DNA-DNAオリガミ複合体を介して、ネットワークを構成する。<br>
-When the concentration of cholesterol-conjugated DNA is low (0.018, 0.069µM), DNA origamis hybridize with the surface of the liposomes relatively stablely. When the concentration is middle (1.8µM), more DNA origamis hybridizes with the surface, and place stress on each lipoosme. It becomes fragile and easy to be broken. When the concentration is high (6.9µM), some liposome exist individually, and others form network via cholesterol-conjugated DNA and DNA origami complexes.<br>
+<h5>Discussion</h5>
-<Img Src="http://openwetware.org/images/7/7c/Experimentinsidefig.png"><br>
+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>
-この仮説によれば、cholesterol修飾DNA濃度が1.8µMの時、DNAオリガミによってリポソームが割れると考えられる。よって、私たちは、この濃度でリポソームが割れるかどうかを調べるため次の実験を行った。<br>
+-->
-According to this hypothesis, when the concentration of cholesterol-conjugated DNA is 1.8µM, DNA origami breaks liposome. Therefore, in the following experiment, we check if liposomes are broken at this concentration of cholesterol-conjugated DNA.<br>
+<!---------------SPRコメントアウトここまで------------------>
-<br>
-<h6>③リポソームの数をカウントする実験 Counting the number of liposomes</h6>
-DNAオリガミを添加することで、リポソームが割れたかどうかを調べるには、DNAオリガミの添加前後のリポソームの数の変化を調べればよい。私たちは、DNAオリガミの添加前後でリポソームの数をカウントする実験を行った。<br>
-リポソームを観察しやすいよう、脂質(DOPC)、脂質を溶かす溶媒(CHCl3)とともに、蛍光色素であるTR-DHPEを加えて、リポソームを作成した。<br>
-次に、cholesterol修飾DNAを1.8µMになるよう加え、蛍光顕微鏡でリポソームの数をカウントした。<br>
-カウント後、DNAオリガミを振りかけ、再度リポソームの数をカウントした。<br>
-To see if DNA origami broke liposome, we counted the number of liposomes before and after adding DNA origami. <br>
-For the sake of observation convenience, we mixed TR-DHPE (red fluorescent dye) with lipid (DOPC) and solvate (CHCl3) and made liposome.<br>
-Then we added cholesterol-conjugated DNA at the final concentration of 1.8µM, and counted the number of liposome with a fluorescent microsopy.<br>
-After counting, we put DNA origami and counted the number of liposome again.<br>
-<br>
-<br>
-<h4>Ⅱフラワーミセルによるアプローチ Approach by flower micelles</h4>
-私たちの実験はリポソームを作製する実験、リポソームにコレステロール修飾DNA及びループDNAを結合させる実験、トリガーDNAを作用させリポソームを割る実験の３つがある。</br>
-<h5>(1)リポソームの作成</h5>
-基本的には上記の膜を湾曲させるアプローチと同じ方法で作成した。</br>
-ここでは相分離リポソームの作り方について説明する。</br></br>
-まずDOPC,DPPC,CholesterolそれぞれのLipidを製作する。</br>
-DOPCを7.8mg.,DPPCを7.3mg, Cholesterolを3.8mg　それぞれとCHCl3を1mlをミクロチューブにいれ、60℃のお湯の中で1時間超音波（43KHz）にかける。</br>
-それぞれ10nMolのDOPC,DPPC,Cholesterol　	Lipidができる</br>
-次にDOPC:DPPC:Cholesterol=10:10:4の割合で混合し相分離リポソームを製作する。</br>
-DOPC(10nM)を4μℓ、DPPC(10nM) を4μℓ、 Cholesterol (10nM)を1.6μℓ、buffer</br></br>
-次に、ミクロチューブに100μℓ観察用Bufferを入れ、３時間ほど湯煎した。</br>
-箱からミクロチューブを取り出しそこから30μℓとり蛍光顕微鏡で観察した。</br>
-蛍光顕微鏡で、リポソームが観察された</br></br>
+<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>
-<h5>(2)コレステロール修飾DNA及びループDNAの結合</h5>
+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>
-フラワーミセルを形成するためにループ構造のＤＮＡをリポソーム表面に結合させなければならない。そのため、まずコレステロール付きのＤＮＡをリポソームに１で製作したリポソームにコレステロール修飾DNAを付ける</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>
-通常のリポソーム、相分離リポソームの二種類で行う。</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>
-</br>
+<h5>Discussion</h5>
-<table>
+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>
- <tr>
+<br>
-  <td>
+<h5 id=12> 2-2-2 Confirming sequence specificity of DNA</h5>
-   完成したリポソーム0.1mM
+<h5>Purpose</h5>
-  </td>
+We demonstrate the selectivity of our Key DNA: the Key DNA only affects the corresponding Flower-anchor DNA and liposomes.<br>
-  <td>
+<br>
-.5μℓ
+<h5>Method</h5>
-  </td>
+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>
- </tr>
+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>
- <tr>
+After adding B-Key DNA, the number of each color liposomes is counted to confirm the selectivity.<br>
-  <td>
+As a control, only buffer is added instead of B-Key DNA.<br>
-   Chol leg 0.1μM
+<A href="http://openwetware.org/wiki/Biomod/2013/Sendai/protocol">Protocol</A><br>
-  </td>
+<br>
-  <td>
+<h5>Result</h5>
-.2μℓ
+Fig.16 shows fluorescent microscope image of liposomes added B-Key DNA (left) and control buffer(right).
-  </td>
+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.
-</table>
+In the right, almost the same number of Green and Red liposomes are seen.<br>
-</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">
-次にコレステロール修飾DNAに相補なループDNAを加える</br>
+<tr bgcolor="moccasin">
-ループDNAは10bp,20bp,40bpの三種類、リポソームが二種類の計6パターンのサンプルで実験を行う。</br>
+<td> Additives </td>
+<td> B-key DNA </td>
-</br>
+<td> Buffer </td>
-<table>
+</tr>
- <tr>
+<tr bgcolor="moccasin">
-  <td>
+<td> Green：Red </td>
-   DNA付リポソーム　0.1mM
+<td>17:2 (n = 19)</td>
-  </td>
+<td>16:17 (n= 33)</td>
-  <td>
+</tr>
-.5μℓ
-  </td>
- </tr>
- <tr>
-  <td>
-   ループDNA        0.1μM
-  </td>
-  <td>
-.0μℓ
-  </td>
- </tr>
 </table>
-</br>
+<div class="captiontable">Table1   Ratio of Green and Red liposomes</div><br>
+Table1 shows the Ratio of Green and Red liposomes.<br>
-確認はＡＦＭを用いる。</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>
-AFMを用いて、DNA付リポソームを観察したところ、リポソーム表面にDNA鎖のようなループを確認することができた。</br>
 <br>
-<h5>(3)DNAループにトリガーストランドのハイブリ</h5>
+<h5>Discussion</h5>
-トリガーストランドを加える前にリポソームの数を数えた</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>
-DNAループができた六種類のリポソームにトリガーストランドを注入しリポソームを割る</br>
+<u>The selectivity of Key DNA has been successfully demonstrated. </u></h6>
-</br>
-<table>
- <tr>
-  <td>
-   ループ付きリポソーム　0.1mM
-  </td>
-  <td>
-.5μℓ
-  </td>
- </tr>
- <tr>
-  <td>
-   トリガーDNA　　      1.0mM
-  </td>
-  <td>
-.2μℓ
-  </td>
- </tr>
-</table>
-</br>
-投入後30分間常温で放置し　蛍光顕微鏡で観察する</br></br>
-トリガーストランド投入前には観察することができたリポソームが、投入後は確認することができなかった。つまりリポソームが予測どうり割れたと考えられる。</br></br>
          </article>