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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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               <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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+<div id="ttop">
+<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>
-<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>
--->
-     </article>
+<table id="toc" class="toc" summary="Contents"><tr><td><div id="toctitle"><h2>Contents</h2></div>
+<ul>
-      <article data-title="アルギン酸">
+<li class="toclevel-1"><a href="#chain">
-<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">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>
-<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</h4></br>
+<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>
-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>
+<li class="toclevel-2"><a href="#11">
-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>
+<span class="tocnumber">2-2-1</span> <span class="toctext">Disruption of liposomes by Flower DNA</span></a></li>
-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>
+<li class="toclevel-2"><a href="#12">
-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>
+<span class="tocnumber">2-2-2</span> <span class="toctext">Confirming sequence specificity of DNA</span></a></li>
-<img src="http://openwetware.org/images/9/9f/Ensin.png"></br>
-     Fig1 アルギン酸ゲルビーズの作製</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>
+</li>
- <img src="http://openwetware.org/images/c/cc/Image0032.jpg"></br></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></br>
+</ul>
+</li>
+</ul>
+</td></tr></table>
-<h4>1-2内部にバッファーの入ったアルギン酸膜の作製</h4></br>
+<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>
-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>Result</h5>
-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>
+<img src="http://openwetware.org/images/9/9e/2-1-1fig1.png" width="100%" height="100%"><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。
+<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>
-キャピラリーは外径1mmのものを熱加工した。</br>
+<br>
-</br>
+Fig.1 shows the continuous images before and after the temperature increase. The view sight was the same position. <br>
-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>
+NIPAM polymer turned into globular states with increasing temperature. Liposomes disappeared by increasing temperature (> Tc).<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>
-<img src="http://openwetware.org/images/1/1d/Image_%E4%BB%AE.png"></br>
+<br>
-     Fig3 二重ノズルの構造</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>
-<!--<img src="http://openwetware.org/wiki/Image:%E7%84%A1%E9%A1%8C.png"></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>
+<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>
-<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</h4></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>
-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>
+<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>
-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>
+<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>
-参考</br>
+In all four conditions, liposomes were observed with a fluorescent microscope.
-http://www.sigmaaldrich.com/etc/medialib/docs/SAJ/Brochure/1/j_recipedds2.Par.0001.File.tmp/j_recipedds2.pdf</br></br>
+We used the mixture of uni- and multi-lamella liposomes (Fig.4~7).<br>
+<!--ここは表を使ってコンパクトに-->
+<div align="center"><Img Src="
+http://openwetware.org/images/d/d7/2-2-1fig4.png"></div><br>
+<div class="caption">Fig.4 Fluorescent microscope image of liposomes <br>(Origami-anchor DNA: 0.018µM; Left: without fluorescence, Right: with fluorescence)</div><br>
+<br>
+ <div align="center"><Img Src="
+http://openwetware.org/images/9/94/2-2-1fig5.png"></div><br>
+<div class="caption">Fig.5 Fluorescent microscope image of liposomes <br>(Origami-anchor DNA: 0.069µM; Left: without fluorescence, Right: with fluorescence)</div><br>
+<br>
+In the presence of fluorescent molecules, when the concentration of Origami-anchor DNA was 0.018, 0.069µM, many gleaming (in green color) liposomes were observed. These results confirmed that the fluorescently labeled Origami well hybridized to the liposomal surface but that did not disrupt (Fig.4,5). <br>
+<br>
+<div align="center"><Img Src="
+http://openwetware.org/images/9/94/2-2-1fig6.png"></div><br>
+<div class="caption">Fig.6 Fluorescent microscope image of liposomes <br>(Origami-anchor DNA: 1.8µM; Left: without fluorescence, Right: with fluorescence)</div><br>
+<br>
+On the other hand, when the concentration of Origami-anchor DNA was 1.8µM, few gleaming liposomes could be seen with a fluorescent microscope (Fig.6). This result indicates the possibility that liposomes have broken.<br>
+<br>
+<div align="center"><Img Src="
+http://openwetware.org/images/c/c0/2-2-1fig7.png"></div><br>
+<div class="caption">Fig.7 Fluorescent microscope image of liposomes <br>(Origami-anchor DNA: 6.9µM; Left: without fluorescence, Right: with fluorescence)</div><br>
+<br>
+When the concentration of Origami-anchor DNA is 6.9µM, some liposomes were gleaming and others distorted, forming networks (Fig.7).
+<br>
+<br>
+<h5>Discussion</h5>
+From these results, we put forward the following hypothesis about the interaction of DNA Origami and liposomes. When the concentration of the anchor DNA is low (0.018, 0.069µM), liposomes was still stable. When the concentration is middle (1.8µM), more DNA Origami hybridizes to the surface and loads on it. This loading made liposomes become fragile and easy to break. When the concentration is high (6.9µM), disrupted liposomes were connected with others, and consequently, form networks via Origami-anchor DNA and DNA Origami complex.
+<br>
+Anyway, <u>these data strongly indicated the designed DNA origami disrupted liposomes with high concentration of the anchor DNA.</u>
+<br>
+<br>
+<br>
-まず、PNIPAMに脂質を修飾する。まず、PNIPAMにCHCl3とAr存在下でジシクロヘキシルカーボジイミド（DCC）と、Ｎ-ヒドロキシ-シアナミド（NHS）を反応させる。（下図の１ができる）次に、CHCl3とAr存在下でジミリストイルホスファチジルエタノールアミン（dimyristoylphosphatidylethanolamine、DMPE）を反応させる。（下図の２ができる）このようにしてPNIPAMに脂質を修飾して、GUVによりニッパム修飾されたリポソームを作製した。</br></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>
-<img src="http://openwetware.org/images/e/e8/NIPAMgousei.png"></br>
+<h5>Result</h5>
-     Fig4 PNIPAMへの脂質の修飾</br></br>
+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>
-※W/Oエマルション法を用いたGUV(Giant Unilamellar Vesicle)</br></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>
-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></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>
+<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>
-参考</br>
+<h4 id=9>2-2 Flower DNA approach</h4>
-Thermoresponsive Nanostructures by Self-Assembly of a Poly(N-isopropylacrylamide)−Lipid Conjugate
+<!-------------SPRコメントアウトここから-------------------->
-Daniel N. T. Hay ,† Paul G. Rickert ,‡ Sönke Seifert ,§ and Millicent A. Firestone *†
+<!--
-J. Am. Chem. Soc., 2004, 126 (8), pp 2290–229　Publication Date (Web): February 3, 2004</br></br>
+<h5 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>
-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>
+<h5>Principle</h5>
-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>
+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>
-<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</h4></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>
-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>
+<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>
-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>
+<h5>Discussion</h5>
+Fig.13 shows the behavior of materials on the surface of liposomes. The increase of SPR value after injecting Anchored DNA indicates that Anchored DNA successfully combined with liposomes.
+Similarly, it is considered that loop DNA combined with Anchored DNA. <br>
+Thus, <u>we confirmed the formation of the loop structures on liposomes.</i><br>
+<br>
+-->
+<!---------------SPRコメントアウトここまで------------------>
-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>
-<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>
+<h5 id=11>2-2-1 Liposome disruption by flower DNA approach</h5>
-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>
+<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>
-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>
+<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>
-M13pm18(7249nt)と226個のステプルを100μℓの12.5mMの酢酸マグネシウム入りの10×TAEの中に入れ、95℃から20℃に１℃/分でアニーリングしたものとM13pm18と226個のステイプルを300μℓの12.5ｍMの酢酸マグネシウムと85%のホルムアルデヒド入りの10×TAEの中に入れ、透析装置で0.2mℓ/分の割合で次第にホルムアルデヒドの濃度を薄くしていったものとをAFMや電気泳動で調べた。</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>
-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>
+<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>
-<img src="http://openwetware.org/images/a/a8/Ureaannealingsample.png"></br>
+<h5 id=12> 2-2-2 Confirming sequence specificity of DNA</h5>
-     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>
+<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>
-<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>
+<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>
-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>
+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>
-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>
+After adding B-Key DNA, the number of each color liposomes is counted to confirm the selectivity.<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>
+As a control, only buffer is added instead of B-Key DNA.<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>
+<A href="http://openwetware.org/wiki/Biomod/2013/Sendai/protocol">Protocol</A><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>
+<h5>Result</h5>
+Fig.16 shows fluorescent microscope image of liposomes added B-Key DNA (left) and control buffer(right).
-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>
+Green rectangles represent Green liposomes; Red, Red liposomes.<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>
+In the left, only Green liposomes (marked with green rectangles) and no Red liposomes can be seen.
-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>
+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>
-<h4>7全体のシステムの機能確認</h4></br>
+<table border cellspacing="3" bgcolor="lightyellow">
+<tr bgcolor="moccasin">
-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>
+<td> Additives </td>
-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>
+<td> B-key DNA </td>
-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>
+<td> Buffer </td>
+</tr>
-      </article>
+<tr bgcolor="moccasin">
+<td> Green：Red </td>
+<td>17:2 (n = 19)</td>
-      <article data-title="リポソーム">
+<td>16:17 (n= 33)</td>
+</tr>
-<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 <font size="2">リポソーム班</font> </h3>
+</table>
-<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>
+<div class="captiontable">Table1   Ratio of Green and Red liposomes</div><br>
-反応の連鎖は、リポソームの作成・トリガー及び内部構造体の作成・そのトリガーを実際に作用させる、という三段階に分けられる。</br></p>
+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>
-<p><h4>・リポソームの作成</h4>
+<br>
-まず、released trigger DNAにより、割られるリポソームを作成する。</br>
+<h5>Discussion</h5>
-</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>
-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>
+<u>The selectivity of Key DNA has been successfully demonstrated. </u></h6>
-</br>
-脂質(DOPC)、脂質を溶かす溶媒(CHCl3)、TR-DHPE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。リポソームは蛍光顕微鏡で観察した。</br>
-</br>
-<Img Src="http://openwetware.org/images/c/cf/Fig1_liposome.png">
-<Img Src="http://openwetware.org/images/1/1c/Fig2_liposome.png"></br>
-Fig.1,2 separated, uni-lamella liposomes observed by a fluorescent microscopy</br>
-蛍光顕微鏡で、数個のリポソームが観察された(Fig.1,2)。リポソームは、赤く膜のみが染色されており、個々に分かれたユニラメラリポソームが作成できた。</br>
-</p>
-<p><h5>・一本鎖DNAをはやしたリポソームの作成</h5>
-次に、作成したリポソームをDNAをトリガーとして割るには、トリガーDNAをリポソーム表面に多数ハイブリダイゼーションさせる必要がある。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、トリガーDNA構造体の一部と相補的な配列を持つ一本鎖DNAとすれば、トリガーDNAがリポソーム表面に多数ハイブリダイゼーションする。
-ここでは、割る前に、一本鎖DNAをはやしたリポソームを生成する。</br>
-</br>
-まず、ミクロチューブにDOPC、CHCl3を99μℓ加え、この溶液を乾燥させた。ここに観察用バッファーを入れ、整置水和した。</br>
-リポソームにコレステロール修飾したDNAをつけるため、作製したリポソームにcholesterol修飾DNAを加えた。</br>
-(上手く観察できなかったので、お盆明けに再度実験します)</p>
-</br>
-<p><h4>・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</h4>
-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>
-</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。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。</br>
-私たちは、電気泳動により、トリガーDNAオリガミの作成と、その蛍光修飾(蛍光基つきDNAが、DNAオリガミに確かに接着していること)を確かめようとした。</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く、DNAオリガミがうまく蛍光修飾されたことを確かめた。</br>
-</br>
-マイクロチューブにM13mp18, ステイプル, 5xTAE Mg2+, mQ,　蛍光基付きDNAを混合、2時間半アニーリングを行った。</br>
-対照実験として、蛍光基付きDNAの代わりに,mQを入れた、蛍光修飾なしDNAオリガミも作成した。</br>
-</br>
-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、アニーリング後、蛍光修飾なしDNAオリガミ溶液を10μlとり、同様の蛍光基付き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>
-</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>
-<Img Src="http://openwetware.org/images/6/64/Fig3_gel.png" align="left">
-Fig.3 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>
-</br>
-染色後のゲル画像(Fig.3)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 clear="left">
-</br>
-</br>
-<Img Src="http://openwetware.org/images/8/8e/Fig4_gel.png" align="left">
-Fig.4 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>
-次に、染色前のゲル画像(Fig.4)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.3)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 clear="left">
          </article>
-        <article data-title="B-Z">
-<h3>外側からリポソームを破壊するサブプロジェクト</h3><br>
-<h4 id="designsubproject3">ＢＺ班</h4><br>
-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>
-基本的には上記のリポソーム班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>2.コレステロール修飾DNA及びループDNAの結合</h5>
-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>
-通常のリポソーム、相分離リポソームの二種類で行う。</br>
-</br>
-<table>
- <tr>
-  <td>
-   完成したリポソーム0.1mM
-  </td>
-  <td>
-.5μℓ
-  </td>
- </tr>
- <tr>
-  <td>
-   Chol leg 0.1μM
-  </td>
-  <td>
-.2μℓ
-  </td>
- </tr>
-</table>
-</br>
-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>
-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>
-</br>
-<table>
- <tr>
-  <td>
-   DNA付リポソーム　0.1mM
-  </td>
-  <td>
-.5μℓ
-  </td>
- </tr>
- <tr>
-  <td>
-   ループDNA        0.1μM
-  </td>
-  <td>
-.0μℓ
-  </td>
- </tr>
-</table>
-</br>
-確認はＡＦＭを用いる。</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>
-<h5>3.DNAループにトリガーストランドのハイブリ</h5></br>
-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>
-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>
-</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>
      </section>
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