Difference between revisions of "Biomod/2013/Sendai/experiment"

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<ul>
 
<ul>
 
<li class="toclevel-1"><a href="#chain">
 
<li class="toclevel-1"><a href="#chain">
<span class="tocnumber">1</span> <span class="toctext">1st stage: Sensing system</span></a></li>
+
<span class="tocnumber">1</span> <span class="toctext">First stage: Sensing system</span></a></li>
 
<ul>
 
<ul>
 
<li class="toclevel-2"><a href="#bending">
 
<li class="toclevel-2"><a href="#bending">
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</ul>
 
</ul>
 
<li class="toclevel-1"><a href="#Flower">
 
<li class="toclevel-1"><a href="#Flower">
<span class="tocnumber">2</span> <span class="toctext">2nd stage: Amplification system</span></a></li>
+
<span class="tocnumber">2</span> <span class="toctext">Second stage: Amplification system</span></a></li>
 
<ul>
 
<ul>
 
<li class="toclevel-2"><a href="#sensing">
 
<li class="toclevel-2"><a href="#sensing">
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<li class="toclevel-2"><a href="#7">
 
<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>
+
<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">
 
<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>
+
<span class="tocnumber">2-1-4</span> <span class="toctext">Disruption of liposomes by DNA Origami (quantitative analysis)</span></a></li>
  
 
<li class="toclevel-2"><a href="#8">
 
<li class="toclevel-2"><a href="#8">
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</td></tr></table>
 
</td></tr></table>
  
<h3 id=chain>1st stage: Sensing system</h3>
+
<h3 id=chain>First stage: Sensing system</h3>
 
<h4 id=bending>1-1Disruption of temperature sensitive liposomes</h4>
 
<h4 id=bending>1-1Disruption of temperature sensitive liposomes</h4>
<h6>Purpose</h6>
+
<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: 32~40˚C ). <br>
+
In our project, we planned to use liposomes conjugated with NIPAM polymer as a chain reaction initiator. NIPAM (poly-N-isopropyl acrylamide) is a temperature sensitive molecule that has a unique critical temperature (Tc: 32°C ). <br>
 
When the temperature increased over than Tc, the hydrophilic polymer changes its property hydrophobic. It is expected that the change should disrupt the membrane lipid alignment. Here we confirm that the possibility of breaking liposomes with NIPAM by increasing temperature.<br>
 
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 <A href="http://www.sigmaaldrich.com/Graphics/COfAInfo/SigmaSAPQM/SPEC/73/731129/731129-BULK_______ALDRICH__.pdf">Sigma Aldrich</A><br>
+
NIPAM was purchesed from SIGMA ALDRICH.<br>
 
<br>
 
<br>
  
<h6>Method</h6>
+
<h5>Method</h5>
 
The liposomes were prepared by natural swelling method. Obtained sample included a mixture of unilamellar and multilamellar liposomes.<br>
 
The liposomes were prepared by natural swelling method. Obtained sample included a mixture of unilamellar and multilamellar liposomes.<br>
 
Then we added NIPAM-conjugated lipids (dissolved in ultra pure water (Milli-Q)) to the liposomes solution.<br>
 
Then we added NIPAM-conjugated lipids (dissolved in ultra pure water (Milli-Q)) to the liposomes solution.<br>
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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>
 
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>
 
Detailed Protocol<br>
<A href="http://openwetware.org/wiki/Biomod/2013/Sendai/protocol#bending">Protocol</A><br>
+
<A href="http://openwetware.org/wiki/Biomod/2013/Sendai/protocol">Protocol</A><br>
  
<h6>Result</h6>
+
<h5>Result</h5>
  
  
  
<img src="http://openwetware.org/images/thumb/2/2b/Experiment_%E3%83%8B%E3%83%83%E3%83%91%E3%83%A04.png/800px-Experiment_%E3%83%8B%E3%83%83%E3%83%91%E3%83%A04.png" width="60%" height="60%"><br>
+
<img src="http://openwetware.org/images/9/9e/2-1-1fig1.png" width="100%" height="100%"><br>
  
 
+
<div class="caption">Fig.1 Phase contrast images of liposomes in NIPAM solution. Temperature increased from RT to enough over than Tc (left to right).</div><br>
 
 
Fig.1 Phase contrast images of liposomes in NIPAM solution. Temperature increased from RT to enough over than Tc (left to right).<br>
 
 
<br>
 
<br>
 
Fig.1 shows the continuous images before and after the temperature increase. The view sight was the same position. <br>
 
Fig.1 shows the continuous images before and after the temperature increase. The view sight was the same position. <br>
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<h6>Discussion</h6>
+
<h5>Discussion</h5>
 
Thermosensitive polymer NIPAM can disrupt the coexisting liposomes by the polymers phase transition. <br>
 
Thermosensitive polymer NIPAM can disrupt the coexisting liposomes by the polymers phase transition. <br>
On the other hand, some liposomes still present even at the high temperature. In this experiment, some fractions were multi-lamellar liposomes. Since globular states of NIPAM (hydrophobic) at high temperature attack the liposome membrane from the outside, it is not surprising that the multi-lamellar liposomes consist of many lipid bilayers are more difficult to disrupt. Therefore, we suppose that liposomes disrupted by temperature shift in Fig.1 were uni-lamella ones. These results confirmed that triggering by heat disrupted the liposomes.<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>2nd stage: Amplification system</h5>
+
 
 +
<h3 id=Flower>Second stage: Amplification system</h5>
 
<h4 id=sensing>2-1 DNA Origami approach</h4>
 
<h4 id=sensing>2-1 DNA Origami approach</h4>
 
<h5 id=5>2-1-1 Making DNA Origami</h5>
 
<h5 id=5>2-1-1 Making DNA Origami</h5>
<h6>Purpose</h6>
+
<h5>Purpose</h5>
In our project, we use DNA Origami as Key DNA to break liposomes. We design rectangular DNA Origami with a chipped edge and try to make it.<br>
+
In our project, to use DNA Origami as the Key DNA to break liposomes, we design the rectangular DNA Origami with a chipped edge.
 +
<br>
 
   
 
   
<h6>Method</h6>
+
<h5>Method</h5>
We mixed M13mp18, staples, 5xTAE Mg2+, and mQ in a microtube and annealed it for 2.5 hours.<br>
+
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#5">Protocol</A><br>
+
<A href="http://openwetware.org/wiki/Biomod/2013/Sendai/protocol">Protocol</A><br>
  
  
<h6>Result</h6>
+
<h5>Result</h5>
We confirmed that our DNA Origami was well formed by AFM (Atomic Force Microscope) (Fig.2).<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/d/d9/Outsideafm2.png"> <br>
+
<br>
Fig.2 AFM image of DNA Origami (M13: 4nM, staples:20nM)<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>
 
<br>
  
<h6>Discussion</h6>
+
<h5>Discussion</h5>
Just like our design, rectangular DNA Origami with a chipped edge was observed.<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 id=6>2-1-2 Labeling DNA Origami with fluorescent-tagged DNA</h5>
<h6>Purpose</h6>
+
<h5>Purpose</h5>
If Origami is fluorescently labeled, it is much easier to observe the effect of DNA Origami on liposomes. So we labeled our Origami by hybridizing it with fluorescent-tagged DNA strand.<br><br>
+
To observe the fluorescent effect of DNA Origami on liposomes by microscope, we labeled our Origami by hybridizing with the fluorescent-tagged DNA strand.  
 
+
<br><br>
<h6>Method</h6>
 
Our DNA Origami has many staples that can bind to fluorescent-tagged DNA for labeling. We mixed fluorescent-tagged DNA together with DNA Origami staples in annealing solution.<br>
 
In addition, to see if the Origami binds to the fluorescent-tagged DNA in a shorter time, we added the fluorescent-tagged DNA into the control annealing solution, which had contained no fluorescent-tagged DNA, and left it for 40 minutes.<br>
 
To see the Origami was well labeled with fluorescent molecules, we used electrophoresis. <br>
 
Electrophoresis was conducted with a 1% agarose gel, CV100V for 50 minutes.<br>
 
  
 +
<h5>Method</h5>
 +
Our DNA Origami is composed of many staples that can bind to the same fluorescent-tagged DNA for labeling. We mixed fluorescent-tagged DNA with DNA Origami staples before annealing, and after annealing solution. Labeling of the DNA Origami was confirmed by gel-electrophoresis. Gel-electrophoresis was conducted with a 1% Agarose gel, 100V for 50 minutes.<br>
 +
<A href="http://openwetware.org/wiki/Biomod/2013/Sendai/protocol">Protocol</A><br>
 
<br>
 
<br>
By scanning a gel before staining, we can see only the bands of DNA structures with fluorescent molecules; scanning a gel after staining, we can see the bands of all DNA structures. So we scanned a gel before and after staining (we scanned both a non-stained and a stained gel). <br>
+
<h5>Result</h5>
First we saw the bands of our Origami in a non-stained gel. Then, we compared the bands with those in a stained gel. If the bands of Origami in a non-stained gel were at the same height as that in a stained gel, we can say that our Origami was successfully fluorescently labeled.<br>
+
Figure 3 shows florescence detection of the gel before staining (Left) and after SYBR Gold staining (Right). In a non-stained gel, only lane 3 and 4 (*Ori, **Ori) was found. The fluorescent bands of Origami in a non-stained gel were at the same height as that in a stained gel, we conclude that our Origami was successfully fluorescently labeled irrespective of the timing of adding the fluorescent DNA.<br>
<A href="http://openwetware.org/wiki/Biomod/2013/Sendai/protocol#6">Protocol</A><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>
<h6>Result</h6>
 
In a non-stained gel (Fig.3), only bands in lane 3 and 4 from the left (*Ori, **Ori) can be seen. They are fluorescently labeled structures. In addition, as they gave the same result, 40 minutes is long enough for fluorescently labeling.<br>
 
<Img Src="http://openwetware.org/images/8/8e/Fig5and6.jpg" width="450"><br>
 
Fig.3 Non-stained gel image: only bands in two lanes can be seen. From the left, they are DNA Origami with fluorescent molecules in pre-annealing (Ori*), and DNA Origami with fluorescent molecules in post-annealing (Ori**)<br>
 
 
<br>
 
<br>
In a stained gel (Fig.4), marker lane (lane 1) had the longest DNA strand of 20kb. Comparing this band and the band of M13mp18 (lane 2) with annealed DNA Origami (lane 1,2,3), the bands of the Origami are at the higher position. Therefore, we concluded that in lane3~5, DNA Origami structure was made as we had expected. <br>
 
We considered that the bands in lane3~5 are seen as if they were diffused, just because our Origami has many staples binding to the fluorescent-tagged DNA, and each Origami attaches to different number of them, and its molecular weight varies.<br>
 
<Img Src="http://openwetware.org/images/2/2d/S_Outside-gel-2.2.png" width="300"> </br>
 
Fig.4 Stained gel image: from the left, marker, M13mp18, Ori*, Ori**, and DNA Origami with no fluorescent molecule (Ori)<br>
 
 
<br>
 
<br>
 
+
<h5>Discussion</h5>
<h6>Discussion</h6>
+
<u>The results indicate we succeeded to label our Origami by the fluorescence DNA.</u><br>
Combining the results of Fig.3 and 4, the fluorescently labeled bands in lane3 and 4 in Fig.3 are at the same height as those of DNA Origami in Fig.4. Thus, we concluded our Origami was successfully fluorescently labeled.<br>
 
 
<br>
 
<br>
  
<h5 id=7>2-1-3 Disrupting liposomes by DNA Origami(microscopic analysis)<h5>
+
<h5 id=7>2-1-3 Disrupting liposomes by DNA Origami (microscopic analysis)<h5>
<h6>Purpose</h6>
+
<h5>Purpose</h5>
To break liposomes with our Origami, first we investigate how our DNA Origami affect liposomes.<br>
+
To break liposomes with our Origami, first we investigate how our DNA Origami affects liposomes.<br>
 
<br>
 
<br>
<h6>Principle</h6>
+
<h5>Principle</h5>
 
To break liposomes with our Origami, a lot of Origami has to hybridize to the surface of the liposomes.<br>
 
To break liposomes with our Origami, a lot of Origami has to hybridize to the surface of the liposomes.<br>
 
To begin with, we added cholesterol-conjugated single-stranded DNA (in the rest of this document, referred to as 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>
 
To begin with, we added cholesterol-conjugated single-stranded DNA (in the rest of this document, referred to as Origami-anchor DNA) into liposomes, and made it float on the surface. The Origami-anchor DNA has a complementary part to our Origami, so the Origami is expected to hybridize to Origami-anchor DNA on the liposomes. In this way, lots of Origami would hybridize to liposomes via Origami-anchor DNA.<br>
 
<br>
 
<br>
<h6>Method</h6>
+
<h5>Method</h5>
We added Origami-anchor DNA into liposomes at the final concentration of 0.018, 0.069, 1.8, and 6.9µM. Then we observed the samples with a phase microscope. <br>
+
To begin with, we mixed cholesterol-conjugated single-stranded DNA (in the rest of this document, referred to as Origami-anchor DNA) into liposomes, and made it float on the surface. Origami-anchor DNA has a complementary part to our Origami. We should note that the anchor DNA was added after liposome formation to avoid the anchor DNA are inserted on inner side of liposome. <br>
Next, adding fluorescently labeled DNA Origami into the above liposomes, we saw if some change would happen with a fluorescent microscope.<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).  
<A href="http://openwetware.org/wiki/Biomod/2013/Sendai/protocol#7">Protocol</A><br>
+
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>
 
<br>
<h6>Result</h6>
+
<h5>Result</h5>
In all four conditions, liposomes were observed with a phase microscope. We confirmed the formation of multi-lamella liposomes (Fig.5~8).<br>
 
 
 
  
 +
In all four conditions, liposomes were observed with a fluorescent microscope.
 +
We used the mixture of uni- and multi-lamella liposomes (Fig.4~7).<br>
  
 
<!--ここは表を使ってコンパクトに-->
 
<!--ここは表を使ってコンパクトに-->
  
<Img Src="http://openwetware.org/images/7/72/Lipofig4.png" width="400"></br>
+
<div align="center"><Img Src="
Fig.5 Phase microscope image of liposomes (Origami-anchor DNA: 0.018µM)<br>
+
http://openwetware.org/images/d/d7/2-2-1fig4.png"></div><br>
 +
<div class="caption">Fig.4 Fluorescent microscope image of liposomes <br>(Origami-anchor DNA: 0.018µM; Left: without fluorescence, Right: with fluorescence)</div><br>
 +
<br>
 +
<div align="center"><Img Src="
 +
http://openwetware.org/images/9/94/2-2-1fig5.png"></div><br>
 +
<div class="caption">Fig.5 Fluorescent microscope image of liposomes <br>(Origami-anchor DNA: 0.069µM; Left: without fluorescence, Right: with fluorescence)</div><br>
 +
<br>
 +
In the presence of fluorescent molecules, when the concentration of Origami-anchor DNA was 0.018, 0.069µM, many gleaming (in green color) liposomes were observed. These results confirmed that the fluorescently labeled Origami well hybridized to the liposomal surface but that did not disrupt (Fig.4,5). <br>
 
<br>
 
<br>
<Img Src="http://openwetware.org/images/d/d0/Lipofig5.png" width="400"></br>
+
<div align="center"><Img Src="
Fig.6 Phase microscope image of liposomes (Origami-anchor DNA: 0.069µM)<br>
+
http://openwetware.org/images/9/94/2-2-1fig6.png"></div><br>
 +
<div class="caption">Fig.6 Fluorescent microscope image of liposomes <br>(Origami-anchor DNA: 1.8µM; Left: without fluorescence, Right: with fluorescence)</div><br>
 
<br>
 
<br>
<Img Src="http://openwetware.org/images/d/de/Lipofig6.png" width="400"></br>
+
On the other hand, when the concentration of Origami-anchor DNA was 1.8µM, few gleaming liposomes could be seen with a fluorescent microscope (Fig.6). This result indicates the possibility that liposomes have broken.<br>
Fig.7 Phase microscope image of liposomes (Origami-anchor DNA: 1.8µM)<br>
 
 
<br>
 
<br>
<Img Src="http://openwetware.org/images/d/d7/Lipofig7.png" width="400"></br>
+
<div align="center"><Img Src="
Fig.8 Phase microscope image of liposomes (Orgami-anchor DNA: 6.9µM)<br>
+
http://openwetware.org/images/c/c0/2-2-1fig7.png"></div><br>
 +
<div class="caption">Fig.7 Fluorescent microscope image of liposomes <br>(Origami-anchor DNA: 6.9µM; Left: without fluorescence, Right: with fluorescence)</div><br>
 
<br>
 
<br>
Adding fluorescently labeled DNA Origami into the above liposomes, we saw if some change would happen with a fluorescent microscope.<br>
+
When the concentration of Origami-anchor DNA is 6.9µM, some liposomes were gleaming and others distorted, forming networks (Fig.7).
When the concentration of Origami-anchor DNA was 0.018, 0.069µM, many gleaming (in green color) liposomes were observed. We confirmed that the fluorescently labeled Origami well hybridized to the liposomal surface (Fig.9,10,11). <br>
 
<table>
 
<tr>
 
  <td>
 
  <Img Src="http://openwetware.org/images/6/6c/Lipofig8.png" width="400">
 
  </td>
 
  <td>
 
  <Img Src="http://openwetware.org/images/a/a6/Lipofig9.png" width="400">
 
  </td>
 
</tr>
 
</table>
 
Fig.9,10 fluorescent microscope image of liposomes (Origami-anchor DNA: 0.018µM)<br>
 
<Img Src="http://openwetware.org/images/b/b4/Lipofig10.png" width="400"></br>
 
Fig.11 fluorescent microscope image of liposomes (Origami-anchor DNA: 0.069µM)<br>
 
 
<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.12). This result indicates the possibility that liposomes have broken.<br>
 
<Img Src="http://openwetware.org/images/1/18/Lipofig11.png" width="400"></br>
 
Fig.12 fluorescent microscope image of liposomes (Origami-anchor DNA: 1.8µM)<br>
 
 
<br>
 
<br>
When the concentration of Origami-anchor DNA is 6.9µM, some liposomes were gleaming and others distorted, forming networks (Fig.13).<br>
+
<h5>Discussion</h5>
+
From these results, we put forward the following hypothesis about the interaction of DNA Origami and liposomes. When the concentration of the anchor DNA is low (0.018, 0.069µM), liposomes was still stable. When the concentration is middle (1.8µM), more DNA Origami hybridizes to the surface and loads on it. This loading made liposomes become fragile and easy to break. When the concentration is high (6.9µM), disrupted liposomes were connected with others, and consequently, form networks via Origami-anchor DNA and DNA Origami complex.
<Img Src="http://openwetware.org/images/8/88/Lipofig12.png" width="400"></br>
 
Fig.13 fluorescent microscope image of liposomes (Origami-anchor DNA: 6.9µM)<br>
 
 
<br>
 
<br>
<h6>Discussion</h6>
+
Anyway, <u>these data strongly indicated the designed DNA origami disrupted liposomes with high concentration of the anchor DNA.</u>
From these results, we put forward the following hypothesis about the interaction of DNA Origami and liposomes.<br>
 
When the concentration of Origami-anchor DNA is low (0.018, 0.069µM), DNA Origami hybridizes to the surface of liposomes relatively stablely. When the concentration is middle (1.8µM), more DNA Origami hybridizes to the surface and loads on it. The liposomes become fragile and easy to break. When the concentration is high (6.9µM), some liposomes exist individually, and others form networks via Origami-anchor DNA and DNA Origami complex.<br>
 
<Img Src="http://openwetware.org/images/7/7c/Experimentinsidefig.png"><br>
 
 
<br>
 
<br>
According to this hypothesis, when the concentration of Origami-anchor DNA is 1.8µM, DNA Origami breaks liposomes. <br>
 
 
<br>
 
<br>
 
<br>
 
<br>
  
<h5 id=13>2-1-4 Disrupting liposomes by DNA Origami(quantitative analysis)<h5>
+
<h5 id=13>2-1-4 Disrupting liposomes by DNA Origami (quantitative analysis)<h5>
<h6>Purpose</h6>
+
<h5>Purpose</h5>
To break liposomes with our Origami, first we investigate how our DNA Origami affect liposomes.<br>
+
The above experiments in 2-1-3 microscopic analysis suggest that our DNA Origami disrupted liposomes. Thus, we performed more quantitative analysis.<br>
 +
<br>
 +
<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>
 
<br>
  
<h6>Method</h6>
+
<h5>Result</h5>
1. Making liposomes that contain GFP in the interior, by an oil/water interface. <br>
+
As figure below, we were able to observe liposomes containing GFP, by the confocal microscope.<br>
2. Observing only liposomes by the confocal microscope. <br>
+
<div align="center">
3. Sample 1. Liposomes + Flower-anchor DNA + Key DNA<br>
+
<img src="http://openwetware.org/images/8/87/Lipo-scalebar-2.png"></div>
Sample 2. Liposomes + Flower-anchor DNA<br>
+
<br><div class="caption">Fig.10 Liposomes conteaining GFP(confocal microscope)</div>
Sample 3. Liposomes + Flower-anchor DNA + Surfactant (2%NP)<br>
+
<br>
Measuring each sample’s fluorescence intensity of 7-13㎛ diameter liposomes by Cell Lab Quanta SC Flow Cytometer<br>
+
<div align="center">
We used 50㎕ from each sample.<br>
+
<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>
内部にGFP(緑の蛍光)を含むリポソームを界面通過法によって作成した。リポソームのみを共焦点顕微鏡により観察した。<br>
 
次に、<br>
 
サンプル1 リポソーム+フラワーアンカーDNA+KeyDNA<br>
 
サンプル2 リポソーム+フラワーアンカーDNA<br>
 
サンプル3 リポソーム+フラワーアンカーDNA+界面活性剤(2%NP)<br>
 
を用意してそれぞれをCell Lab Quanta SC Flow Cytometerで直径が7~13μm??(片山さん確認)のリポソームの個数蛍光強度を計測する。サンプルは各50ul使用する。<br>
 
  
<h6>Result</h6>
+
<br>
As figure below, we was able to observe liposomes containing GFP, by the confocal microscope.<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>
共焦点顕微鏡によって観察したGFP入りのリポソームは下図のようになり、リポソームが確認できた。<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>
(共焦点の図)
+
<u>These results confirmed that our designed DNA origami actually disrupted liposomes. (The efficiency was higher than 2% surfactant!)</u>
 
+
<br>
 
+
<br>
The abscissa of the following graph is the fluorescence intensity of only liposomes, and the ordinate represents the number of liposomes.<br>
+
<h5 id=8>2-1-5 Confirming sequence specificity of DNA flow cytometer</h5>
以下のグラフの横軸はリポソームのみの蛍光強度、縦軸はリポソームの個数を表している。<br>
+
<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>
<img src="http://openwetware.org/images/b/bc/%E3%81%90%E3%82%89%E3%81%B5%EF%BC%91.png"><br>
+
<br>
グラフ1 Lipo-leg-tae-graph<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>
<img src="http://openwetware.org/images/9/9c/%E3%82%B0%E3%83%A9%E3%83%95%EF%BC%92.png"><br>
+
Sample 2 (no binding pair). Liposomes + Orgiami-anchor DNA(A) + Key DNA(B)<br>
グラフ2 Lipo-leg-origami-graph<br>
+
<A href="http://openwetware.org/wiki/Biomod/2013/Sendai/protocol">Protocol</A><br>
 
 
 
 
<img src="http://openwetware.org/images/4/4a/%E3%81%90%E3%82%89%E3%81%B5%EF%BC%93.png"><br>
 
グラフ3 Lipo-leg-origami2-graph<br><br>
 
 
 
 
 
Figure 1 shows that liposomes having high fluorescence intensity have a wide distribution.<br>
 
グラフ1は蛍光強度が高いリポソームの分布が多い。<br>
 
 
 
Figure 2 shows the result of liposomes including Origami-anchor DNA and DNA origami. Fluorescence intensity was not detected at all.<br>
 
グラフ2はオリガミアンカーDNAとDNAオリガミをくわえたものである。蛍光強度がまったく観測されなかった。<br>
 
 
 
 
 
Figure 3 a surfactant shows that liposomes with positive-control surfactant have almost no fluorescence intensity.<br>
 
グラフ3はポジティブコントロールの界面活性剤を加えたものである。こちらも蛍光強度がまったく観測されなかった。<br>
 
 
 
 
 
<h6> Discussion </h6>
 
Figure 1 indicates the distribution map when liposomes surely exist. Figure 3 shows the distribution as liposomes surely do not exist. Figure 2 is similar to Figure 3. Therefore, it is supposed that liposomes are broken in Figure 2. Judging from this experiment, Origami DNA can disrupt liposomes.<br>
 
グラフ1はリポソームが確実にあるときの分布、グラフ3は界面活性剤によりほぼ確実にリポソームないときの分布である。グラフ2はグラフ3と似ている。そのためグラフ2ではリポソームが割れていると考えられる。本実験によりDNAオリガミによってリポソームが破壊できたと考えられる。<br>
 
 
 
 
 
 
 
 
 
<h5 id=8>2-1-5 Confirming sequence specificity of DNA </h5>
 
<h6>Purpose</h6>
 
In this project, we adopt DNA for the Key of chain reaction because DNA has a significant characteristic: sequence specificity. Utilizing this sequence specificity, we aim to select liposomes that will be broken by Key DNA, induce chain-reactive liposomal disruption by some Key DNA, and make connections between liposomes.<br>
 
Corresponding to the liposomes, we arrange two kinds of Origami-anchor DNA of different sequences and attach the anchor to the liposomes. Then we mix both kinds of liposomes together. <br>
 
Into the mixture, Key DNA for just one kind of liposomes is added. We confirm that the Key DNA breaks only the corresponding kind of liposomes.<br>
 
 
<br>
 
<br>
 +
<h5>Result</h5>
 +
The results were shown in figure 12.<br>
 +
<div align="center">
 +
<img src="http://openwetware.org/images/6/6d/2-1-5matometagazou-deka.png" style="
 +
    width: 90%;
 +
"></div>
 +
<br><div class="caption">Fig.12 Results of complementary key DNA and no binding Key DNA</div>
  
<h6>Method</h6>
 
We made two kinds of liposomes: liposomeA and liposomeB by water-in-oil emulsion process. LiposomeA contains GFP (Green Fluorescent Protein) inside, and liposomeB has Rhodamine (red fluorescent dye) in itself. <br>
 
<!--脂質の組成は?-->
 
 
<br>
 
<br>
Origami-anchor DNA for liposome A has the sequence of 5'-CCAGAAGACG-chol-3'. The anchor for liposome B has the sequence of 5'-TCCACTAACG-chol-3'. Both Origami-anchor DNA was mixed with the corresponding liposomes.<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>
Each liposome was centrifuged for one minute to remove the excess Origami- anchor DNA.<br>
 
Then we mixed 1µl of each liposome and observed it with a phase-contrast microscope.
 
Next, 4µl refined DNA Origami was added to the mixture (of liposomeA and B). The sample was also observed with a phase-contrast microscope. <br>
 
<!--蛍光ついている??-->
 
Protocol<br>
 
<A href="http://openwetware.org/wiki/Biomod/2013/Sendai/protocol#8">Protocol</A><br>
 
 
 
<h6>Result</h6>
 
Fig.16 is the phase-contrast microscope image of the mixture of liposome A and B before the addition of Key DNA Origami.
 
 
<br>
 
<br>
<img src="http://openwetware.org/images/6/68/LegA%2BB_10.jpg"><br>
+
<h5>Discussion</h5>
Fig.16 Phase contrast microscope image of the mixture of liposome A (Green) and B (Red)
+
<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>
<h6>Discussion</h6>
 
(要加筆)
 
 
<br>
 
<br>
 +
 +
 
<h4 id=9>2-2 Flower DNA approach</h4>
 
<h4 id=9>2-2 Flower DNA approach</h4>
(SPRコメントアウトしておきます)
+
<!-------------SPRコメントアウトここから-------------------->
 
<!--
 
<!--
 
<h5 id=10> 2-2-1 Confirming the formation of the loop structure by SPR</h5>
 
<h5 id=10> 2-2-1 Confirming the formation of the loop structure by SPR</h5>
<h6>Purpose</h6>
+
<h5>Purpose</h5>
 
To break liposomes by flower DNA method, we aim to attach many loop strands to the surface of liposomes. <br>
 
To break liposomes by flower DNA method, we aim to attach many loop strands to the surface of liposomes. <br>
 
To achieve this, we adopt the same hybridization method via Anchored DNA as used in i)Bending approach into liposomes: the Anchored DNA has a complementary part to our loop strand and the loop strand is expected to hybridize to 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>
Line 344: Line 294:
 
<br>
 
<br>
  
<h6>Principle</h6>
+
<h5>Principle</h5>
 
As our loop strand is too small to observe with an AFM or a fluorescent microscope, we used an apparatus called SPR.<br>
 
As our loop strand is too small to observe with an AFM or a fluorescent microscope, we used an apparatus called SPR.<br>
 
SPR is a Surface Plasmon Resonance assay that estimates the weight of molecules attached to membrane surface, by the change of the reflection of the laser beam.<br>
 
SPR is a Surface Plasmon Resonance assay that estimates the weight of molecules attached to membrane surface, by the change of the reflection of the laser beam.<br>
Line 351: Line 301:
 
<br>
 
<br>
  
<h6>Method</h6>
+
<h5>Method</h5>
 
<ur><li>1. Inject 45µl DOPC (100mM) into SPR</li>
 
<ur><li>1. Inject 45µl DOPC (100mM) into SPR</li>
 
<li>2. Inject 5µl NAOH to SPR in order to stabilize the point</li>
 
<li>2. Inject 5µl NAOH to SPR in order to stabilize the point</li>
Line 358: Line 308:
 
<li>5. Inject 10 µl Key DNA of 40 bp (0.1µM) to SPR</li>
 
<li>5. Inject 10 µl Key DNA of 40 bp (0.1µM) to SPR</li>
 
<br>
 
<br>
<A href="http://openwetware.org/wiki/Biomod/2013/Sendai/protocol#11">Protocol</A><br>
+
<A href="http://openwetware.org/wiki/Biomod/2013/Sendai/protocol">Protocol</A><br>
  
<h6>Result</h6>
+
<h5>Result</h5>
 
The result was shown in Fig.15 below.<br>
 
The result was shown in Fig.15 below.<br>
 
 
 
 
Line 370: Line 320:
 
<br>
 
<br>
  
<h6>Discussion</h6>
+
<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.
 
Fig.13 shows the behavior of materials on the surface of liposomes. The increase of SPR value after injecting Anchored DNA indicates that Anchored DNA successfully combined with liposomes.
 
Similarly, it is considered that loop DNA combined with Anchored DNA. <br>
 
Similarly, it is considered that loop DNA combined with Anchored DNA. <br>
Thus, we confirmed the formation of the loop structures on liposomes.<br>
+
Thus, <u>we confirmed the formation of the loop structures on liposomes.</i><br>
 
<br>
 
<br>
 
-->
 
-->
<h5 id=11>2-2-1 Disruption of liposomes by Flower DNA</h5>
+
<!---------------SPRコメントアウトここまで------------------>
<h6>Purpose</h6>
 
In Flower DNA approach, Key DNA should attach to Flower-anchor DNA on liposomes and break them. This experiment is conducted for the confirmation of it. <br>
 
フラワーミセルアプローチでは鍵DNAがリポソーム表面に生えているフラワーアンカーDNAにハイブリしてリポソームが割れる必要がある。それを確かめるために本実験を行った。<br>
 
<h6>Method</h6>
 
We made phase-separated liposomes (DOPC: DPPC: cholesterol= 1: 1: 1) with rhodamine dye inside by water-in-oil emulsion process. Then flower-anchor DNA (stained with SYBR Gold) was added into the liposomes. <br>
 
Next, we added Key DNA into the liposomes. The liposomes were observed in a chamber on a slide glass with a fluorescent microscope. <br>
 
内部にローダミンを含む相分離リポソーム(DOPC:DPPC:cholesterol=1:1:1)を界面通過法により作製して、リポソームにサイバーゴールドで染色したフラワーアンカーDNAを加えて、そのあとキーDNAを加えた。チャンバーinスライドガラスを使って位相差顕微鏡で観察した。<br>
 
<A href="http://openwetware.org/wiki/Biomod/2013/Sendai/protocol#11">Protocol</A><br>
 
  
  
<h6>Result</h6>
 
<img src="http://openwetware.org/images/4/47/%EF%BC%91%EF%BC%8D%EF%BC%91.png"><br>
 
<img src="http://openwetware.org/images/d/d6/%EF%BC%91%EF%BC%8D%EF%BC%92.png"><br>
 
<img src="http://openwetware.org/images/0/05/%EF%BC%91%EF%BC%8D%EF%BC%93.png"><br>
 
蛍光顕微鏡(赤 波長後で聞く)で観察したところ、リポソームが縮んでいる様子が観察された。波長を??(緑)に変更するとサイバーゴールドで染色されたフラワーアンカーDNAが光る。縮んだリポソームの周りが緑に発光しているのを確認できた。<br>
 
<img src="http://openwetware.org/images/5/5e/%EF%BC%92%EF%BC%8D%EF%BC%91.png"><br>
 
<img src="http://openwetware.org/images/e/ea/%EF%BC%92%EF%BC%8D%EF%BC%92.png"><br>
 
チャンバーを使って観察したのでトリガー溶液とリポソーム溶液が混合している様子を観察した。上図のさかい目の右側がトリガー溶液、左側がリポソーム溶液である。両者の境目で発光するネットワークのようなものが確認できた。<br>
 
  
<img src="http://openwetware.org/images/f/f6/%EF%BC%93%EF%BC%8D%EF%BC%91.png"><br>
+
<h5 id=11>2-2-1 Liposome disruption by flower DNA approach</h5>
<img src="http://openwetware.org/images/5/53/%EF%BC%93%EF%BC%8D%EF%BC%92.png"><br>
+
<h5>Purpose</h5>
<img src="openwetware.org/images/e/e0/3-3.png"><br>
+
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>
  
  
境目のネットワークの部分を拡大すると中がデキストリンTXR(赤)で染色されていないリポソームが確認できた。波長を緑に変えるとリポソームの周りが緑に染色されていた。<br>
+
<h5>Result</h5>
 
+
<div align="center">
<h6>Discussion</h6>
+
<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>
1段目リポソームが縮んだのでフラワーによるリポソーム破壊が起こっている可能性がある<br>
+
<div class="caption">Fig.13 Results of complementary key DNA and no binding Key DNA</div><br>
2段目境目のネットワーク構造はリポソームが割れた残骸かもしれない。(緑に光るのはフラワーアンカーDNAのみであるため)<br>
 
3段目なかが染色されていないリポソームは膜の一部が割れて内部の蛍光が漏れたものではないかと考えられる。<br>
 
<h5 id="12">2-2-2 Confirming sequence specificity of DNA</id>
 
  
 +
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>
  
 
         </article>
 
         </article>

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            <a href="http://openwetware.org/wiki/Biomod/2013/Sendai"><h1 style="color:white;" ><b>Biomod<span>2013<br>&emsp; Team</span>Sendai</b></h1></a> 
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<h2>Experiment</h2>

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

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

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

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


</li>


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

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

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

<h5>Result</h5>


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

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


<br>


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


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

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


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

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

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

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

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

<!--ここは表を使ってコンパクトに-->

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

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

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

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

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

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

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

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

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

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

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

   width: 90%;

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

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


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

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

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

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

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

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


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


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

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

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

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