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<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">1st 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">2nd 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>


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

<h3 id=chain>1st stage: Sensing system</h3> <h4 id=bending>1-1Disruption of temperature sensitive liposomes</h4> <h6>Purpose</h6> 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> 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="">Sigma Aldrich</A><br> <br>

<h6>Method</h6> 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="">Protocol</A><br>


<img src="" width="60%" height="60%"><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> 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>


<h6>Discussion</h6> 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>

<h3 id=Flower>2nd stage: Amplification system</h5> <h4 id=sensing>2-1 DNA Origami approach</h4> <h5 id=5>2-1-1 Making DNA Origami</h5> <h6>Purpose</h6> 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> Mixing M13mp18, staples, 5xTAE Mg2+, and mQ in a microtube and annealing for 2.5 hours.<br> <A href="">Protocol</A><br>

<h6>Result</h6> We obtain DNA Origami same as our design. The result was confirmed by AFM (Atomic Force Microscope.)<br> <Img Src=""> <br> Fig.2 AFM image of DNA Origami (M13: 4nM, staples:20nM)<br> <br>

<h6>Discussion</h6> As shown in Fig. 2, DNA Origami was well-formed.<br>

<h5 id=6>2-1-2 Labeling DNA Origami with fluorescent-tagged DNA</h5> <h6>Purpose</h6> Since it is much easier to observe the fluorescent effect of DNA Origami on liposomes, we labeled our Origami by hybridizing with the fluorescent-tagged DNA strand. <br><br>

<h6>Method</h6> Our DNA Origami is composed of many staples that can bind to fluorescent-tagged DNA for labeling. We mixed fluorescent-tagged DNA together with DNA Origami staples in the last annealing solution.<br> In addition, to observe the binding of Origami staples and fluorescent-tagged DNAs faster, 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 confirm the Origami was well-labeled with fluorescent molecules, we used gel-electrophoresis.<br> Gel-electrophoresis was conducted with a 1% Agarose gel, CV100V for 50 minutes.<br>

<br> By scanning a gel before staining, we can see only the bands of DNA structures with fluorescent molecules. While scanning a gel after staining, we can see the bands of all DNA structures. So we scanned a gel before and after staining (we scanned both a non-stained and a stained gel). <br> First we saw the bands of our Origami in a non-stained gel. Then, we compared the bands with those in a stained gel. If the bands of Origami in a non-stained gel were at the same height as that in a stained gel, we can say that our Origami was successfully fluorescently labeled.<br> <A href="">Protocol</A><br>

<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, that are fluorescently labeled structures. In addition, as we got the same result, 40 minutes is long enough for fluorescently labeling.<br> <div align="center"> <Img Src="" width="450"><br> Fig.3 Non-stained gel image: only two lanes show the band: 3rd lane - DNA Origami with fluorescent molecules in pre-annealing (Ori*), 4th lane - and DNA Origami with fluorescent molecules in post-annealing (Ori**)<br></div> <br> For the stained gel (Fig.4), lane 1 is a DNA marker. Comparing the band of M13mp18 (lane 2) with annealed DNA Origami (lane 3,4,5), the bands of the Origami are at the higher position. Thus, we concluded that DNA Origami structure in lane 3~5, was made as we had expected. <br> We considered that the bands in lane 3~5 are diffused since our Origami has many staples binding to the fluorescent-tagged DNA, and each Origami attaches to different number of them. Thus its molecular weight varies.<br>

<Img Src="" 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>

<h6>Discussion</h6> From the results shown in Fig. 3 and 4, the fluorescently labeled bands in 3rd and 4th lanes 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>

<h5 id=7>2-1-3 Disrupting liposomes by DNA Origami (microscopic analysis)<h5> <h6>Purpose</h6> To break liposomes with our Origami, first we investigate how our DNA Origami affect liposomes.<br> <br> <h6>Principle</h6> 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> <h6>Method</h6> 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> Next, adding fluorescently labeled DNA Origami into the above liposomes, we saw if some change would happen with a fluorescent microscope.<br> <A href="">Protocol</A><br> <br> <h6>Result</h6> In all four conditions, liposomes were observed with a phase microscope. We confirmed the formation of multi-lamella liposomes (Fig.5~8).<br>


<Img Src="" width="400"></br> Fig.5 Phase microscope image of liposomes (Origami-anchor DNA: 0.018µM)<br> <br> <Img Src="" width="400"></br>

Fig.6 Phase microscope image of liposomes (Origami-anchor DNA: 0.069µM)<br>

<br> <Img Src="" width="400"></br>

Fig.7 Phase microscope image of liposomes (Origami-anchor DNA: 1.8µM)<br>

<br> <Img Src="" width="400"></br>

Fig.8 Phase microscope image of liposomes (Orgami-anchor DNA: 6.9µM)<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 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>

  <Img Src="" width="400">
  <Img Src="" width="400">

</table> Fig.9,10 fluorescent microscope image of liposomes (Origami-anchor DNA: 0.018µM)<br> <Img Src="" width="400"></br> Fig.11 fluorescent microscope image of liposomes (Origami-anchor DNA: 0.069µM)<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="" width="400"></br> Fig.12 fluorescent microscope image of liposomes (Origami-anchor DNA: 1.8µM)<br> <br> When the concentration of Origami-anchor DNA is 6.9µM, some liposomes were gleaming and others distorted, forming networks (Fig.13).<br>

<Img Src="" width="400"></br>

Fig.13 fluorescent microscope image of liposomes (Origami-anchor DNA: 6.9µM)<br>

<br> <h6>Discussion</h6> 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=""><br> <br> According to this hypothesis, when the concentration of Origami-anchor DNA is 1.8µM, DNA Origami breaks liposomes. <br> <br> <br>

<h5 id=13>2-1-4 Disrupting liposomes by DNA Origami (quantitative analysis)<h5> <h6>Purpose</h6> As DNA Origami is likely to have disrupted liposomes in 2-1-3 microscopic analysis, we investigate how our DNA Origami affects liposomes quantitatively.<br> We make phase-separatied liposomes made of DOPC, DPPC, DOPE and cholesterol.<br> Phase-separated liposomes are liposomes consisting of several kinds of lipids. It has less fluidity and its membranee is more stiff than normal liposomes.<br> Due to the above reasons, we consider that phase-separated liposomes are more suitable to be disrupted. Thus, this time, we used phase-separated liposomes.<br> <br> <h6>Method</h6> 1. Making liposomes that contain GFP in the interior, by an oil/water interface. <br> 2. Observing only liposomes by the confocal microscope. <br> 3.<br> Sample 1. Liposomes + Origami-anchor DNA<br> Sample 2. Liposomes + Origami-anchor DNA + Surfactant (2%NP)<br> Sample 3. Liposomes + Origami-anchor DNA + Key DNA<br>

<div class="caption-right">

<Img Src="" style="padding-left:10mm"><span>Fig.みぎ EV-SS(Sample 3) </span>

</div> We used 50㎕ from each sample.<br> Measuring each sample’s fluorescence intensity ofliposomes by Cell Lab Quanta SC Flow Cytometer.<br> Only 7-13㎛ diameter liposomes were analyzed (cut off by EV value). Liposomes showing over 100 SS value (the indicator of sample complexity) were also omitted because of reliability of the data(fig.みぎ).<br>

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+アンカーDNA<br> サンプル2 リポソーム+アンカーDNA+界面活性剤(2%NP)<br> サンプル3 リポソーム+アンカーDNA+keyDNA<br> を用意してそれぞれをCell Lab Quanta SC Flow Cytometerで直径が7~13μm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> As figure below, we was able to observe liposomes containing GFP, by the confocal microscope.<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>


The abscissa of the following graph is the fluorescence intensity of only liposomes, and the ordinate represents the number of liposomes.<br> 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>

<div class="caption-left">

<Img Src="" style="padding-left:5mm"><span>Fig.1 Adding nothing<br>(Sample1) </span>


<div class="caption-left">

<Img Src="" style="padding-left:5mm"><span>Fig.2 Adding Surfactant<br>(Sample2) </span>


<div class="caption-left">

<Img Src="" style="padding-left:5mm"><span>Fig.3 Adding KeyDNA<br>(Sample3) </span>

</div> <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> IWe confirm the selectivity of Key DNAs to the anchor DNA. We compare the effect of the complementary Key DNA and the no-binding Key DNA.<br> <br>

<h6>Method</h6> 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). Liposomes showing over 100 SS value (the indicator of sample complexity) were also omitted because of reliability of the data.<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> Protocol<br> <A href="">Protocol</A><br>

<h6>Result</h6> The results are shown in figures XXXX.<br> <br> <div class="caption-left">

<Img Src="" style="padding-left:10mm"><span>Fig.1 Sample1(complement)<br>(Sample1) </span>

</div> <div class="caption-left">

<Img Src="" style="padding-left:10mm"><span>Fig.2 Sample2(No binding pair)<br>(Sample1) </span>

</div> <br> <div class="caption-left">

<Img Src="" style="padding-left:5mm"><span>Fig.3 Adding Complementary key DNA<br>(Sample1) </span>

</div> <div class="caption-left">

<Img Src="" style="padding-left:5mm"><span>Fig.4 Adding no binding Key DNA<br>(Sample1) </span>

</div> <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>

<h6>Discussion</h6> These results demonstrated the selectivity of the Key DNA. This selectivity will be used to control the order to disrupt target liposomes. <br>

<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> <h6>Purpose</h6> 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>

<h6>Principle</h6> 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>

<h6>Method</h6> <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="">Protocol</A><br>

<h6>Result</h6> The result was shown in Fig.15 below.<br>

<Img Src=""></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>

<h6>Discussion</h6> 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, we confirmed the formation of the loop structures on liposomes.<br> <br> --> <!---------------SPRコメントアウトここまで------------------>

<h5 id=11>2-2-1フラワーミセルによりリポソームを破壊する実験</h5> <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> 内部溶液をTXR-デキストランで染色した相分離リポソーム(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="">Protocol</A><br>

<h6>Result</h6> <img src=""><br> <img src=""><br> <img src=""><br> We observed shrunk liposomes by red filter. When we observed them by green filter, Flower-anchor DNA (dyed with SYBR Gold) was bright. There was green fluorescence around shrunk liposomes.<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=""><br> <img src=""><br> We observed the contact surface of Key DNA and liposomes. The right side of the boundary is Key DNA and the left side of it is liposome. There are something bright like a network on the boundary.<br>


<img src=""><br> <img src=""><br> <img src=""><br>

When magnifying the network, we observed liposomes undyed with Texas-Red dextran. As we observed them by green filter, liposomes were dyed green.<br>


<h6>Discussion</h6> As liposomes in Figure ? were shrunk, Flower-anchor DNA probably broke liposomes. The network on the boundary in Figure ? may have been the wreck of liposomes (, because only Flower-anchor DNA is dyed green).<br> We suppose that liposomes in Figure ? were undyed, because liposome membrane partly broke and the inside fluorescence had leaked.<br>

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> 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>



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