Biomod/2013/Sendai/experiment

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       <article data-title="Chain Reaction">
       <article data-title="Chain Reaction">
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<h3 id="experimentsubproject2">外側からリポソームを破壊するサブプロジェクト</h3>
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<h3>Step 2 Chain-reactive burst</h3>
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<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>
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<p>Once the trigger DNA, which begins the interaction, is released, the next is the chain-reactive burst. If a liposome containing new triggers and active ingredients is broken, the released triggers come to destroy the surrounding liposomes one after another.<br>
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Once the trigger DNA, which begins the interaction, is released, the next is the chain reaction. If a liposome containing new triggers and active ingredients is broken, the released triggers come to break the surrounding liposomes one after another.<br>
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We tackled the problem of destroying liposomes by the following two approaches.</p>
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We tackled the problem of breaking liposomes by the following two approaches.<br>
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<ur>
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<li>Ⅰ膜を湾曲させるアプローチ ⅠBending membranes</li>
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<li>Ⅱフラワーミセルによるアプローチ ⅡUtilizing flower micelles</li></ur>
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</br></p>
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<h4>Ⅰ膜を湾曲させるアプローチ ⅠBending membranes</h4>
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<h5>(1)リポソームを破壊するトリガーであるDNAオリガミの作成 (1)Making DNA origami that acts as a trigger for breaking liposomes</h5>
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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>
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私たちのプロジェクトでは、リポソームを破壊するトリガーとしてDNA origamiを利用する。AFMによりDNAオリガミの作成を、電気泳動によりその蛍光標識(蛍光基つきDNAが、DNAオリガミに確かに接着していること)を確かめた。<br>
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DNA origami is a method applied to making nano-structures of various shapes. DNA origami consists of two kinds of strands: scaffold and staples. Scaffold is a long round single-stranded DNA, and staples are short linear single-stranded DNAs.<br>
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In our project, we used DNA origami as triggers for breaking liposomes. <br>
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We confirmed that it was well formed by AFM (Atomic Force Microscope) and that it was also fluorescently labeled (fluorescent molecules were successfully attached to the origamis) by electrophoresis.<br>
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<br>
<br>
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<h6>①アニーリング Annealing</h6>
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<ur><li>ⅠBending membranes</li>
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マイクロチューブにM13mp18, ステイプル, 5xTAE Mg2+, mQを混合、2時間半アニーリングを行った。<br>
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<li>ⅡUtilizing flower micelles</li></ur>
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We mixed M13mp18, staples, 5xTAE Mg2+, and mQ in a microtube and annealed it for 2.5 hours.<br>
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<br>
<br>
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<h4>ⅠBending membranes</h4>
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<h4>Experiment list</h4>
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The experiment necessary for realization of Bending membrane is following.<br>
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1)Making DNA origami<br>
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1-1)Making DNA origami<br>
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1-2)Labeling DNA origami<br>
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2)Destroying liposomes<br>
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2-1)Making liposomes<br>
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2-2)Investigating the interaction of DNA origami and liposomes<br>
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2-3)Counting liposomes<br>
<br>
<br>
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<h6>②AFM observation</h6><br>
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<br>
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DNAオリガミが設計図通りにできたことを確かめるため、AFM観察を行った(Fig.1)。設計図通り、端の欠けた長方形のDNAオリガミが確認できた。<br>
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<h4>1)Making DNA origami</h4>
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To see the origami formation, we observed the sample by AFM (Fig.1). Just like our design, rectangle origamis chipped in its edges were observed.<br>
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<h4>1-1)Making DNA origami<h4>
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<h5>Purpose</h5>
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In our project, we used DNA origami as triggers for destroying liposomes. We designed a rectangular DNA origami with a chipped edge and tried to make it.<br>
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<br>
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<h5>Principle</h5>
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DNA origami is a method applied to making nano-structures of various shapes. DNA origami consists of two kinds of strands: scaffold and staples. Scaffold is a long round single-stranded DNA, and staples are short linear single-stranded DNAs.<br>
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<br>
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<h5>Method</h5>
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We mixed M13mp18, staples, 5xTAE Mg2+, and mQ in a microtube and annealed it for 2.5 hours.<br>
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<A href=”http://openwetware.org/wiki/Biomod/2013/Sendai/protocol”>Protocol</A><br>
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<br>
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<h5>Result</h5>
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We confirmed that our DNA origami was well formed by AFM (Atomic Force Microscope) (Fig.1).<br>
<Img Src="http://openwetware.org/images/d/d9/Outsideafm2.png"> <br>
<Img Src="http://openwetware.org/images/d/d9/Outsideafm2.png"> <br>
Fig.1 AFM image of DNA origami (M13: 4nM, staples:20nM)<br>
Fig.1 AFM image of DNA origami (M13: 4nM, staples:20nM)<br>
<br>
<br>
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<h6>③電気泳動による蛍光標識の確認 Confirmation of fluorescently labeling of origamis by electrophoresis</h6>
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<h5>Discussion</h5>
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DNAオリガミをリポソームに作用させるとき、そのDNAオリガミが蛍光標識されていると、確認が容易である。DNAオリガミがうまく蛍光標識されているかを確かめるため、電気泳動を行った。<br>
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Just like our design, rectanglar origamis with chipped edges were observed.<br>
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If the origami is fluorescently labeled, it is much easier to observe the effect of DNA origami on liposomes. To see the origami was well labeled with fluorescent molecules, we used electrophoresis.<br>
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<br>
<br>
<br>
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このDNAオリガミは、標識のための蛍光基付きDNAが付くことのできるステイプルをもっている。同上の条件で、蛍光基付きDNAを混合し、2時間半アニーリングを行った。
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<h4>1-2)Labeling DNA origami<h4>
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また、蛍光基付きDNAがDNAオリガミに接着するのに、より早い時間でもすむかを調べるため、蛍光修飾なしDNAオリガミ溶液を10μlとり、蛍光基付きDNA0.6µlを加え、40分間放置した(これを、蛍光後付け修飾DNAオリガミとした)。<br>
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<h5>Purpose</h5>
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Our DNA origami has many staples that can bind to fluorescent tagged DNAs for labeling. We mixed fluorescent tagged DNAs with other staples in ① Annealing solution.<br>
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If the origami is fluorescently labeled, it is much easier to observe the effect of DNA origami on liposomes. So we labeled our origami by hybridizing it with fluorescent tagged DNA strands.<br>
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In addition, to see if the origami binds to the fluorescent tagged DNA in shorter time, we added the fluorescent tagged DNA into control annealing solution, which contains no fluorescent tagged DNA, and left it for 40 minutes.<br>
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<br>
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<h5>Method</h5>
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Our DNA origami has many staples that can bind to fluorescent tagged DNAs for labeling. We mixed fluorescent tagged DNAs together with DNA origami staples in annealing solution.<br>
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In addition, to see if the origami binds to the fluorescent tagged DNA in shorter time, we added the fluorescent tagged DNA into control annealing solution, which contained no fluorescent tagged DNA, and left it for 40 minutes.<br>
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To see the origami was well labeled with fluorescent molecules, we used electrophoresis. <br>
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Electrophoresis was conducted with a 1% agarose gel, CV100V for 50 minutes.<br>
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<a href=”http://openwetware.org/wiki/Biomod/2013/Sendai/protocol”>Protocol</a><br>
<br>
<br>
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染色前にゲルスキャンを行うと、蛍光基のついたストランドのみが観察されるので、蛍光修飾された構造体の位置を確認することができる。次に、染色後、ゲルスキャンを行うと、全てのDNA構造体の位置が確認できる。これらを合わせて、まず、染色後にDNAオリガミが正しく作られたことを確かめ、さらに、それが染色前にみられた蛍光修飾された構造体の位置とほぼ等しいと示すことで、DNAオリガミがうまく蛍光修飾されたことを確かめた。<br>
 
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>
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>
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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 are at the same height as those in a stained gel, we can say that our origami is successfully fluorescently labeled. <br>
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First we saw the bands of our origami in a non-stained gel. Then, we compared the bands with those in a stained gel. If the bands of origami in a non-stained gel were at the same height as those in a stained gel, we can say that our origami is successfully fluorescently labeled.<br>
<br>
<br>
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電気泳動は、1%アガロースゲルを用い、CV100Vで50分行った。<br>
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<h5>Result</h5>  
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Electrophoresis was conducted with a 1% agarose gel, CV100V for 50 minutes.<br>
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In a non-stained gel (Fig.2), only bands in lane 3 and 4 from the left (*Ori, **Ori) can be seen. They are fluorescent labeled structures. In addition, as they gave the same result, 40 minutes is long enough for fluorescent labeling.<br>
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<Img Src="http://openwetware.org/images/0/01/Outside-gel-3.png" width="300"><br>
<Img Src="http://openwetware.org/images/0/01/Outside-gel-3.png" width="300"><br>
Fig.2 Non-stained gel image: only bands in two lanes can be seen. From the left, they are DNA origami with fluorescent molecules in pre-annealing (Ori*), and DNA origami with fluorescent molecules in post-annealing (Ori**)<br>
Fig.2 Non-stained gel image: only bands in two lanes can be seen. From the left, they are DNA origami with fluorescent molecules in pre-annealing (Ori*), and DNA origami with fluorescent molecules in post-annealing (Ori**)<br>
<br>
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染色前のゲル画像(Fig.2)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>
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In a stained gel (Fig.3), maker (lane 1) had the longest DNA strand of 20kb. Comparing this and M13mp18 (lane 2) with annealed DNA origamis (lane 3,4,5), the bands of the origamis are at the higher position. Therefore, we concluded that in lane3~5, DNA origami structure made of M13 and staples were made as we had expected. <br>
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In a non-stained gel (Fig.2), only bands in lane 3 and 4 from the left (*Ori, **Ori) can be seen. They are fluorescent labeled structure. In addition, as they gave the same result, 40 minutes is enough for fluorescent labeling.<br>
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We considered that the bands in lane3~5 are seen as if they were diffused, just because our origami has many staples binding to the fluorescent tagged DNAs, and each origami attaches to different number of them, and its molecular weight varies.<br>
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<br>
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<Img Src="http://openwetware.org/images/c/c6/Outside-gel-2.png" width="300"> </br>
<Img Src="http://openwetware.org/images/c/c6/Outside-gel-2.png" width="300"> </br>
Fig.3 Stained gel image: from the left, maker, M13mp18, Ori*, Ori**, and DNA origami with no fluorescent molecule (Ori)<br>
Fig.3 Stained gel image: from the left, maker, M13mp18, Ori*, Ori**, and DNA origami with no fluorescent molecule (Ori)<br>
<br>
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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>
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<h5>Discussion</h5>
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In a stained gel (Fig.3), maker (lane 1) had the longest DNA strand of 20kb. Comparing this and M13mp18 (lane 2) with annealed DNA origamis (lane 3,4,5), the bands of the origamis were at the higher position. Therefore, we concluded that in lane3~5, DNA origami structure made of M13 and staples were made as we had expected. <br>
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Combining the results of Fig.2 and 3, the fluorescent labeled bands in lane3 and 4 in Fig.2 are at the same height as those of DNA origami in Fig.3. Thus, we concluded our origami was successfully fluorescently labeled.<br>
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We considered that the bands in lane3~5 were seen as if they were diffused, just because our origami had many staples binding to the fluorescent tagged DNA, and each origami attached to different number of them, and its molecular weight varied.<br>
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<br>
<br>
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Combining the results of Fig.2 and 3, the fluorescent labeled bands in lane3 and 4 in Fig.2 are at the same height as those of DNA origami in Fig.3. Thus, we concluded our origami was successfully fluorescently labeled.<br>
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<h4>2)Destroying liposomes</h4>
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<h4>2-1) Making liposomes</h4>
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<h5>Purpose</h5>
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We make liposomes that are to be broken by DNA origami.<br>
<br>
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<h5>(2)DNAオリガミでリポソームを割る実験 Breaking liposomes with DNA origami</h5>
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<h5>Principle</h5>
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<h6>①リポソームの作成 Making liposomes</h6>
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Phospholipids, which compose liposomes, are amphipathic molecules. They have hydrophilic and hydrophobic groups, and when they touch water, they make micelles: some hydrophilic groups take water inside. At the same time, other hydrophilic groups touch the water outside. So they form the innermost and outermost part of a micelle. On the other hand, the hydrophobic groups form the intermediate part of a micelle. <br>
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DNAオリガミで割るためのリポソームを作成した。<br>
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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>
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We made liposomes that were to be broken by DNA origami.<br>
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Phospholipids, which compose liposomes, are amphipathic molecules. They have hydrophilic and hydrophobic groups, and when they touches water, they make micelles: some hydrophilic groups take water inside. At the same time, other hydrophilic groups touch the water outside. So they form the innermost and outermost part of a micelle. On the other hand, the hydrophobic groups form the intermediate part of a micelle. <br>
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In this way, spherical liposomes are made.<br>
In this way, spherical liposomes are made.<br>
<br>
<br>
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脂質(DOPC)、脂質を溶かす溶媒(CHCl3)をミクロチューブにいれ、Arガスで乾かした。次に、観察用Bufferを加えたのち、湯煎してリポソームを作成した。<br>
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<h5>Method</h5>
To make liposomes, first we mixed lipid (DOPC) and solvent (CHCl3) in a microtube, and desiccate it with Argon gas. Then, adding some buffer (1xTAE Mg2+), we heated it in warm water for a few hours.<br>
To make liposomes, first we mixed lipid (DOPC) and solvent (CHCl3) in a microtube, and desiccate it with Argon gas. Then, adding some buffer (1xTAE Mg2+), we heated it in warm water for a few hours.<br>
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<a href=”http://openwetware.org/wiki/Biomod/2013/Sendai/protocol”>Protocol</a><br>
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<h6>②Investigating the interaction of DNA origami and liposomes</h6>
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次に、作成したリポソームとDNAオリガミとの相互作用を調べた。<br>
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リポソームを、DNAオリガミをトリガーとして割るには、DNAオリガミをリポソーム表面に多数ハイブリさせる必要がある。そこで、リポソームにコレステロール修飾DNAを添加し、表面に一本鎖DNAが現れるようにする。これを、DNAオリガミの一部と相補的な配列を持つ一本鎖DNAとすれば、DNAオリガミがリポソーム表面に多数ハイブリする。 <br>
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Next, we investigated how our DNA origami affected liposomes.<br>
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To break liposomes with triggers of DNA origami, many origamis have to hybridize with the surface of liposomes. To begin with, we added cholesterol-conjugated single-stranded DNAs (in the rest of this document, referred to as ccDNAs) into liposomes, and made them float on the surface. If the ccDNAs have some complementary parts to our origami, the origami is expected to hybridize with the surface.<br>
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<br>
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作製したリポソームにコレステロール修飾したDNAをつけるため、このリポソームにcholesterol修飾DNAを0.018, 0.069, 1.8, 6.9µMになるように加え、位相差顕微鏡でリポソームを確認した。<br>
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The result and discussion are integrated in the next passage of (2-2) Investigating the interaction of DNA origami and liposomes.<br>
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We added ccDNAs 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>
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<br>
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<br>
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<h4>2-2) Investigating the interaction of DNA origami and liposomes<h4>
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<h5>Purpose</h5>
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To destroy liposome with our origami, first we investigated how our DNA origami affected liposomes.<br>
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<br>
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<h5>Principle</h5>
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To destroy liposomes with our origami, many origamis have to hybridize with the surface of liposomes.<br>
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To begin with, we added cholesterol-conjugated single-stranded DNAs (in the rest of this document, referred to as ccDNAs) into liposomes, and made them float on the surface. If the ccDNA have a complementary part to our origami, the origami is expected to hybridize with the surface. In this way, many origamis would hybridize with liposome via ccDNAs.<br>
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<br>
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<h5>Method</h5>
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We added ccDNAs into liposomes at the final concentration of 0.018, 0.069, 1.8, and 6.9µM. Then we observed the samples with a phase microscope. Next, adding fluorescently labeled DNA origamis into the above liposomes, we saw if some change would happen with a fluorescent microscope.<br>
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<a href=”http://openwetware.org/wiki/Biomod/2013/Sendai/protocol”>Protocol</a><br>
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<br>
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<h5>Result</h5>
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In all four conditions, liposomes were observed with a phase microscope. We confirmed the formation of multilamella liposomes (Fig.4~7).<br>
<br>
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<Img Src="http://openwetware.org/images/7/72/Lipofig4.png" width="400"></br>
<Img Src="http://openwetware.org/images/7/72/Lipofig4.png" width="400"></br>
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Fig.4 Phase microscope image of liposomes (cholesterol-conjugated DNA: 0.018µM)<br>
Fig.4 Phase microscope image of liposomes (cholesterol-conjugated DNA: 0.018µM)<br>
<br>
<br>
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<Img Src="http://openwetware.org/images/d/d0/Lipofig5.png" width="400"></br>
<Img Src="http://openwetware.org/images/d/d0/Lipofig5.png" width="400"></br>
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Fig.5 Phase microscope image of liposomes (cholesterol-conjugated DNA: 0.069µM)<br>
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Fig.5 Phase microscope image of liposomes (cholesterol-conjugated DNA: 0.069µM)<br>
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<br>
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<Img Src="http://openwetware.org/images/d/de/Lipofig6.png" width="400"></br>
<Img Src="http://openwetware.org/images/d/de/Lipofig6.png" width="400"></br>
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Fig.6 Phase microscope image of liposomes (cholesterol-conjugated DNA: 1.8µM)<br>
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Fig.6 Phase microscope image of liposomes (cholesterol-conjugated DNA: 1.8µM)<br>
<br>
<br>
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<Img Src="http://openwetware.org/images/d/d7/Lipofig7.png" width="400"></br>
<Img Src="http://openwetware.org/images/d/d7/Lipofig7.png" width="400"></br>
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Fig.7 Phase microscope image of liposomes (cholesterol-conjugated DNA: 6.9µM)<br>
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Fig.7 Phase microscope image of liposomes (cholesterol-conjugated DNA: 6.9µM)<br>
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4つの条件全てにおいて、位相差顕微鏡で、リポソームが観察され、マルチラメラリポソームの作成が確認できた(Fig.4~7)。<br>
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In all four conditions, liposomes were observed with a phase microscope. We confirmed the formation of multilamella liposomes (Fig.4~7).<br>
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<br>
<br>
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次に、DNAオリガミを加え、蛍光顕微鏡で観察した。<br>
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Adding fluorescently labeled DNA origamis into the above liposomes, we saw if some change would happen with a fluorescent microscope.<br>
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cholesterol修飾DNA濃度が0.018, 0.069µMの時、多数のリポソームの膜が緑色に光っており、リポソーム膜に蛍光標識したDNAオリガミがハイブリしていることが確認できた(Fig.8,9,10)。<br>
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When the concentration of ccDNA was 0.018, 0.069µM, many gleaming (in green color) liposomes were observed. We confirmed that the fluorescently labeled origamis well hybridized with the liposomal surface (Fig.8,9,10). <br>
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Next, adding fluorescently labeled DNA origamis into the above liposomes, we saw if some change would happen with a fluorescent microscope.<br>
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When the concentration of ccDNA was 0.018, 0.069µM, many gleaming (in green color) liposomes were observed. We confirmed that the fluorescently labeled origamis well hybridized with the liposomal surface (Fig.8,9,10). <br>
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<table>
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Line 378: Line 386:
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</table>
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Fig.8,9 fluorescent microscope image of liposomes (cholesterol-conjugated DNA: 0.018µM)<br>
Fig.8,9 fluorescent microscope image of liposomes (cholesterol-conjugated DNA: 0.018µM)<br>
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<Img Src="http://openwetware.org/images/b/b4/Lipofig10.png" width="400"></br>
<Img Src="http://openwetware.org/images/b/b4/Lipofig10.png" width="400"></br>
Fig.10 fluorescent microscope image of liposomes (cholesterol-conjugated DNA: 0.069µM)<br>
Fig.10 fluorescent microscope image of liposomes (cholesterol-conjugated DNA: 0.069µM)<br>
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<br>
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一方で、cholesterol修飾DNA濃度が1.8µMの時は、蛍光顕微鏡では光るリポソームがほとんど確認できなかった(Fig.11)。この結果は、リポソームが割れた可能性を示唆している。<br>
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On the other hand, when the concentration of ccDNA was 1.8µM, few gleaming liposomes could be seen with a fluorescent microscope (Fig.11). This result indicates the possibility that liposomes were broken.<br>
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On the other hand, when the concentration of ccDNAs is 1.8µM, few gleaming liposomes could be seen with a fluorescent microscope (Fig.11). This result indicates the possibility that liposomes were broken.<br>
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+
<Img Src="http://openwetware.org/images/1/18/Lipofig11.png" width="400"></br>
<Img Src="http://openwetware.org/images/1/18/Lipofig11.png" width="400"></br>
Fig.11 fluorescent microscope image of liposomes (cholesterol-conjugated DNA: 1.8µM)<br>
Fig.11 fluorescent microscope image of liposomes (cholesterol-conjugated DNA: 1.8µM)<br>
-
 
-
 
-
 
<br>
<br>
-
cholesterol修飾DNA濃度が6.9µMの時、膜が緑色に光っているリポソームのほか、脂質が変形し、ネットワーク状に広がっているのが観察された(Fig.12)。<br>
+
When the concentration of ccDNA is 6.9µM, some liposomes were gleaming and others distorted, forming networks (Fig.12).<br>
-
When the concentration of ccDNAs is 6.9µM, some liposomes were gleaming and others distorted, forming networks (Fig.12).<br>
+
-
 
+
-
 
+
-
 
+
-
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<Img Src="http://openwetware.org/images/8/88/Lipofig12.png" width="400"></br>
<Img Src="http://openwetware.org/images/8/88/Lipofig12.png" width="400"></br>
-
 
+
Fig.12 fluorescent microscope image of liposomes (cholesterol-conjugated DNA: 6.9µM)<br>
-
 
+
-
 
+
-
Fig.12 fluorescent microscope image of liposomes (cholesterol-conjugated DNA: 6.9µM)<br>
+
<br>
<br>
-
以上の結果から、私たちはリポソームとDNAオリガミの相互作用について、以下の仮説を考えた。<br>
+
<h5>Discussion</h5>
From these results, we put forward the following hypothesis about the interaction of DNA origami and liposomes.<br>
From these results, we put forward the following hypothesis about the interaction of DNA origami and liposomes.<br>
-
<br>
 
-
cholesterol修飾DNAが低濃度(0.018, 0.069µM)の時、リポソームにはDNAオリガミが安定してハイブリしている。中濃度(1.8µM)の時、リポソームにより多くのcholesterol修飾DNAを介してDNAオリガミが突き刺さるので、個々のリポソームに負荷がかかり、リポソームは割れやすくなる。cholesterol修飾DNAが十分多い(6.9µM)時、一部のリポソームは互いに独立して存在しているが、一部のリポソームは、cholesterol修飾DNA-DNAオリガミ複合体を介して、ネットワークを構成する。<br>
 
When the concentration of ccDNA is low (0.018, 0.069µM), DNA origamis hybridize with the surface of the liposomes relatively stablely. When the concentration is middle (1.8µM), more DNA origamis hybridizes with the surface and place stress on it. Then, liposomes become fragile and easy to be broken. When the concentration is high (6.9µM), some liposomes exist individually, and others form networks via ccDNA and DNA origami complexes.<br>
When the concentration of ccDNA is low (0.018, 0.069µM), DNA origamis hybridize with the surface of the liposomes relatively stablely. When the concentration is middle (1.8µM), more DNA origamis hybridizes with the surface and place stress on it. Then, liposomes become fragile and easy to be broken. When the concentration is high (6.9µM), some liposomes exist individually, and others form networks via ccDNA and DNA origami complexes.<br>
-
 
<Img Src="http://openwetware.org/images/7/7c/Experimentinsidefig.png"><br>
<Img Src="http://openwetware.org/images/7/7c/Experimentinsidefig.png"><br>
<br>
<br>
-
この仮説によれば、cholesterol修飾DNA濃度が1.8µMの時、DNAオリガミによってリポソームが割れると考えられる。よって、私たちは、この濃度でリポソームが割れるかどうかを調べるため次の実験を行った。<br>
+
According to this hypothesis, when the concentration of ccDNA is 1.8µM, DNA origami destroys liposomes. Therefore, in the following experiment, we checked if DNA origami would destroy liposomes at this concentration of ccDNA.<br>
-
According to this hypothesis, when the concentration of ccDNA is 1.8µM, DNA origami breaks liposomes. Therefore, in the following experiment, we checked if DNA origami would break liposomes at this concentration of ccDNA.<br>
+
<br>
<br>
-
<h6>③リポソームの数をカウントする実験 Counting the number of liposomes</h6>
+
<br>
-
DNAオリガミを添加することで、リポソームが割れたかどうかを調べるには、DNAオリガミの添加前後のリポソームの数の変化を調べればよい。私たちは、DNAオリガミの添加前後でリポソームの数をカウントする実験を行った。<br>
+
<h4>2-3)Counting liposomes</h4>
-
リポソームを観察しやすいよう、脂質(DOPC)、脂質を溶かす溶媒(CHCl3)とともに、蛍光色素であるTR-DHPEを加えて、リポソームを作成した。<br>
+
<h5>Purpose</h5>
-
次に、cholesterol修飾DNAを1.8µMになるよう加え、蛍光顕微鏡でリポソームの数をカウントした。<br>
+
To see if DNA origami destroys liposomes, we counted the number of liposomes before and after adding DNA origami. <br>
-
カウント後、DNAオリガミを振りかけ、再度リポソームの数をカウントした。<br>
+
<br>
-
To see if DNA origami breaks liposomes, we counted the number of liposomes before and after adding DNA origami. <br>
+
<h5>Method</h5>
For the sake of observation convenience, we mixed TR-DHPE (red fluorescent dye) with lipid (DOPC) and solvate (CHCl3), and made liposomes. The liposomal surfaces were dyed by TR-DHPE.<br>
For the sake of observation convenience, we mixed TR-DHPE (red fluorescent dye) with lipid (DOPC) and solvate (CHCl3), and made liposomes. The liposomal surfaces were dyed by TR-DHPE.<br>
Then we added ccDNAs at the final concentration of 1.8µM, and counted the number of liposomes with a fluorescent microscope.<br>
Then we added ccDNAs at the final concentration of 1.8µM, and counted the number of liposomes with a fluorescent microscope.<br>
After counting, we put DNA origami and counted the number of liposomes again.<br>
After counting, we put DNA origami and counted the number of liposomes again.<br>
 +
<a href=”http://openwetware.org/wiki/Biomod/2013/Sendai/protocol”>Protocol</a><br>
 +
<Img Src="http://openwetware.org/images/a/a3/Addingimage.png"></br>
 +
<br>
 +
<br>
 +
<br>
 +
<h4>ⅡUtilizing flower micelles</h4>
 +
<h4>Experiment list</h4>
 +
The experiment necessary for realization of Bending membrane is following.<br>
 +
1) Making liposome <br>
 +
2) Confirming the formation of loop structure by SPR<br>
 +
3) Confirming the hybridization of trigger and loop DNA.<br>
 +
4) Destroying liposome<br>
 +
<br>
<br>
<br>
 +
<h4>1)Making liposome</h4>
 +
<h5>Purpose</h5>
 +
We make liposomes that are to be destroyed by flower micelle method.<br>
<br>
<br>
-
<h4>Ⅱフラワーミセルによるアプローチ Approach by flower micelles</h4>
+
<h5>Principal</h5>
-
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>
+
We made normal liposomes made of DOPC and phase-separatied liposomes made of  DOPC, DPPC and cholesterol.<br>
 +
Phase-separated liposomes are liposomes made by several kinds of lipids. On the surface of phase-separated liposomes several kinds of lipids separate and the liposomes are formed by some layers.<br>
 +
As the surface lipids of the phase-separated liposomes are not so changeable as the normal liposomes, It is considered that power produced by the hybridization of the loop and trigger strands reaches the liposome more effectively.<br>
 +
So the phase separation liposome was used for experiments this time.<br>
 +
<br>
 +
<h5>Method</h5>
 +
<ur><li>1. Making DOPC, DPPC, and Cholesterol lipid Lipid<br>
 +
1-1 Put 7.8 mg DOPC, 7.3mg DPPC , and 3.8mg Cholesterol into each microtube, and add 1ml CHCl3.<br>
 +
1-2 Put it in a ultrasonic bath of 60 degrees Celsius for one hour.<br>
 +
1-3 10mM DOPC, DPPC, Cholesterol lipid is made.<br></li>
 +
          <br>
 +
            <li>2. Making phase-separated liposomes<br>
 +
            2-1 Mix DOPC,DPPC, and Cholesterol at the ratio of 1:1:1 to make phase-separated liposomes. In this experiment, mix 4µl DOPC (10mM), 4µl DPPC (10mM),4µl Cholesterol (10mM) and 88l buffer well.<br>
 +
            2-2 Add 12µl Texas red (10M) <br>
 +
            2-3 Dry the sample using Argon gas<br>
 +
            2-4 Hydrated the dried sample with by 100ml 1xTAE<br>
 +
            2-5 Put the sample in hot water for three hours. Then leave it at low temperature for one hour to let the surface lipid separate.</il></ur><br>
 +
<br>
 +
<h5>Result</h5>
 +
As is shown in Fig.13, phase-separated liposomes were observed by a fluorescent microscope. They are basically multi-lamella liposomes.<br>
 +
We confirmed the formation of phase-separated liposomes with a fluorescent microscope.<br>
 +
 +
<Img Src="http://openwetware.org/images/7/79/Flowerex1.png"></br>
 +
Fig.13 Fluorescent microscope image of phase-separated liposomes<br>
 +
<br>
 +
<h5>Discussion</h5>
 +
Using the above-mentioned method, we successfully made phase-separated liposomes.
-
<h5>(1)リポソームの作成</h5>
+
<h4>2) Confirming the formation of loop structure by SPR</h4>
-
基本的には上記の膜を湾曲させるアプローチと同じ方法で作成した。</br>
+
<h5>Purpose</h5>
-
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>
+
To destroy liposomes by flower micelle method, we aim to attach many loop strands to the surface of liposomes. <br>
-
まずDOPC,DPPC,CholesterolそれぞれのLipidを製作する。</br>
+
To achieve this, we adopt the same hybridization method via ccDNAs as used in Ⅰbending membranes into liposomes: the ccDNA has a complementary part to our loop strand and the loop strand is expected to hybridize with liposomes.<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>
+
We checked the hybridization of liposomes and ccDNAs, and that of ccDNAs and our loop strands. <br>
-
それぞれ10nMolのDOPC,DPPC,Cholesterol  Lipidができる</br>
+
<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>
+
<h5>Principle</h5>
-
DOPC(10nM)を4μℓ、DPPC(10nM) を4μℓ、 Cholesterol (10nM)を1.6μℓ、buffer</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 ccDNA attaches to a liposome, and then loop strand attaches to it, SPR value increases after each step.<br>
 +
We measured SPR value after each step of adding DOPC into liposomes, and loop DNAs into it.<br>
 +
<br>
 +
<h5>Method</h5>
 +
<ur><li>1. Inject 45µl DOPC (100mM) into SPR</li>
 +
<li>2. Inject 5µl NAOH to SPR in order to stabilize the point</li>
 +
<li>3. Inject 10µl ccDNA (0.1M) to SPR</li>
 +
<li>4. Inject 10µl loop DNA of 40 bp (0.1M) to SPR</li>
 +
<li>5. Inject 10 µl trigger DNA of 40 bp (0.1M) to SPR</li>
 +
<br>
 +
<br>
 +
<h5>Result</h5>
 +
The result was shown in Fig.14 below.<br>
 +
 +
<Img Src="http://openwetware.org/images/f/fd/Flowerex2.png"></br>
 +
Fig.14 The transition of SPR value<br>
 +
<br>
 +
As the first injection of ccDNAs caused no change of SPR value, we injected ccDNAs for two times. <br>
 +
Fig 14 shows that SPR value increased after injecting ccDNAs and loop DNAs. Moreover, we should note that after injecting trigger DNA, some changes of SPR value were observed.<br>
 +
<br>
 +
<h5>Discussion</h5>
 +
Fig.14 shows the behavior of materials on the surface of liposomes. The increase of SPR value after injecting ccDNAs indicates that ccDNAs successfully combined with liposomes.
 +
Similarly, it is considered that loop DNAs combined with ccDNAs. <br>
 +
Thus, we confirmed the formation of the loop structures on liposomes.<br>
 +
<br>
 +
<br>
 +
<h4>3) Confirming the hybridization of trigger and loop DNA</h4>
 +
<h5>Purpose</h5>
 +
We checked whether trigger DNA hybridizes with loop DNA at normal temperature by electrophoresis. <br>
 +
<br>
 +
<h5>Method</h5>
 +
<ur><li>1. Prepare three microtubes and put three kinds of trigger DNAs (10, 20, 40b; 5µl, 100nM) into each tube.</li>
 +
<li>2. Add three kinds of loop DNAs (10, 20, 40b; 5µl, 100nM) into corresponding tube (tube that contains trigger DNA of corresponding number of nucleotides) and leave them at normal temperature for approximately one hour.</li>
 +
<li>3. Add 6x loading buffer with the quantity of 20% of the samples.</li>
 +
<li>4. Make an acrylic amide gel.</li>
 +
<li>5. Load samples (including maker) into 10 lanes</li>
 +
The electrophoresis was conducted with CV 100V for one hour.<br>
 +
<br>
 +
<h5>Result</h5>
 +
The result was shown in Fig.15.<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>
+
<Img Src="http://openwetware.org/images/3/37/Flowerex3.png"></br>
-
蛍光顕微鏡で、リポソームが観察された</br></br>
+
Fig.15 Stained gel image<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>
+
-
  0.5μℓ
+
-
  </td>
+
-
</tr>
+
-
<tr>
+
-
  <td>
+
-
  Chol leg 0.1μM
+
-
  </td>
+
-
  <td>  
+
-
  0.2μℓ
+
-
  </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>
+
-
  0.5μℓ
+
-
  </td>
+
-
</tr>
+
-
<tr>
+
-
  <td>
+
-
  ループDNA        0.1μM 
+
-
  </td>
+
-
  <td>  
+
-
  1.0μℓ
+
-
  </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>
+
<br>
<br>
-
<h5>(3)DNAループにトリガーストランドのハイブリ</h5>
+
The lane of 20 base loop and trigger shows a strong band at different height from the band of only 20 base loop and trigger. As for the samples of 40 base, the result was the same. <br>
-
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>
+
On the other hand, the lane of 10 base loop and trigger shows a band at the same height as the band of only 10 base loop. No band was seen in the lane of only 10 base trigger.<br>
-
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>
-
 
+
<h5>Discussion</h5>
-
</br>
+
The fact that the band of 20 base loop and trigger was at the different height from the band of only 20 base loop or trigger indicates that 20 base loop and trigger DNA hybridized and made a double strand. As the samples of 40 base showed the same result, we concluded that 20 and 40base loops and triggers hybridize at normal temperature.<br>
-
<table>
+
However, as for the samples of 10 bases, there was no difference between the two band height. Therefore, 10 base loop and trigger had not hybridized. <br>
-
<tr>  
+
It is estimated that no band was seen in the lane of only 10 base trigger because of some kind of mistakes. Therefore we do not take this into consideration.<br>
-
  <td>  
+
From the above, we find that the 20 and 40nt trigger hybridizes with a loop at normal temperature.<br>
-
  ループ付きリポソーム 0.1mM
+
<br>
-
  </td>
+
<br>
-
  <td>
+
<h4>4) Destroying liposome</h4>
-
  0.5μℓ
+
<h5>Purpose</h5>
-
  </td>
+
It was tested if liposomes would be destroyed by adding trigger DNA.<br>
-
</tr>
+
<br>
-
  <tr>
+
<h5>Principle</h5>
-
  <td>
+
Whether liposomes are destroyed or not can be decided by counting the number of liposomes before and after the trigger addition. As a control, we added the same amount of buffer instead of trigger. Liposomes are observed by a fluorescent microscope.<br>
-
  トリガーDNA        1.0mM 
+
<br>
-
  </td>
+
<h5>Method</h5>
-
  <td>  
+
<ur><li>1. Make liposomes with loop DNAs<br>
-
   0.2μℓ
+
1-1 Mix 2µl liposome (0.2mM) with 2µl ccDNA (10µM) at normal temperature<br>
-
  </td>  
+
1-2 Add 2µl loop DNA (20µM)</li><br>
-
  </tr>
+
<li>2. Destroy the liposomes with the loop DNAs<br>
-
</table>
+
2-1      Add 2µl trigger DNA (20µM) </li></ur><br>
-
</br>
+
<br>
-
 
+
<h5>Result</h5>
-
投入後30分間常温で放置し 蛍光顕微鏡で観察する</br></br>
+
Fig.16 is the result of the control experiment; Fig17, the result of adding trigger DNAs.<br>
 +
   <Img Src="http://openwetware.org/images/4/4f/Flowerex4.png">
 +
  <Img Src="http://openwetware.org/images/1/1e/Flowerex5.png">
 +
Fig.16,17 Fluorescent microscope image of liposomes
 +
<br>(Fig.16: control, Fig.17: sample added trigger DNAs)<br>
 +
   
 +
As it was difficult to count the number of liposomes in both cases, we did not count them.<br>
 +
<br>
 +
<h5>Discussion</h5>
 +
As we were not able to see a clear numerical change, we did not see whether liposomes were destroyed by this experiment.<br>
 +
Two ideas why liposomes were not destroyed are come up:<br>
 +
1. The lipid ratio for making liposomes was not appropriate. We should investigate the most appropriate and effective ratio for destroying liposomes.<br>
 +
2. Liposomes in this experiment were multi-lamella ones: Multi-lamella liposomes have some leaflets piled up. It is considered that more power is needed to destroy them. We would try other methods except the hydration method in future to make uni-lamella liposomes (which is relatively easy to destroy).<br>
 +
Solving the above- mentioned problems, liposomes would be destroyed<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>
         </article>

Revision as of 14:14, 30 August 2013

Biomod2013 Sendai ver2.0

Biomod2013
  Team
Sendai

Experiment

Step1 Egg-type trigger

ⅠExperiment list

The experiment necessary for realization of Egg-type trigger is following
1-1) Making liposome in alginate hydro gel beads
1-2 ) preparing of alginate hydro gel membrane containig buffer
2) Function confirmation of PNIPAM
3) Measurement of density of EGTA and time necessary to dissolve alginc acid gel
4) Urea diluting annealing

1-1) Making liposome in alginate hydro gel beads

Purpose
We need to put liposome in alginate hydrogel beads for chelating agent which in the liposome destroy alginate hydrogel membrane after liposome is broken. at first We make liposome and put it in a sodium alginate water solution and make alginate hydrogel beads with the solution
Method
We made liposome by passing droplet which covered one fold of phosphatide films in one fold of phosphatide film which was formed in the interface of two things not mixing well like oil and water We made outer buffer by adding oil 70μℓ to glucose 100μℓ.Then, we made inner buffer by adding oil 40µℓ to BSG(fluorescent substance) 1µℓ,and mixed it until it is muddy white by pipetting and tapping. We poured the inner buffer on outer buffer and centrifuged it for 70 seconds.After that, took out the liposome which collected at the bottom of the tube. Then, we added the liposome to 1.5%sodium alginate solution, and we put it in a capillary and dropped it in 0.4M CaCl2 aq by centrifuging it in a device such as Fig1 for 2-3 minutes
Fig1 Experimental device of alginic acid gel beads

Result
Because fluorescence protein is in the liposome, liposome shines. We observed the alginate hydrogel beads by cofocus laser microscope. (Fig 2) because the globe which glittered in alginate hydrogel beads is confirmed, it showed that liposome was in the alginate hydrogel beads.


Fig2 Cofocus laser microscope image of alginic acid gel beads with liposome

Discussion
Fig2を見るとリポソームが非常に小さくなってしまっている。これは浸透圧の影響を受けてリポソーム内の水分子が外部に漏れてしまっているためだと考えられる。これはアルギン酸水溶液の濃度やリポソーム内の溶液を調整することで改善できると考えれる。
From Fig2, liposome became very small. It was thought that this is because H2O molecule in the liposome leak under the influence of osmotic pressure outside. This problem may be improved by adjusting the density of sodium alginate solution and solution in the liposome.

1-2 ) preparing of alginate hydro gel membrane containing buffer

Purpose
Viscosity of beads made from alginate hydrogel is so thick that DNA can't be annealed because appulse numbers is decreased. So, we have to make alginate hydrogel membrane containing buffer. To make alginate hydrogel membrane containing buffer, we made double nosepiece and did following experiments.
Principle
When we provided enhanced gravity to double capillary by a centrifuge rotor, enclosed content fluid and extra solution (sodium alginate) effuse from front edge of nosepiece in the state of granularities, content fluid wrapped in sodium alginate. These granularities fall in drops to the solution of sodium alginate and only surface turn into gel and we got membranal gel beads.
Method
We used double capillary(Fig2), made from outer thick one and inner fine one. To confirm solution in the gel beads, we used 1.5% sodium alginate solution as outer solution, fluorescent stuff and 0.4M CaCl2 aq as inner one. Finally, we put capillary containing solution into centrifuge rotor for a few minutes and fell in drops to 0.4M CaCl2 aq.

Fig3 二重ノズルの構造

Result
We observed alginate hydrogel by confocal laser microscope and we could confirm fluorescence in the alginate hydrogel.
Discussion
Fig4を見てわかるように球体にならずカエルの卵状のゲルができた。これは遠心の速度を変えることで改善できると考えられる。
また今回、内管溶液に0.4M CaCl2 aqを使用しているためDNAが凝縮してしまう。これを改善するために濃度の低いMgCl2 aq などを使用する必要がある。 Alginate hydrogel did not become the globe, tube-formed gel was made(like frog spawn) It is thought that this problem can be improved by changing centrifugal speed.
In addition, this time, DNA will aggregate because there is Ca2+ in inner solution. It is necessary to use low density MgCl2 aq to improve this problem.

2) Function confirmation of PNIPAM

Purpose
In this project, it is necessary that liposome destroys surely when we increase temperature to 32℃.So, we confirmed whether liposome with PNIPAM destroys when the temperature become 32℃.
Principle
NIPAM is hydrophilic at less than 32℃, but it become hydrophobic and shrinks when it becomes the high temperature than 32℃. Therefore, the liposome that modified NIPAM becomes unstable and is broken at the time of high temperature than 32℃.
Reference
http://www.sigmaaldrich.com/etc/medialib/docs/SAJ/Brochure/1/j_recipedds2.Par.0001.File.tmp/j_recipedds2.pdf


Method
・Cork both of them and put in a refrigerator in half day.
We put observation buffer and fluorescence reagent in their tubes, and perform operation of ① and ② respectively.
① Setting a supersonic wave device to 20℃ and scratching the supersonic wave for 15 minutes.
② Setting a supersonic wave device to 40℃ and scratching the supersonic wave for 15 minutes.
We took 5μL of each mixed things and dilute them in observation buffer of 195μL and collected appropriate amount of object and observing them.

Fig4 PNIPAM付き脂質

Result
From Fig (?) and Fig (? + 1) when the temperature is 32℃ or low, liposome were observed. But when the temperature is higher than 32℃, liposome were not observed.
Discussion
This time we observed liposomes respectively on the same condition excepting temperature.
So the next time we will try to observe liposomes that are made at lower temperature than 32℃. After observation, heat them at higher temperature than 32℃ and then, observe them whether there are any liposomes or not.

3) Measurement of density of EGTA and time necessary to dissolve alginc acid gel

Purpose
It is necessary for the liposome in the alginic acid membrane to hold enough EGTA (one of the chelating agents) to dissolve alginic acid gel. In addition, when liposome is broken by an effect of NIPAM, Urea annealing and the destruction of the alginic acid film happen at the same time. But Time for Urea diluting Annealing must be shorter than Time for destruction of the alginic acid membrane. Therefore We measure density and time of the EGTA necessary to dissolve alginic acid gel beads.
Method
We make alginic acid gel beads and add 50mM,100mM,500mM EGTA to the solution. In addition, we prepare the thing which does not put EGTA in solution(control experiment)
We measure how many alginic acid gel beads decrease as time passes about the density of four kinds of EGTA.We take out the sample 0 minutes later, five minutes, ten minutes later・・・and count the number of alginic acid gel beads.We did the same experiment several times.
Result
From these results, the density of EGTA necessary to dissolve alginic acid gel beads is ?mM. and it took about ? minutes until alginic acid gel beads melted
Discussion

4) Urea diluting annealing

Purpose
It is necessary for trigger DNA origami to be formed by Urea diluting Annealing. In addition, Time for Urea diluting Annealing must be shorter than Time for destruction of the alginic acid membrane to realize this system (system of the alginic acid group) . Therefore we measured the time that Urea diluting Annealing takes.
Principle
Polarity of H2O molecular becomes weak in the presence of urea. So urea interrupts the hydrogen bond of DNA base. For that, the melting point of DNA decreases. This enables hybridization at low temperature by decreasing the concentration of urea gradually. So, we can do annealing by diluting urea.
Method
We added M13mp18 and staples at the rate of 1:20 in TAE buffer with urea (6M) and Mg2+ (12.5mM).Then, we set the filter to floater and float it on TAE buffer with Mg2+(12.5mM).The devise was mixed by stirrer for 4 hours. By doing that, urea passes the filter and escape to outside buffer but DNA remains in filter, so we can do urea diluting annealing. Then, we observed sample remained in the filter by AFM and electrophoresis.
We observed structures as we designed by AFM imaging.

Result
The result is Fig5 as below. The scale of DNA origami is similar to our design. (for details of DNA origami design click here).

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)

Discussion
We observed DNA origami like we designed as you can see Fig? .However, we also observed a lot of sheet structures like fragment. It is expectable that the reason why these fragments formed is that any staples didn’t hybridize with M13. We think this low yield of objective DNA origami was caused by high speed of diluting urea because in this experiment, urea easily passes filter. In our objective system, urea is diluted by destruction of liposome caused by the effect of PNIPAM. So, the speed of diluting urea is later than the speed of this experiment. It is necessary to measure the yield of DNA origami when the speed of diluting urea is later.

Step 2 Chain-reactive burst

Once the trigger DNA, which begins the interaction, is released, the next is the chain-reactive burst. If a liposome containing new triggers and active ingredients is broken, the released triggers come to destroy the surrounding liposomes one after another.
We tackled the problem of destroying liposomes by the following two approaches.


  • ⅠBending membranes
  • ⅡUtilizing flower micelles

  • ⅠBending membranes

    Experiment list

    The experiment necessary for realization of Bending membrane is following.
    1)Making DNA origami
    1-1)Making DNA origami
    1-2)Labeling DNA origami
    2)Destroying liposomes
    2-1)Making liposomes
    2-2)Investigating the interaction of DNA origami and liposomes
    2-3)Counting liposomes


    1)Making DNA origami

    1-1)Making DNA origami

    Purpose
    In our project, we used DNA origami as triggers for destroying liposomes. We designed a rectangular DNA origami with a chipped edge and tried to make it.

    Principle
    DNA origami is a method applied to making nano-structures of various shapes. DNA origami consists of two kinds of strands: scaffold and staples. Scaffold is a long round single-stranded DNA, and staples are short linear single-stranded DNAs.

    Method
    We mixed M13mp18, staples, 5xTAE Mg2+, and mQ in a microtube and annealed it for 2.5 hours.
    Protocol

    Result
    We confirmed that our DNA origami was well formed by AFM (Atomic Force Microscope) (Fig.1).

    Fig.1 AFM image of DNA origami (M13: 4nM, staples:20nM)

    Discussion
    Just like our design, rectanglar origamis with chipped edges were observed.


    1-2)Labeling DNA origami

    Purpose
    If the origami is fluorescently labeled, it is much easier to observe the effect of DNA origami on liposomes. So we labeled our origami by hybridizing it with fluorescent tagged DNA strands.

    Method
    Our DNA origami has many staples that can bind to fluorescent tagged DNAs for labeling. We mixed fluorescent tagged DNAs together with DNA origami staples in annealing solution.
    In addition, to see if the origami binds to the fluorescent tagged DNA in shorter time, we added the fluorescent tagged DNA into control annealing solution, which contained no fluorescent tagged DNA, and left it for 40 minutes.
    To see the origami was well labeled with fluorescent molecules, we used electrophoresis.
    Electrophoresis was conducted with a 1% agarose gel, CV100V for 50 minutes.
    Protocol

    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).
    First we saw the bands of our origami in a non-stained gel. Then, we compared the bands with those in a stained gel. If the bands of origami in a non-stained gel were at the same height as those in a stained gel, we can say that our origami is successfully fluorescently labeled.

    Result
    In a non-stained gel (Fig.2), only bands in lane 3 and 4 from the left (*Ori, **Ori) can be seen. They are fluorescent labeled structures. In addition, as they gave the same result, 40 minutes is long enough for fluorescent labeling.

    Fig.2 Non-stained gel image: only bands in two lanes can be seen. From the left, they are DNA origami with fluorescent molecules in pre-annealing (Ori*), and DNA origami with fluorescent molecules in post-annealing (Ori**)

    In a stained gel (Fig.3), maker (lane 1) had the longest DNA strand of 20kb. Comparing this and M13mp18 (lane 2) with annealed DNA origamis (lane 3,4,5), the bands of the origamis are at the higher position. Therefore, we concluded that in lane3~5, DNA origami structure made of M13 and staples were made as we had expected.
    We considered that the bands in lane3~5 are seen as if they were diffused, just because our origami has many staples binding to the fluorescent tagged DNAs, and each origami attaches to different number of them, and its molecular weight varies.

    Fig.3 Stained gel image: from the left, maker, M13mp18, Ori*, Ori**, and DNA origami with no fluorescent molecule (Ori)

    Discussion
    Combining the results of Fig.2 and 3, the fluorescent labeled bands in lane3 and 4 in Fig.2 are at the same height as those of DNA origami in Fig.3. Thus, we concluded our origami was successfully fluorescently labeled.

    2)Destroying liposomes

    2-1) Making liposomes

    Purpose
    We make liposomes that are to be broken by DNA origami.

    Principle
    Phospholipids, which compose liposomes, are amphipathic molecules. They have hydrophilic and hydrophobic groups, and when they touch water, they make micelles: some hydrophilic groups take water inside. At the same time, other hydrophilic groups touch the water outside. So they form the innermost and outermost part of a micelle. On the other hand, the hydrophobic groups form the intermediate part of a micelle.
    In this way, spherical liposomes are made.

    Method
    To make liposomes, first we mixed lipid (DOPC) and solvent (CHCl3) in a microtube, and desiccate it with Argon gas. Then, adding some buffer (1xTAE Mg2+), we heated it in warm water for a few hours.
    Protocol

    The result and discussion are integrated in the next passage of (2-2) Investigating the interaction of DNA origami and liposomes.


    2-2) Investigating the interaction of DNA origami and liposomes

    Purpose
    To destroy liposome with our origami, first we investigated how our DNA origami affected liposomes.

    Principle
    To destroy liposomes with our origami, many origamis have to hybridize with the surface of liposomes.
    To begin with, we added cholesterol-conjugated single-stranded DNAs (in the rest of this document, referred to as ccDNAs) into liposomes, and made them float on the surface. If the ccDNA have a complementary part to our origami, the origami is expected to hybridize with the surface. In this way, many origamis would hybridize with liposome via ccDNAs.

    Method
    We added ccDNAs into liposomes at the final concentration of 0.018, 0.069, 1.8, and 6.9µM. Then we observed the samples with a phase microscope. Next, adding fluorescently labeled DNA origamis into the above liposomes, we saw if some change would happen with a fluorescent microscope.
    Protocol

    Result
    In all four conditions, liposomes were observed with a phase microscope. We confirmed the formation of multilamella liposomes (Fig.4~7).


    Fig.4 Phase microscope image of liposomes (cholesterol-conjugated DNA: 0.018µM)


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


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


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

    Adding fluorescently labeled DNA origamis into the above liposomes, we saw if some change would happen with a fluorescent microscope.
    When the concentration of ccDNA was 0.018, 0.069µM, many gleaming (in green color) liposomes were observed. We confirmed that the fluorescently labeled origamis well hybridized with the liposomal surface (Fig.8,9,10).
    Fig.8,9 fluorescent microscope image of liposomes (cholesterol-conjugated DNA: 0.018µM)

    Fig.10 fluorescent microscope image of liposomes (cholesterol-conjugated DNA: 0.069µM)

    On the other hand, when the concentration of ccDNA was 1.8µM, few gleaming liposomes could be seen with a fluorescent microscope (Fig.11). This result indicates the possibility that liposomes were broken.

    Fig.11 fluorescent microscope image of liposomes (cholesterol-conjugated DNA: 1.8µM)

    When the concentration of ccDNA is 6.9µM, some liposomes were gleaming and others distorted, forming networks (Fig.12).

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

    Discussion
    From these results, we put forward the following hypothesis about the interaction of DNA origami and liposomes.
    When the concentration of ccDNA is low (0.018, 0.069µM), DNA origamis hybridize with the surface of the liposomes relatively stablely. When the concentration is middle (1.8µM), more DNA origamis hybridizes with the surface and place stress on it. Then, liposomes become fragile and easy to be broken. When the concentration is high (6.9µM), some liposomes exist individually, and others form networks via ccDNA and DNA origami complexes.


    According to this hypothesis, when the concentration of ccDNA is 1.8µM, DNA origami destroys liposomes. Therefore, in the following experiment, we checked if DNA origami would destroy liposomes at this concentration of ccDNA.


    2-3)Counting liposomes

    Purpose
    To see if DNA origami destroys liposomes, we counted the number of liposomes before and after adding DNA origami.

    Method
    For the sake of observation convenience, we mixed TR-DHPE (red fluorescent dye) with lipid (DOPC) and solvate (CHCl3), and made liposomes. The liposomal surfaces were dyed by TR-DHPE.
    Then we added ccDNAs at the final concentration of 1.8µM, and counted the number of liposomes with a fluorescent microscope.
    After counting, we put DNA origami and counted the number of liposomes again.
    Protocol




    ⅡUtilizing flower micelles

    Experiment list

    The experiment necessary for realization of Bending membrane is following.
    1) Making liposome
    2) Confirming the formation of loop structure by SPR
    3) Confirming the hybridization of trigger and loop DNA.
    4) Destroying liposome


    1)Making liposome

    Purpose
    We make liposomes that are to be destroyed by flower micelle method.

    Principal
    We made normal liposomes made of DOPC and phase-separatied liposomes made of DOPC, DPPC and cholesterol.
    Phase-separated liposomes are liposomes made by several kinds of lipids. On the surface of phase-separated liposomes several kinds of lipids separate and the liposomes are formed by some layers.
    As the surface lipids of the phase-separated liposomes are not so changeable as the normal liposomes, It is considered that power produced by the hybridization of the loop and trigger strands reaches the liposome more effectively.
    So the phase separation liposome was used for experiments this time.

    Method
  • 1. Making DOPC, DPPC, and Cholesterol lipid Lipid
    1-1 Put 7.8 mg DOPC, 7.3mg DPPC , and 3.8mg Cholesterol into each microtube, and add 1ml CHCl3.
    1-2 Put it in a ultrasonic bath of 60 degrees Celsius for one hour.
    1-3 10mM DOPC, DPPC, Cholesterol lipid is made.

  • 2. Making phase-separated liposomes
    2-1 Mix DOPC,DPPC, and Cholesterol at the ratio of 1:1:1 to make phase-separated liposomes. In this experiment, mix 4µl DOPC (10mM), 4µl DPPC (10mM),4µl Cholesterol (10mM) and 88l buffer well.
    2-2 Add 12µl Texas red (10M)
    2-3 Dry the sample using Argon gas
    2-4 Hydrated the dried sample with by 100ml 1xTAE
    2-5 Put the sample in hot water for three hours. Then leave it at low temperature for one hour to let the surface lipid separate.

    Result
    As is shown in Fig.13, phase-separated liposomes were observed by a fluorescent microscope. They are basically multi-lamella liposomes.
    We confirmed the formation of phase-separated liposomes with a fluorescent microscope.

    Fig.13 Fluorescent microscope image of phase-separated liposomes

    Discussion
    Using the above-mentioned method, we successfully made phase-separated liposomes.

    2) Confirming the formation of loop structure by SPR

    Purpose
    To destroy liposomes by flower micelle method, we aim to attach many loop strands to the surface of liposomes.
    To achieve this, we adopt the same hybridization method via ccDNAs as used in Ⅰbending membranes into liposomes: the ccDNA has a complementary part to our loop strand and the loop strand is expected to hybridize with liposomes.
    We checked the hybridization of liposomes and ccDNAs, and that of ccDNAs and our loop strands.

    Principle
    As our loop strand is too small to observe with an AFM or a fluorescent microscope, we used an apparatus called SPR.
    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.
    If ccDNA attaches to a liposome, and then loop strand attaches to it, SPR value increases after each step.
    We measured SPR value after each step of adding DOPC into liposomes, and loop DNAs into it.

    Method
  • 1. Inject 45µl DOPC (100mM) into SPR
  • 2. Inject 5µl NAOH to SPR in order to stabilize the point
  • 3. Inject 10µl ccDNA (0.1M) to SPR
  • 4. Inject 10µl loop DNA of 40 bp (0.1M) to SPR
  • 5. Inject 10 µl trigger DNA of 40 bp (0.1M) to SPR


  • Result
    The result was shown in Fig.14 below.

    Fig.14 The transition of SPR value

    As the first injection of ccDNAs caused no change of SPR value, we injected ccDNAs for two times.
    Fig 14 shows that SPR value increased after injecting ccDNAs and loop DNAs. Moreover, we should note that after injecting trigger DNA, some changes of SPR value were observed.

    Discussion
    Fig.14 shows the behavior of materials on the surface of liposomes. The increase of SPR value after injecting ccDNAs indicates that ccDNAs successfully combined with liposomes. Similarly, it is considered that loop DNAs combined with ccDNAs.
    Thus, we confirmed the formation of the loop structures on liposomes.


    3) Confirming the hybridization of trigger and loop DNA

    Purpose
    We checked whether trigger DNA hybridizes with loop DNA at normal temperature by electrophoresis.

    Method
  • 1. Prepare three microtubes and put three kinds of trigger DNAs (10, 20, 40b; 5µl, 100nM) into each tube.
  • 2. Add three kinds of loop DNAs (10, 20, 40b; 5µl, 100nM) into corresponding tube (tube that contains trigger DNA of corresponding number of nucleotides) and leave them at normal temperature for approximately one hour.
  • 3. Add 6x loading buffer with the quantity of 20% of the samples.
  • 4. Make an acrylic amide gel.
  • 5. Load samples (including maker) into 10 lanes
  • The electrophoresis was conducted with CV 100V for one hour.

    Result
    The result was shown in Fig.15.

    Fig.15 Stained gel image

    The lane of 20 base loop and trigger shows a strong band at different height from the band of only 20 base loop and trigger. As for the samples of 40 base, the result was the same.
    On the other hand, the lane of 10 base loop and trigger shows a band at the same height as the band of only 10 base loop. No band was seen in the lane of only 10 base trigger.

    Discussion
    The fact that the band of 20 base loop and trigger was at the different height from the band of only 20 base loop or trigger indicates that 20 base loop and trigger DNA hybridized and made a double strand. As the samples of 40 base showed the same result, we concluded that 20 and 40base loops and triggers hybridize at normal temperature.
    However, as for the samples of 10 bases, there was no difference between the two band height. Therefore, 10 base loop and trigger had not hybridized.
    It is estimated that no band was seen in the lane of only 10 base trigger because of some kind of mistakes. Therefore we do not take this into consideration.
    From the above, we find that the 20 and 40nt trigger hybridizes with a loop at normal temperature.


    4) Destroying liposome

    Purpose
    It was tested if liposomes would be destroyed by adding trigger DNA.

    Principle
    Whether liposomes are destroyed or not can be decided by counting the number of liposomes before and after the trigger addition. As a control, we added the same amount of buffer instead of trigger. Liposomes are observed by a fluorescent microscope.

    Method
  • 1. Make liposomes with loop DNAs
    1-1 Mix 2µl liposome (0.2mM) with 2µl ccDNA (10µM) at normal temperature
    1-2 Add 2µl loop DNA (20µM)

  • 2. Destroy the liposomes with the loop DNAs
    2-1 Add 2µl trigger DNA (20µM)


  • Result
    Fig.16 is the result of the control experiment; Fig17, the result of adding trigger DNAs.
    Fig.16,17 Fluorescent microscope image of liposomes
    (Fig.16: control, Fig.17: sample added trigger DNAs)
    As it was difficult to count the number of liposomes in both cases, we did not count them.

    Discussion
    As we were not able to see a clear numerical change, we did not see whether liposomes were destroyed by this experiment.
    Two ideas why liposomes were not destroyed are come up:
    1. The lipid ratio for making liposomes was not appropriate. We should investigate the most appropriate and effective ratio for destroying liposomes.
    2. Liposomes in this experiment were multi-lamella ones: Multi-lamella liposomes have some leaflets piled up. It is considered that more power is needed to destroy them. We would try other methods except the hydration method in future to make uni-lamella liposomes (which is relatively easy to destroy).
    Solving the above- mentioned problems, liposomes would be destroyed
    .

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