Biomod/2013/Sendai/design

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<article data-title="Chain-reactive burst">
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  <article data-title="Chain-reactive burst">
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<h3 id="chain">Chain-reactive burst</h3></br>
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<a name="chain-reactive burst"></a>
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<h3>Chain-reactive burst</h3></br>
<br>
<br>
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We designed chain-reactive burst systems as follows.Each liposome contains triggers, drugs, and has receptors of the trigger.  When liposomes are destroyed, new triggers and drugs are released.To achieve liposome's burst by outside trigger, following two approaches are raised. <br>
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We designed “chain-reactive burst” system as follows.<br>
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リポソームが連鎖的に割れるようなシステムをデザインしました。リポソーム内部のtriggerは、リポソームが割れると放出され、周囲のリポソームを割ります。リポソームに外部からの負荷をかけて割ることを目的に、以下の2つのアプローチを考案しました。<br>
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Each liposome contains triggers and drugs inside, and aptamers for the trigger on its surface.  When liposomes are destroyed, new triggers and drugs are released. To achieve liposomal burst by outside triggers, we propose the following two approaches. <br>
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<br>
<br>
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<li>i)膜を湾曲させるアプローチ<br><h5>i)A bending approach</h5></li>
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<li> <h5>i) Bending approach</h5></li>
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<li>ii)フラワーミセルによるアプローチ</br><h5>ii)A flower micelle approach</h5></li>
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<li> <h5>ii) Flower micelle approach</h5></li>
<br>
<br>
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At first, we considered a theory to disrupt liposomes by a trigger DNA signal. About the change of vesicles by destroyed, estimated the difference of free energies of vesicles by calculations about free energies of different kinds of size. And also discuss what kind of size that vesicles are more stable.<br>
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First, we considered a theory to disrupt liposomes by a trigger DNA signal through calculation. If a liposome is destroyed, its size becomes smaller. We estimated the free energy gap between the two liposomal states: a large liposome and a small one. And discuss which size of liposomes is more stable.<br>
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Please see the details (Go to <a href=” http://openwetware.org/wiki/Biomod/2013/Sendai/caluculation”>Calculation</a>).<br>
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Please see the details below link (Go to Calcultion pageリンクを張る).<br>
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<h4> i)Bending Approach</h4><br>
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このリポソームを割るときの理論的アプローチについて考察しました。ベシクルが壊れる変化について、大きさの違いによる自由エネルギーの値から、どのような大きさのときにより安定で、どのくらいのエネルギー差があるのかを計算により見積もった。詳細はCalculationのページへ。(リンクを張る)<br>
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<h4>i)膜を湾曲させるアプローチ<br>i)A bending approach</h4><br>
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<Img Src="http://openwetware.org/images/d/d2/Bending-flow.png" Align="center" width="900px" ><br>
<Img Src="http://openwetware.org/images/d/d2/Bending-flow.png" Align="center" width="900px" ><br>
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<div align="center">Fig2 process of bending membranes</div><br>
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<div align="center">Fig.2 Process of bending approach</div><br>
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<br>
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1.リポソーム表面にccDNAを付着させる。
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Our bending approach consists of the following four steps.<br>
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Cholesterol modified DNA strands attaches surface of liposomes.<br>
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1.Cholesterol-conjugated DNA strands (in the rest of this document, referred to as “aptamer”) attaches to the surfaces of liposomes.<br>
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2.相補的DNA鎖付きのDNAオリガミを加える。
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2. Then, DNA origami complementary to the aptamer is added as triggers.<br>
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Then, DNA origami with complementaly strand are added as triggers.<br>
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3. Triggers bind to the surfaces of liposomes and give a load on the membrane.<br>
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3.表面にbindしたオリガミ構造体によって膜表面に負荷がかかる。(詳細は後述)
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4. Due to the load by triggers, liposomes are destroyed.<br>
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  Triggers bind surface of liposomes and get a load on membrane.<br>
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4.負荷によって膜がburstする。
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  By the load of trigger, liposome was destroyed.<br>
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<br>
<br>
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<h5>a)Mechanism of bending membranes</h5>
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<h5>a) Mechanism of bending membranes</h5>
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To destroy liposomes, we focused on the mechanism the living things use to bend cell membranes. We consider that if we could make use of the mechanism of bending membranes (destabilizing membranes), it would lead to the collapse of membranes. The following three mechanisms have been proposed as of now (<A Href="http://www.ncbi.nlm.nih.gov/pubmed/19780639">Membrane-bending proteins</A>)<br>
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リポソームを割るため、私たちは生物が膜を湾曲させるメカニズムに着目した。膜の湾曲、すなわち不安定化を最大限に利用することが出来れば、膜の崩壊につながると考えたからである。膜を湾曲させるメカニズムには、以下の三つが提案されている。<br>
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To break liposomes, we focused on the mechanism the living things use to bend cell membranes. We consider that if we could make use of the mechanism of bending membranes (destabilizing membranes), it would lead to the collapse of membranes. The following three mechanisms have been proposed as of now (<A Href="http://www.ncbi.nlm.nih.gov/pubmed/19780639">Membrane-bending proteins</A>)<br>
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<br>
<br>
  <Img Src="http://openwetware.org/images/a/ae/Designfig2.png" Align="left" width="280px" height="400px">
  <Img Src="http://openwetware.org/images/a/ae/Designfig2.png" Align="left" width="280px" height="400px">
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Aは、両親媒性基をもつ分子が細胞膜に挿入されることにより、膜が湾曲するというものである。脂質二重膜の内側の強い疎水性部分は、脂質両膜をくっつけて離さない性質をもっている。このため、両親媒性基が片方の膜内に入りこみ、その膜が広がると、もう片方の膜は、少ない表面積でも済むように、内側になるようつられて曲がる。
 
<br>
<br>
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Bは、膜表面に付着した分子が固い足場となり、下の膜を変形したり、あらかじめ湾曲されていた膜を固定化(stabilize)するというものである。<br>
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The mechanism A is that amphipathic molecules are inserted into the cell membrane and the bending is caused. The inner hydrophobic part of the lipid bilayer has a strong adhesive power for the two leaflets. Thus, once the amphipathic molecules are inserted into one leaflet of the membrane and expand it, the other leaflet bends according to it, making its surface area smallest.<br>
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Cは、片方の膜に脂質を群がらせることにより、脂質の量が両膜で不均等になることにより、膜が湾曲するというものである。
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<br>
<br>
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The mechanism B is that the molecule attached to the membrane becomes a rigid scaffold and distort the membrane under itself, or stabilize the already bended membrane.<br>
<br>
<br>
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生体膜を湾曲させるタンパク質のほとんどは、A~Cのメカニズムを組み合わせて使っている。<br>
 
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また、近年、タンパク質同士が密集することで、膜が湾曲されるという考えも提唱されている(Membrane bending by protein-protein crowding). これは、膜結合タンパク質同士の衝突による、横方向の圧力により、膜が曲がるというものである。
 
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<br>
 
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The mechanism A is that amphipathic molecules are inserted into the cell membrane and the bending is caused. The inner hydrophobic part of the lipid bilayer has a strong adhesive power for the two leaflets. Thus, once the amphipathic molecules are inserted into one leaflet of the membrane and expand it, the other leaflet bends according to it, making its surface area smallest.<br>
 
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The mechanism B is that the molecule attached to the membrane becomes a rigid scaffold and distort the membrane under itself, or stabilize the already bended membrane.<br>
 
The mechanism C is that lipid molecules are clustered in one leaflet of the membrane and the inequality of lipid quantity makes the membrane bend.<br>
The mechanism C is that lipid molecules are clustered in one leaflet of the membrane and the inequality of lipid quantity makes the membrane bend.<br>
<br>
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In addition, a theory that protein crowding causes the bending of cell membranes ( <A Href="http://www.ncbi.nlm.nih.gov/pubmed/22902598">Membrane bending by protein- protein crowding</A>) has recently been suggested. This mechanism is that the collision of membrane proteins produces lateral pressure on membranes and distorts them.<br>
In addition, a theory that protein crowding causes the bending of cell membranes ( <A Href="http://www.ncbi.nlm.nih.gov/pubmed/22902598">Membrane bending by protein- protein crowding</A>) has recently been suggested. This mechanism is that the collision of membrane proteins produces lateral pressure on membranes and distorts them.<br>
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<br>
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以上から、膜を不安定にさせるためには、<br>
 
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<ur><li>・固い足場となる</li>
 
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<li>・足場の影響を最大にするため、表面積が大きい</li>
 
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<li>・衝突により大きな圧力が生じる</li>
 
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構造が有効であると考えられる。
 
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<br>
 
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Due to the above reasons, the efficient design for destabilizing membranes is the structures that :<br>
Due to the above reasons, the efficient design for destabilizing membranes is the structures that :<br>
<ur><li>have rigid scaffolds</li>
<ur><li>have rigid scaffolds</li>
<li>have large surface areas to maximize the effect of the scaffold on the membrane</li>
<li>have large surface areas to maximize the effect of the scaffold on the membrane</li>
<li>produce a large pressure by collisions</li></ur>
<li>produce a large pressure by collisions</li></ur>
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<br>
<h5>b) Rigid scaffolds</h5>
<h5>b) Rigid scaffolds</h5>
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私たちは、固い足場となる分子を実現するために、DNAオリガミに着目した。DNAオリガミは任意の形に固い構造を作ることができるからである。
 
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そして、表面積の大きい構造として、平面構造を、
 
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<br>
 
To make rigid scaffolds, we took note of DNA origami, because DNA origami is a method for making rigid structures of any shape. Moreover, we adopted a 2D structure to make the surface area largest.<br>
To make rigid scaffolds, we took note of DNA origami, because DNA origami is a method for making rigid structures of any shape. Moreover, we adopted a 2D structure to make the surface area largest.<br>
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<br>
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衝突により横方向に最大の圧力が生ずるような構造として、長方形や三角形を考えた。<br>
 
We also designed rectangle and triangle to make the pressure of the collision highest.<br>
We also designed rectangle and triangle to make the pressure of the collision highest.<br>
<Img Src="http://openwetware.org/images/6/63/Outsidefig3.png" Align="left">
<Img Src="http://openwetware.org/images/6/63/Outsidefig3.png" Align="left">
<br>  
<br>  
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We suppose that rectangle and triangle structures are most effective for the following reasons. <br>
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Rectangle is expected to work as one scaffold in itself; triangle (the most efficient figure that covers a sphere) structures, to gather and work as one big rigid scaffold.<br>
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長方形はそれ自体で一つの足場として働き、三角形(球面をもっとも効率よく覆う図形)は沢山集合して一つの固い足場を作ればもっとも効率が良いと考えられる。<br>
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We suppose that rectangle and triangle structures are most effective for the following reasons. Rectangle is expected to work as one scaffold in itself; triangle (the most efficient figure that covers a sphere) structures, to gather and work as one big rigid scaffold.<br>
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<br>
<br>
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長方形オリガミの設計は以下の様である。<br>
 
The design of our rectangular DNA origami is as below.<br>
The design of our rectangular DNA origami is as below.<br>
<Img Src="http://openwetware.org/images/6/6e/Outsidefig4.png" Align="left">
<Img Src="http://openwetware.org/images/6/6e/Outsidefig4.png" Align="left">
  <Img Src="http://openwetware.org/images/a/a7/Lipo5.png" Align="right">
  <Img Src="http://openwetware.org/images/a/a7/Lipo5.png" Align="right">
<br>
<br>
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DNAオリガミは、縦67.6nm(26らせん)、横127nm(374塩基)の、長方形である。AFMでの観察時に裏表の区別が付けられるよう、右上で縦10らせん、横161塩基の長方形を切り取った形とした。設計はcaDNAno2で行った。<br>
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We used caDNAno for our DNA origami design.<br>
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さらに、膜が不安定になるよう、このオリガミの中心部分のステイプル141本を、コレステロール付きDNAと結合させ(両親媒性基をもたせ)、膜に突き刺すことが出来るようにした。<br>
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The DNA origami has a rectangle shape of 67.6nm (26 helixes) by 127 nm (374 bases).<br>
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つまり、このコレステロール修飾DNAは、リポソームと足場をつなぐ役割をするだけでなく、膜に突き刺さり、膜を不安定化する両親媒性分子としても働く。<br>
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We cut out a smaller rectangle of 10 helixes by 161 bases at one edge of this origami, so that we could distinguish the two sides during AFM (Atomic Force Microscope) observation.<br>
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We used caDNAno for our DNA origami design. The DNA origami has a rectangle shape of 67.6nm (26 helixes) by 127 nm (374 bases). We cut out a smaller rectangle of 10 helixes by 161 bases at one edge of this origami, so that we could distinguish the two sides during AFM (Atomic Force Microscope) observation. Besides, to destabilize the membrane by inserting this origami, we designed 141 staples at the center of the origami to hybridize with cc DNAs (in the rest of this document, referred to as ccDNA. It gives our origami amphipathicity) and enabled it to insert into the membrane. To sum up, the cc DNA not only connects DNA origami and liposomes but also inserts into the membrane and destabilizes it.<br>
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Besides, to destabilize the membrane by inserting this origami, we designed 141 staples at the center of the origami to hybridize with aptamers (These aptamers give our origami amphipathicity), and enabled it to insert into the membrane. <br>
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To sum up, the aptamer not only connects DNA origami and liposomes but also inserts into the membrane and destabilizes it.<br>
<Img Src="http://openwetware.org/images/c/c5/Outsidefig5.png" Align="right">
<Img Src="http://openwetware.org/images/c/c5/Outsidefig5.png" Align="right">
<br>  
<br>  
<br>
<br>
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<h4>ii)Flower micelle approach</h4><br>
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<h4>Ⅱフラワーミセルによるアプローチ</br>ii)Utilizing flower micelles</h4><br>
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<Img Src="http://openwetware.org/images/1/17/Designflowerflow.png" Align="center" width="900px" ><br>
<Img Src="http://openwetware.org/images/1/17/Designflowerflow.png" Align="center" width="900px" ><br>
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<div align="center">Fig2 process of flower miceles burst</div><br>
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<div align="center">Fig.3 Process of flower micelle approach</div><br>
<Img Src="http://openwetware.org/images/b/b2/Flower1.png" style="height:300px; width:425px; float:right;">
<Img Src="http://openwetware.org/images/b/b2/Flower1.png" style="height:300px; width:425px; float:right;">
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リポソームを割るには、フラワーミセルという方法がある。これはミセルに隙間なくコポリマーによる輪を取り付け、その輪の温度による形状の変化によりミセルに負荷をかけ、割るというものである。<br>
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There is a method called flower micelles for collapsing liposomes. <br>
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There is a method called flower micelles for breaking liposomes. In this method, we cover the surface of the micelles with many copolymer rings, heat and distort the rings, and produce pressure on the micelle and break them.<br>
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In this method, we cover the surface of the micelles with many copolymer rings, heat and distort the rings, and produce pressure on the micelle and collapse them.<br>
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<br>
<br>
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今回はこのフラワーミセルの原理を応用しリポソームとDNAによってリポソームを割ることを試みる。<br>
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We tried to collapse liposomes by applying the basis of flower micelles.<br>
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We tried to break liposomes by applying the basis of flower micelles.<br>
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<br>
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1. First, we mix aptamer (the same strand as used in i) Bending approach), loop strands, and liposomes.<br>
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1.リポソームにコレステロール修飾されているDNA一本鎖ストランドと、これに相補なDNA一本鎖ストランドを加える。このDNAは両端が相補に結合するように設計されているため、リポソーム表面でDNAのループが形成されるようになる。<br>
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The loop strand is designed to have two complementary parts to the aptamers at its both ends. So when it binds to the aptamers, it is expected to make a loop between its both ends. <br>
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1.First, we mixed cc DNAs, loop strands, and liposomes. The loop strand is designed to have two complementary parts to the cc DNAs at its both ends. So when it binds to the cc DNAs, it is expected to make a loop between its both ends. The complex of the cc DNAs and loop strand floats on the surface of the liposomes.<br>
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The complex of the aptamers and loop strand floats on the surface of the liposomes.<br>
<Img Src="http://openwetware.org/images/a/aa/Flower2.png">  
<Img Src="http://openwetware.org/images/a/aa/Flower2.png">  
<br>
<br>
<br>
<br>
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2.次にループを形成しているDNAに相補なトリガーストランドを加える。これとループDNAがハイブリタイゼーションし結合する。<br>
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2. Next, we add complementary trigger strand to the loop strand. The trigger strand hybridizes with the loop strand.<br>
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2.Next, we added complementary trigger strand to the loop strand. The trigger strand hybridizes with the loop strand.<br>
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<br>
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この際DNAが持続長以下の長さに設計してあるため、DNAはまっすぐに保とうとする。<br>
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3. And then the strands keep straight, because we designed the trigger strand shorter than its persistence length.<br>
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And then strands keep straight, because we designed the trigger strand shorter than persistence length.<br>
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<img src="http://openwetware.org/images/0/03/Flower3.png">
<img src="http://openwetware.org/images/0/03/Flower3.png">
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4.その際に生じる力でリポソームに負荷がかかり、リポソームが割れるはずである。<br>
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4.This process gives pressure on the liposome and collapses them.<br>
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4.This process gives pressure on the liposome and breaks them.<br>
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<Img Src="http://openwetware.org/images/3/3b/Flower4.png">
<Img Src="http://openwetware.org/images/3/3b/Flower4.png">
<br>
<br>
<br>
<br>
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リポソーム内部の溶液中にトリガーを入れておくことができるので、Ⅰ・Ⅱを使って、外側からリポソームを破壊し連鎖反応を引き起こすことも容易であると考えられる。<br>
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We consider if some triggers are kept inside the liposomes and the liposomal membrane is broken by the above i) and ii) methods from the outside, it would be much easy to begin the chain reaction. <br>
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We consider if some triggers are kept inside the liposomes and the liposomal membrane is broken by the above and methods from the outside, it would be much easy to begin the chain reaction. <br>
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<br>
<br>
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このシステムで使用するDNAの配列はDNAdesignを使って設計しました。<br>
 
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プログラムのソースはこちら。ループの部分が40nt, 20nt,10ntのDNAを設計しました。<br>
 
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赤色の部分がリポソームから生えているコレステロール付きのDNAと相補になっています。青色の部分が相補的になっていてこれらがハイブリタイゼーションすることでリポソームに負荷がかかり、リポソームが割れます。<br>
 
We designed the DNA sequence for this approach by <A Href="http://www.dna.caltech.edu/DNAdesign/">DNA design</A>, software for designing DNA sequences. <br>
We designed the DNA sequence for this approach by <A Href="http://www.dna.caltech.edu/DNAdesign/">DNA design</A>, software for designing DNA sequences. <br>
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We arranged three kinds of DNA strands that hybridize with the surface of liposomes via cc DNA. Each has 40nt, 20nt, and 10nt loop parts (shown below as blue parts). The blue parts are complementary to the blue trigger strands, and when they hybridize, they place some stress on the liposome and break it. <br>
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We arranged three kinds of DNA strands that hybridize with the surface of liposomes via aptamer. <br>
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The red parts are for hybridizing with liposomes. They are complementary to the cc DNA on the surface of liposomes. The cc DNA is the same as that used in Ⅰ Approach by bending membrane (see <A Href="http://openwetware.org/wiki/Biomod/2013/Sendai/protocol">Protocol</A>). <br>
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Each has 40nt, 20nt, and 10nt loop parts (shown below as blue parts). <br>
 +
The blue parts are complementary to the blue trigger strands, and when they hybridize, they place some stress on the liposome and collapse it. <br>
 +
The red parts are for hybridizing with liposomes. They are complementary to the aptamer on the surface of liposomes. <br>
 +
The aptamer is the same as that used in i)Bending approach.<br>
<font size="-2">
<font size="-2">
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The sequence of cholesterol-conjugated DNA<br>
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Aptamer DNA<br>
<font color="red">CCAGAAGACG</font> -cholesterol<br>
<font color="red">CCAGAAGACG</font> -cholesterol<br>
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A loop is 40nt<br>
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40nt loop DNA<br>
<font color="red">CGTCTTCTGG</font>TTTTTTTTTT<font color="blue">GCGAACCACGGTTCCCAGCGTGACCTTCATGCTTAAGTTT</font><font color="red">CGTCTTCTGG</font><br>
<font color="red">CGTCTTCTGG</font>TTTTTTTTTT<font color="blue">GCGAACCACGGTTCCCAGCGTGACCTTCATGCTTAAGTTT</font><font color="red">CGTCTTCTGG</font><br>
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trigger Strand of coping in loop 40nt<br>
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Trigger DNA for 40 nt loop DNA<br>
<font color="blue">AAACTTAAGCATGAAGGTCACGCTGGGAACCGTGGTTCGC</font><br>
<font color="blue">AAACTTAAGCATGAAGGTCACGCTGGGAACCGTGGTTCGC</font><br>
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A loop is 20nt<br>
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20nt loop DNA<br>
<font color="red">CGTCTTCTGG</font>TTTTTTTTTTTT<font color="blue">CATAACATGAGGCGCCGT</font><font color="red">CGTCTTCTGG</font><br>
<font color="red">CGTCTTCTGG</font>TTTTTTTTTTTT<font color="blue">CATAACATGAGGCGCCGT</font><font color="red">CGTCTTCTGG</font><br>
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trigger Strand of coping in loop 20nt<br>
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Trigger DNA for 20 nt loop DNA<br>
<font color="blue">ACGGCGCCTCATGTTATGAA</font><br>
<font color="blue">ACGGCGCCTCATGTTATGAA</font><br>
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A loop is 10nt<br>
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10nt loop DNA<br>
<font color="red">CGTCTTCTGG</font>TTTTTTTTTT<font color="blue">CTGTAACTAA</font><font color="red">CGTCTTCTGG</font><br>
<font color="red">CGTCTTCTGG</font>TTTTTTTTTT<font color="blue">CTGTAACTAA</font><font color="red">CGTCTTCTGG</font><br>
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trigger Strand of coping in loop 10nt<br>
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Trigger DNA for 10 nt loop DNA<br>
<font color="blue">TTAGTTACAG</font><br>
<font color="blue">TTAGTTACAG</font><br>
</font>
</font>

Revision as of 04:50, 31 August 2013

Biomod2013 Sendai ver2.0

Biomod2013
  Team
Sendai

Design

Egg-type initiator


example-tab2
Fig1 process of Egg-type initiator


Egg-type initiator consists of two layers.
The first layer: “alginate gel membrane”
The alginate gel membrane has a solution phase inside them, and resembles artificial salmon caviars (JINKOH-IKURA in Japanese).

The second layer: “temperature-sensitive liposomes”
The temperature-sensitive liposomes contain PNIPAM lipids in their membrane. Temperature shift causing hydrophobicity change of the PNIPAM induces collapse of the liposomes (See “Characters of the PNIPAM molecular” ). The temperature-sensitive liposomes encapsulate a chelate compound (EGTA) and DNAs.

The two layers realize a dual disruption system as follows (Fig1). The first is “liposome disruption” by increasing temperature. The second is “disruption of alginate membrane” by chelating reagent (EGTA) of calcium released from the liposomes disrupted. This system enables to release many trigger DNAs at a limited point.

1.
The alginate gel membrane encapsulates many temperature-sensitive liposomes.
Please refer following URL.
http://www.sigmaaldrich.com/etc/medialib/docs/SAJ/Brochure/1/j_recipedds2.Par.0001.File.tmp/j_recipedds2.pdf
2.
Warming the temperature-sensitive liposomes from room temperature to over 32 ºC causes disruption of the liposomes.
3.
The liposome disruption release DNAs and EGTA (and urea) which chelate calcium. Lowering calcium concentration starts to melt the alginate gel membrane. During this process, DNA origami is formed when urea is added.
4.
As a result of the melting, trigger DNAs (DNA origami structure or DNA strands for the flower micelle approach (See design: chain-reactive burst )) are released from the melted alginate membrane.
An application to DNA origami formation using the dual disruption system
This dual structure enables to spontaneous formation of DNA origami through environmental stimulation. Three components, Urea (a denature reagent of DNA nanostructure) and materials of DNA origami and EGTA, can be encapsulated in liposomes. It takes times (several hours) to melt the gel membrane by EGTA released from the temperature-sensitive liposomes. Because small molecules can diffuse in alginate gels, urea inside alginate is expected to be gradually diluted during the melting. DNA origami could be formed through the dilution of urea (See “DNA origami formation through urea dilution” ).

※Principle of this system
Alginate gels
Alginate is widely used in foods additives and drug stabilizers. Alginate becomes gel under certain concentrations of calcium, but lowering or chelating calcium melts alginate gels (alginate become sols under the condition).

Characters of the PNIPAM molecular
Hydrophobicity of NIPAM varies at temperatures. NIPAM is hydrophilic at less than 32 ºC, but it become hydrophobic and shrinks at > 32 ºC. Therefore, liposomes containing a modified NIPAM (poly(NIPAM-co-AA-co-ODA) in their membranes become unstable at high temperature (temperature-sensitive liposomes). Consequently, increasing temperature disrupt the liposomes.
Reference
http://www.sigmaaldrich.com/etc/medialib/docs/SAJ/Brochure/1/j_recipedds2.Par.0001.File.tmp/j_recipedds2.pdf

Fig.6 Function of PNIPAM
A schematic image how liposome containing PNIPAM disrupt at high temperature is shown.

DNA origami formation through urea dilution
Polarity of water molecular becomes weak in the presence of urea. Thus, urea interrupts the hydrogen bond of DNA bases. For the reason, the melting point of DNA hybridization decreases. Thus, gradually decreasing the concentration of urea enables to form DNA origami structure under isothermal condition.

Chain-reactive burst



We designed “chain-reactive burst” system as follows.
Each liposome contains triggers and drugs inside, and aptamers for the trigger on its surface. When liposomes are destroyed, new triggers and drugs are released. To achieve liposomal burst by outside triggers, we propose the following two approaches.

  • i) Bending approach
  • ii) Flower micelle approach

  • First, we considered a theory to disrupt liposomes by a trigger DNA signal through calculation. If a liposome is destroyed, its size becomes smaller. We estimated the free energy gap between the two liposomal states: a large liposome and a small one. And discuss which size of liposomes is more stable.
    Please see the details (Go to Calculation).

    i)Bending Approach



    Fig.2 Process of bending approach


    Our bending approach consists of the following four steps.
    1.Cholesterol-conjugated DNA strands (in the rest of this document, referred to as “aptamer”) attaches to the surfaces of liposomes.
    2. Then, DNA origami complementary to the aptamer is added as triggers.
    3. Triggers bind to the surfaces of liposomes and give a load on the membrane.
    4. Due to the load by triggers, liposomes are destroyed.

    a) Mechanism of bending membranes
    To destroy liposomes, we focused on the mechanism the living things use to bend cell membranes. We consider that if we could make use of the mechanism of bending membranes (destabilizing membranes), it would lead to the collapse of membranes. The following three mechanisms have been proposed as of now (Membrane-bending proteins)


    The mechanism A is that amphipathic molecules are inserted into the cell membrane and the bending is caused. The inner hydrophobic part of the lipid bilayer has a strong adhesive power for the two leaflets. Thus, once the amphipathic molecules are inserted into one leaflet of the membrane and expand it, the other leaflet bends according to it, making its surface area smallest.

    The mechanism B is that the molecule attached to the membrane becomes a rigid scaffold and distort the membrane under itself, or stabilize the already bended membrane.

    The mechanism C is that lipid molecules are clustered in one leaflet of the membrane and the inequality of lipid quantity makes the membrane bend.

    Most membrane bending proteins combine the above three mechanisms.
    In addition, a theory that protein crowding causes the bending of cell membranes ( Membrane bending by protein- protein crowding) has recently been suggested. This mechanism is that the collision of membrane proteins produces lateral pressure on membranes and distorts them.

    Due to the above reasons, the efficient design for destabilizing membranes is the structures that :
  • have rigid scaffolds
  • have large surface areas to maximize the effect of the scaffold on the membrane
  • produce a large pressure by collisions

  • b) Rigid scaffolds
    To make rigid scaffolds, we took note of DNA origami, because DNA origami is a method for making rigid structures of any shape. Moreover, we adopted a 2D structure to make the surface area largest.

    We also designed rectangle and triangle to make the pressure of the collision highest.

    We suppose that rectangle and triangle structures are most effective for the following reasons.
    Rectangle is expected to work as one scaffold in itself; triangle (the most efficient figure that covers a sphere) structures, to gather and work as one big rigid scaffold.

    The design of our rectangular DNA origami is as below.

    We used caDNAno for our DNA origami design.
    The DNA origami has a rectangle shape of 67.6nm (26 helixes) by 127 nm (374 bases).
    We cut out a smaller rectangle of 10 helixes by 161 bases at one edge of this origami, so that we could distinguish the two sides during AFM (Atomic Force Microscope) observation.
    Besides, to destabilize the membrane by inserting this origami, we designed 141 staples at the center of the origami to hybridize with aptamers (These aptamers give our origami amphipathicity), and enabled it to insert into the membrane.
    To sum up, the aptamer not only connects DNA origami and liposomes but also inserts into the membrane and destabilizes it.


    ii)Flower micelle approach



    Fig.3 Process of flower micelle approach

    There is a method called flower micelles for collapsing liposomes.
    In this method, we cover the surface of the micelles with many copolymer rings, heat and distort the rings, and produce pressure on the micelle and collapse them.

    We tried to collapse liposomes by applying the basis of flower micelles.

    1. First, we mix aptamer (the same strand as used in i) Bending approach), loop strands, and liposomes.
    The loop strand is designed to have two complementary parts to the aptamers at its both ends. So when it binds to the aptamers, it is expected to make a loop between its both ends.
    The complex of the aptamers and loop strand floats on the surface of the liposomes.


    2. Next, we add complementary trigger strand to the loop strand. The trigger strand hybridizes with the loop strand.

    3. And then the strands keep straight, because we designed the trigger strand shorter than its persistence length.
    4.This process gives pressure on the liposome and collapses them.


    We consider if some triggers are kept inside the liposomes and the liposomal membrane is broken by the above i) and ii) methods from the outside, it would be much easy to begin the chain reaction.

    We designed the DNA sequence for this approach by DNA design, software for designing DNA sequences.
    We arranged three kinds of DNA strands that hybridize with the surface of liposomes via aptamer.
    Each has 40nt, 20nt, and 10nt loop parts (shown below as blue parts).
    The blue parts are complementary to the blue trigger strands, and when they hybridize, they place some stress on the liposome and collapse it.
    The red parts are for hybridizing with liposomes. They are complementary to the aptamer on the surface of liposomes.
    The aptamer is the same as that used in i)Bending approach.
    Aptamer DNA
    CCAGAAGACG -cholesterol
    40nt loop DNA
    CGTCTTCTGGTTTTTTTTTTGCGAACCACGGTTCCCAGCGTGACCTTCATGCTTAAGTTTCGTCTTCTGG
    Trigger DNA for 40 nt loop DNA
    AAACTTAAGCATGAAGGTCACGCTGGGAACCGTGGTTCGC
    20nt loop DNA
    CGTCTTCTGGTTTTTTTTTTTTCATAACATGAGGCGCCGTCGTCTTCTGG
    Trigger DNA for 20 nt loop DNA
    ACGGCGCCTCATGTTATGAA
    10nt loop DNA
    CGTCTTCTGGTTTTTTTTTTCTGTAACTAACGTCTTCTGG
    Trigger DNA for 10 nt loop DNA
    TTAGTTACAG

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