Biomod/2013/Sendai/design: Difference between revisions

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<img src="http://openwetware.org/images/a/af/NIPAM%E3%83%AA%E3%83%9D%E3%81%A1%E3%82%83%E3%82%93.png"></div><br>
<img src="http://openwetware.org/images/2/24/NIpam-%E3%83%AA%E3%83%9D%E3%81%A1%E3%82%83%E3%82%93%EF%BC%92.png"></div><br>
Fig.3 A schematic image how liposome containing PNIPAM collapsed at high temperature is shown.<br><br>図、親水性→疎水性が分かるように文字を入れる<br>
Fig.3 A schematic image how liposome containing PNIPAM collapsed at high temperature is shown.<br><br>図、親水性→疎水性が分かるように文字を入れる<br>


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

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        <h2>Design</h2>

<table id="toc" class="toc" summary="Contents"><tr><td><div id="toctitle"><h2>Contents</h2></div> <ul> <li class="toclevel-1"><a href="#chain"> <span class="tocnumber">1</span> <span class="toctext">Our Target(Lipo HANABI)</span></a></li> <li class="toclevel-1"><a href="#bending"> <span class="tocnumber">2</span> <span class="toctext">How to break</span></a></li> <ul> <li class="toclevel-2"><a href="#Flower"> <span class="tocnumber">2-1</span> <span class="toctext">step1 温度感受性リポソームの破壊</span></a></li> <li class="toclevel-2"><a href="#sensing"> <span class="tocnumber">2-2</span> <span class="toctext">step2 DNAによる連鎖的リポソームの破壊</span></a></li> <ul> <li class="toclevel-2"><a href="#5"> <span class="tocnumber">2-2-1</span> <span class="toctext">DNAオリガミによるアプローチ</span></a></li> <li class="toclevel-2"><a href="#6"> <span class="tocnumber">2-2-2</span> <span class="toctext">anchored DNAによるアプローチ</span></a></li> </li>


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


<h3 id=chain>1. Our target(Lipo-HANABI) </h3> 今回のプロジェクトでは生物的な分子放出システム構築の基本的な例として次の２段階の分子放出システムの構築を目指す。 温度感受性リポソームを使って温度上昇を感知し、その内部の反応を開始するDNAを放出する。（１段階目）そして、そのDNAがもととなって、温度感受性リポソーム周辺の、内部に同じDNAをもつリポソームが連鎖的に割れていく（２段階目）という系だ。<br> この系は一点から周囲へとリポソームの破壊が連鎖的に広がっていく系であるため、その様子が日本のHANABI（Japanese Firework）に似ていることから私たちはこの系をLipo-HANABIと呼ぶことにした。<br>

このように２段階にするメリットは、１種類目のリポソームが特定の刺激（ここでは温度上昇）に対して反応するように作ることができれば、２種類目のリポソームは同じものでも連鎖反応が起こせることである。そうすることで増幅率や、耐ノイズ性を２段階目で保証しやすい。異なる刺激に反応するシステム（例えば光やpHなど）をつくるには、１段階目だけ設計しなおせばよい。<br> 今回のプロジェクトで第一段階目の入力として、「温度」を用いる理由は、比較的条件の設定が簡便であり、局所的な加熱が可能であり、（顕微鏡下でスポットを加熱することができる）温熱療法など、生体組織でも使われており、本システムを例えばDDSとして利用する場合には入力として使えると考えたためである。<br>

このLipo-HANABIというシステムを実現するために私たちは温度感受性リポソーム（for１段階目）とDNAによって破壊することができるリポソーム（for２段階目）の２種類をつくり、１種類目のリポソームが外部刺激（温度上昇）により壊れると、２種類目のリポソームを壊す鍵DNAが放出される。２種類目のリポソームにはそれ自身を壊す鍵DNAが入っているため、連鎖的にリポソームが壊れ、大量の内容分子と鍵DNAが２種類目のリポソームの連鎖的な破壊により放出される。<br><br> In our project, we aim to construct a molecular releasing system as a basic example of biological one. We designed the system as follows. <br> (1)A temperature-sensitive liposome sense increase in temperature and release DNA to start the next reaction. <br> (2)The first DNA chain-reactively collapse liposomes which have the same DNA sequences in its inside. <br> In this system, the collapse of liposome prevail from one point to surround chain-reactively. So we call this system Lipo-HANABI because the shape of this system is look like Hanabi, Japanese fireworks. <br> The merit of constructing two phases is to use the same type of liposome in phase (2) when we would like to take chain reaction. <br> The above system confirm amplification factor and prevention of noise. When we would like to change what the system senses, we have only to make the first phase liposome. The reason to select temperature as the first phase input is as follows. <br> (1)We can easily make the condition. <br> (2)We can heat locally in using microscope. <br> (3)Heat is used in our body tissue and the system is likely to be used as input when we apply to DDS. <br> To realize the Lipo-HANABI system, we have to make temperature-sensitive liposome and the other one to be collapsed by DNA. <br> The first liposome is collapsed by rising temperature. It releases DNA to collapse the other ones. <br> There are DNA to collapse the same kind of ones in the liposomes. So we can take chain-reactive collapse of liposome. <br>


<h3 id=bending>2. How to collapse liposomes</h3>

このシステムで最も重要なのは、まず特定の刺激に対してリポソームを壊し、DNAによるリポソームの破壊が連鎖的に起こることである。<br> １段階目は温度で不安定化するリポソームを用いる。(温度感受性リポソーム) <br> ２段階目は、一段階目のリポソームに内包されていた鍵DNAで不安定化されるリポソームを用いる。この２段階目のリポソームに、鍵DNA と一緒にpayloadを加えることで分子の放出が行える。しかもそれは指数関数的に分子の濃度が増加していく放出である。<br><br> The most important point for this project is to collapse liposome from a specific stimulus and take chain-reactive collapse of liposomes. We made liposomes to be unstable in heating them. We call this liposome “the first liposomes”. (temperature-sensitive liposome) We used liposomes to be unstable when they are attached to DNA which were confined in the first-phase liposomes.We call this liposome "the first liposome". We can release molecules by confining DNA and payload in the second- phase liposome. Payload means the materials to function in the specific place. In addition, this system raise the solution of the molecules exponentially.


<h4 id=Flower>2-1）Step1 The collapse of temperature-senstive loposomes</h4> We used temperature-sensitive liposomes in the first phase. Warming the temperature-sensitive liposomes causes collapse of the liposomes. We used NIPAM to collapse liposome. Nipam is a polymer and there are various kind of NIPAM. we selected PNIPAM because it can be a switch because it is hydrophobic by raising temperature. Characters of the PNIPAM molecular are as below.<br> 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 collapse the liposomes.<br> Reference( <a href= "http://www.sigmaaldrich.com/etc/medialib/docs/SAJ/Brochure/1/j_recipedds2.Par.0001.File.tmp/j_recipedds2.pdf">pdf</a>) <br>


<div align="center"> <img src="http://openwetware.org/images/2/24/NIpam-%E3%83%AA%E3%83%9D%E3%81%A1%E3%82%83%E3%82%93%EF%BC%92.png"></div><br> Fig.3 A schematic image how liposome containing PNIPAM collapsed at high temperature is shown.<br><br>図、親水性→疎水性が分かるように文字を入れる<br>


<h4 id=sensing>2-2）Step2 DNAによる連鎖的リポソームの破壊 <br>The chain-reactive collaption by DNA </h4> Each liposome in 2nd phase contains DNA strand and payload inside. We call this DNA strand "key DNA". On its surface, there are anchored DNA.Anchored DNA mean the ones attached to liposome by cholesterol modification. When liposomes are collapsed, new key DNA and payload are released. To achieve the burst of liposome by key DNA outside, we propose the following two approaches: DNA origami approach and anchored DNA approach.<br>


<h5 id=5>2-2-1) DNA origami approach</h5> <h6>The abstract of this approach</h6>


一段目から放出される鍵分子はDNAオリガミでそのDNAオリガミにより2段目のリポソーム表面に大量のDNAオリガミを吸着させ、リポソーム膜面に「曲げ」ストレスを与えることにより、リポソームを壊す。<br><br> In this approach, the key DNA refer to DNA origami. A large amount of key DNA attach to the surface of the second-phase liposomes. Liposomes are globe, however, DNA origami is plane surface. So DNA origami put a load on a liposome and they collapse it.<br>


<Img Src="http://openwetware.org/images/e/ed/%E3%83%AA%E3%83%9D%E3%81%A1%E3%82%83%E3%82%93%EF%BC%91%EF%BC%93.jpg" ><br>

リポソームを作製した後にコレステロール修飾したDNAをリポソームに振り掛けて、DNAオリガミに足をつける。こうすることでリポソームの内側にコレステロール修飾したDNAが生えないようにして、リポソームにDNAオリガミをつけることができる。<br><br> After making liposome, we mixed liposomes modified by cholesterol with DNA.　In addition, we attached strand to DNA origami. In this method, we can put DNA strand put liposome only outside of the liposomes and we can attach DNA origami to liposomes.<br>

<Img Src="http://openwetware.org/images/c/ce/%E3%83%AA%E3%83%9D%E3%81%A1%E3%82%83%E3%82%93%E2%91%A7.png" Align="center" width="900px" hight="800" >

<div align="center">Fig.1 Process of bending approach</div><br> <br> Three steps to collapse liposomes by DNA origami<br> 1. DNA origami with strand attach to liposome modified by complementary strand to DNA origami's leg.<br> 2. Triggers bind to the surfaces of liposomes and give a load on the membrane.<br> 3. Due to the load by DNA origami,liposomes are collapsed.<br> <br>


<h6>このアプローチの原理的根拠<br>The fundamental reason of the approach</h6>


We have to design the most suited DNA origami to collapse liposomes. 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>

<div class="caption-left"> 

<Img Src="http://openwetware.org/images/a/ae/Designfig2.png" width="280px" height="400px">

<span>Fig.2 Mechanism of bending membranes</span></div> <br> 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> <br> 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> 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> <div class="c-both"></div>

Most membrane bending proteins combine the above three mechanisms.<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> <br>

<h6>このアプローチのDNAデザイン<br>The DNA design of the approach</h6>


Due to the above reasons, the efficient design for destabilizing membranes is the structures that :<br> <ur><li>have rigid scaffolds</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> <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> <br>

We also designed rectangle and triangle to make the pressure of the collision highest.<br> <Img Src="http://openwetware.org/images/4/49/%E3%83%AA%E3%83%9D%E3%81%A1%E3%82%83%E3%82%93%EF%BC%91%EF%BC%92.png"> <div align="center">Fig.3 Rectangle origami</div><br> We suppose that rectangle and triangle structures are most effective for the following reasons. <br> 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> <br> The design of our rectangular DNA origami is as below.<br> <Img Src="http://openwetware.org/images/4/45/Outsidefig8.png"> <div align="center">Fig.4 Rectangular origami</div> <br> <div class="caption-right"> 

<Img Src="http://openwetware.org/images/a/a7/Lipo5.png" ><span>Fig.5 DNA origami designed by caDNAno</span>

</div> We used <A Href="http://cadnano.org/">caDNAno2</A> for our DNA origami design.<br> The DNA origami has a rectangle shape of 67.6nm (26 helixes) by 127 nm (374 bases).<br> 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> 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> <div class="c-both"></div> 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/3/37/%E3%83%AA%E3%83%9D%E3%81%A1%E3%82%83%E3%82%93%EF%BC%93.png"> <div align="center">Fig.6 Unstable liposome</div> <br>


<h5 id=6>2-2-2) Flower DNAによるアプローチ</h5> <h6>このアプローチの概要<br>The abstraction of the approach</h6>


このアプローチでは鍵DNAはDNA鎖であり、我々はDNAの持続長の変化を利用した。DNAはハイブリダイゼーションして２本鎖になると持続長が長くなる。具体的な方法は次の通りである。一段階目の温度感受性リポソームから放出されるDNAストランドはそのDNAストランドは二段階目のリポソーム表面に埋め込んであるフラワーDNAにハイブリダイゼーションする。フラワーDNAはコレステロール修飾されてリポソームの表面にあるDNAのことである。ハイブリダイゼーションによって持続長が長くなったフラワーDNAはリポソーム膜面に「引っ張り（引き裂き）」ストレスを与え、リポソームを壊すのである。<br><br> In this approach, the key DNA refer to DNA strands and we used the change of persistence length of DNA. The persistence length of DNA become longer when it hybridize. The specific way is as bellow. The DNA strands released from the first-phase liposomes hybridize flower DNA on the surface of liposome.　Flower DNA refer to the ones conjugated on liposome with cholesterol. Flower DNA, whose persistence length get longer by hybridization, claw the membrane of the liposome and cause a collapse of liposome.<br>


<Img Src="http://openwetware.org/images/8/8b/Flower-new.png" Align="center" ><br>


<Img Src="http://openwetware.org/images/6/69/%E3%83%AA%E3%83%9D%E3%81%A1%E3%82%83%E3%82%93%EF%BC%92.png" Align="center" width="900px" hight="800" ><br> <div align="center">Fig.10 How to straighten loop</div>

1. We add a Key DNA strand corresponding (complementary) to the loop strand on the liposome. The Key DNA strand hybridizes Flower DNA strand, making it change to be straight.<br> 2. The double-stranded part keeps straight (though it was originally a flower DNA), because the Key DNA strand is designed to be shorter than its persistence length.<br> 3. In this process, some stress is placed on the liposome, and it is collapsed.<br> <br>


<img src="http://openwetware.org/images/0/03/Flower3.png"><br> <div align="center">Fig.7 Process of flower micelle approach</div><br><br>


<h6>このアプローチの原理的根拠<br>The fundamental reason of the approach</h6>


<div <!--class="caption-right"-->> <Img Src="http://openwetware.org/images/e/ed/%E3%83%AA%E3%83%9D%E3%81%A1%E3%82%83%E3%82%93%E2%91%A5.png" style="width:425px;"><br> <span>Fig.8 Flower micelle method</span></div>


There is a method called <a href="http://pubs.acs.org/doi/ipdf/10.1021/jp104711q">flower micelles</a>.<br> In this method, Many copolymer rings cover the surfaces of liposomes. The rings can be distorted by heating, place some stress on the liposomes, and collapse them.<br>


<h6>このアプローチのDNAデザイン<br>The design of the approach</h6>



We tried to collapse liposomes by applying the mechanism of flower micelles.<br> フラワーミセルをリポソームに応用するためには、<br> ・多くのコレ付きDNAを表面に埋め込むこと<br> ・鍵DNAのハイブリによってDNAの性質が変化すること<br> といった要素が重要になってくる。<br><br> To apply the basis of flower micelles to the liposomes<br> ・attach many DNA conjugated by cholesterol to the surface of the liposome<br> ・change the behavior of DNA　by hybridizing Key DNA<br> To meet above conditions,we designed proper placement of DNA sequence.<br> <div class="c-both"></div> <br>


We designed the DNA sequences for this approach by <A Href="http://www.dna.caltech.edu/DNAdesign/">DNA design</A>, software for designing DNA sequences. <br> <br> 私たちは3種類のDNAを用意しました。Anchored DNAとFlower DNA、Key DNAです。それぞれのDNAは50塩基対と10塩基対、40塩基対です。詳しい配列は下の表にあります。<br> 最初に、Flower DNA をつくるために、私たちは50塩基対と10塩基対の鎖をアニーリングしました。それから、私たちはフラワーDNA、リポソームを混成しました。この状態では、リポソームは上の絵に示しているとおりDNAが修飾されていて花のようになっています。Flower DNAには２本鎖のほかに１本鎖のところがあり、持続長は短いのでリポソームには負荷が掛かっていません。アンカーDNAはコレステロール修飾されており、リポソームに対して高い親和性を有しています。アンカーDNAはリポソームの上を浮動しています。<br>最初のリポソームによって放出されたKey　DNAがFlower DNA にハイブリダイゼーションするとき、DNAは持続長が長くなるのでリポソームに負荷をかけます。最終的に、フラワーDNAはリポソームを割るのです。<br><br>

We arranged three kinds of DNA: flower DNA, key DNA and Each DNA strand has 50nt, 10nt and 40nt. There is a list of the each DNA sequence below.<br> First, to make flower　DNA, we annealed 50nt DNA and 10nt one. Then we mixed Flower DNA with liposome. In this state, liposomes look like flowers as we show the above picture. There are double strand point and single strand point in the Flower DNA. At this point, Flower DNA don’t put a load on liposomes because the persistence length of single-strand DNA is short. 

Flower DNA are conjugated by cholesterol and have high affinity for liposome. They float on a liposome. When Key DNA from the first-phase DNA hybridize Flower DNA, Flower DNA put a load on liposome because the persistence length become longer. Finally,　Flower DNA　can collapse liposome.<br><br>


<br> <table border cellspacing="3" bgcolor="lightyellow"> <tr bgcolor="lightyellow"> <td> The kinds of DNA strands </td> <td> Its sequence </td> </tr> <tr bgcolor="moccasin"> <td> Aptamer DNA </td> <td> CCAGAAGACG -cholesterol </td> </tr> <tr bgcolor="moccasin"> <td> 40nt loop DNA </td> <td> CGTCTTCTGGTTTTTTTTTTGCGAACCACGGTT<br>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;CCCAGCGTGACCTTCATGCTTAAGTTTCGTCTTCTGG </td> </tr> <tr bgcolor="moccasin"> <td> Trigger DNA for 40 nt loop DNA </td> <td> AAACTTAAGCATGAAGGTCACGCTGGGAACCGTGGTTCGC </td> </tr> <tr bgcolor="moccasin"> <td> 20nt loop DNA </td> <td> CGTCTTCTGGTTTTTTTTTTTTCATAACATGAGGCGCCGTCGTCTTCTGG </td> </tr> <tr bgcolor="moccasin"> <td> Trigger DNA for 20 nt loop DNA </td> <td> ACGGCGCCTCATGTTATGAA </td> </tr> <tr bgcolor="moccasin"> <td> 10nt loop DNA </td> <td> CGTCTTCTGGTTTTTTTTTTCTGTAACTAACGTCTTCTGG </td> </tr> <tr bgcolor="moccasin"> <td> Trigger DNA for 10 nt loop DNA </td> <td> TTAGTTACAG </td> </tr> </table>

<br> <br> 


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