Biomod/2014/Kashiwa/Design: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
No edit summary
No edit summary
 
(223 intermediate revisions by 2 users not shown)
Line 5: Line 5:
<head>
<head>
<style>
<style>
h1.sub {
 
   font-size:20px;
h1.big{
   font-size :24px;
  display: block;
  text-decoration: none;
  color: #000000;
　font-weight:bolder;
  margin: 25px 15px;
  }
 
p.menu {
  font-size :100%;
  line-height: 135%;
  margin: 0px 40px;
}
 
div.imagebox {
  border: 1.7px dashed #2E2EFE; /* 1.枠線 */
  background-color: #F0F8FF;  /* 2.背景色 */
  width: auto; /* 横幅 */
  float: right; /* 右に配置 */
  margin: 0px 10px;
}
}
p.image {
  text-align: center;
  width: auto;
  height: auto;
  margin: 5px;
}
p.caption {
  margin: 5px 10px;
  font-size: 90%;  /* 5.文字サイズ */
  color: black; /* 6.文字色 */
}
</style>
</style>
</head>
</head>


<body>
<body>
<a name="1">&nbsp;</a>
<font face="Futura,Arial,Frutiger" font size="24px">DESIGN</font>
<font face="Futura,Arial,Frutiger" font size="24px">DESIGN</font>
<br>
<br>
<br>
<br>
<p class="paragraph">IIn order to achieve the <a href=Project#3>project goal</a>, we designed two constructs using DNA origami; the Receptor for the sensing system and the Motor-Monomer for the moving system.</p>
<p class="paragraph">
In order to achieve the project goal, we designed two constructs; the Receptor for the sensing system and the Motor-Monomer for the moving system. The Receptor and Motor models were based on the DNA origami structure from the earlier studies (see <a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Trials">Early Trial</a> section). We especially focused to improve the DNA origami structure to create motility upon a signal reception.
<a name="2">&nbsp;</a></p>
 
<h1 class="title"><a name="receptor">&nbsp;The Receptor</a></h1>
 
<p class="paragraph">
The Receptor is developed to be inserted in the liposome with two functions: recognition of an outside signal and activation of the Motor-Monomers inside to start polymerization.
</p>
 
<table align="right">
<td>
<img src="http://openwetware.org/images/c/c5/Project1Kashiwa.png" width="200px" height="225px">
</td>
<td>
<img src="http://openwetware.org/images/c/c9/Project2Kashiwa.png" width="200px" height="225px">
</td>
</table>
 
<p class="paragraph">
For this, we designed the Receptor that consists of the Activator and the Wall surrounding the Activator. The Activator stimulates the Motor-Monomers to polymerize. In the absence of the outside signal, the Wall component, which is connected to the Activator with a linker, physically blocks the Activator to associate with Motor-Monomers. Upon outside signal recognition, the Wall separates from the Activator and the Activator is released into the liposome, resulting the activation of Motor-Monomers (the activation mechanism is described in <a href="#1">the Motor-Monomer</a> part).
</p>
 
<br clear="right">
<br>
<h1 class="big">Requirements</h1>
<p class="paragraph">To develop the Receptor, the following five mechanisms have to be implemented:</p>
 
<p class="paragraph">
<p class="menu">1. Embedding the Wall in the liposome
<p class="menu">2. Linking the Activator to the liposome</p>
<p class="menu">3. Surrounding the Activator with the Wall</p>
<p class="menu">4. Separating the Wall from the Activator upon signal reception</p>
<p class="menu">5. Releasing the Activator into the liposome</p>
</p>
 
<br>
<h1 class="big">Structure (The Wall)</h1>
<img src="http://openwetware.org/images/1/15/WallKashiwa.gif" width="325px" height="309px" align="right">
 
 
<br>
<br>
<h1 class="title"><a name="receptor">&nbsp;The Receptor</a></h1>
<p class="paragraph">The Wall is constructed using DNA origami building block of 18 honeycomb structures. It is composed of the main body and the extension bar. The body part is U-shaped to surround the receptor, and the extension bar part is 16 nm long to penetrate into the liposomal membrane.
<p class="paragraph">The Receptor was developed to recognize an external signal on the liposome surface upon which it releases the Polymerization Imitator inside. For this, we designed a pair of heterounits for the Receptor, the Receptor E and the Receptor A. They dimerize when an external signal is recognized and cause the release of the Polymerization Initiator (In this case, the single-stranded DNA).</p>
</p>


<h1 class="sub">Requirements</h1>
<p class="paragraph">
<p class="paragraph">To develop the Receptor, the following three mechanisms have to be considered  :</p>
The upper ends of the honeycomb in the main body are smooth in order to attach to the liposomal membrane, while the other ends are rough to self-
aggregation of the Wall.
</p>
<br clear="right">


<p class="paragraph">1. Embedding of Receptor on the liposome
<br>
<br>2. Recognization of the external signal
<h1 class="big" clear="both">Strategy</h1>
<br>3. Release of the Polymerization Initiator internally</p>
<p class="paragraph">The details of our strategy for the Receptor to fulfill the five aforementioned requirements are described below.</p>


<h1 class="sub">Structure</h1>
<br>
<p class="paragraph">1. Embedding the Wall in the liposome</p>
<div class="imagebox">
  <p class="image"><img src="http://openwetware.org/images/9/96/Wall.png" width="100px" height="110px"></p>
  <p class="caption">Fig.1. Cholesterol modification<br>of the Wall. Four yellow ellipses<br>show cholesterol-modified staples.</p>
</div>
<p class="paragraph">
The whole Wall structure is so big that it cannot penetrate into the phospholipid bilayers of the liposome. We therefore insert only one honeycomb structure extended from the body part of the Wall into the membrane of liposome. Moreover, the upper side of the Wall is modified with cholesterol to decrease the polarity of the inserted part, supporting the embedding into the liposome.
</p>
<br clear="right">


<h1 class="sub">Strategy</h1>
<p class="paragraph">The details of our strategy to fulfill the requirements are described below.</p>
<br>
<br>
<p class="paragraph">1. Embedding of Receptor on the liposome</p>
<p class="paragraph">2. Linking the Activator to the liposome</p>
<div class="imagebox">
  <p class="image"><img src="http://openwetware.org/images/7/7a/Raft_for_wiki.png" width="200px" height="200px"></p>
  <p class="caption">Fig.2. Structure of the Activator and the Anchor.</p>
</div>
<p class="paragraph">
The Activator is a restriction enzyme Hind&#8546;. Hind&#8546; is an restriction endonuclease that recognizes and cleaves base sequence 5’-AAGCTT-3’. The Activator is associated with the liposome via linkage to the following Anchor embedded in the liposomal membrane.
</p>
<p class="paragraph">
The Anchor is composed of a double-stranded DNA chain and MISTIC protein (Membrane Integrating Sequence for Translation of Integral Membrane Protein Constructs). The reason we chose MISTIC was that it folds into the liposomal membrane easily compared to other membrane proteins<sup>1</sup>. The double-stranded DNA chain links the MISTIC and the Activator.
</p>
<br clear="right">
<br>
<p class="paragraph">3. Connecting the Activator and the Wall</p>
<div class="imagebox">
  <p class="image"><img src="http://openwetware.org/images/f/f1/Bivalent_SA.png" width="150px" height="100px"></p>
  <p class="caption">Fig.3. simplified model of divalent streptavidin.</p>
</div>
<p class="paragraph">
To prevent the Motor-Monomers from associating the Activator before the signal induction, the Activator has to be inactivated by the Wall.</p>
<p class="paragraph">
For this, we bind biotin-modified staple strands to the Activator and the Wall. When divalent streptavidin (mutant of wild type tetravalent streptavidin) is added, the Activator and the Wall are combined by the biotin-streptavidin binding.
</p>
<br clear="right">


<p class="paragraph">The natural Receptor often ends up sticking on the outside of the liposome and rarely penetrates it. Therefore, we modified the Receptor with cholesterol so that it penetrates the liposome and can access the interior.</p>
<br>
<br>
<p class="paragraph">2. Recognization of the external signal. </p>
<p class="paragraph">4. Separating the Wall from the Activator</p>
<div class="imagebox">
  <p class="image"><img src="http://openwetware.org/images/1/1d/Receptormechanism.png" width="400px" height="100px"></p>
  <p class="caption">Fig.4. Separating the Wall from the Activator upon recognition<br> of the outside signal.</p>
</div>
<p class="paragraph">
To activate the polymerization of Motor-Monomers, the Wall has to separate from the Activator upon recognition of the outside signal. Our ultimate goal is to start PoLICe patrolling when it recognizes the cancer markers. However, it is complicated and difficult to use a native cancer marker as a signal. We therefore used a simplified model signal: the restriction enzyme.</p>
 
<p class="paragraph">
When the restriction enzyme is added, it cuts the recognition site on the DNA staple strand that connects the Wall and the Anchor. The Wall consequently separates from the Anchor and the Motor-Monomers are able to associate with the Activator.
</p>
<br clear="right">


<p class="paragraph">We used Thrombin as an external signal in this experiment. Thrombin, a serine protease, has specific sites where different DNA aptamers bind to. We inserted thrombin-binding DNA aptamer sequences into the Receptor heterounits. Furthermore, we bound a staple strand to each heterounit, two strands to be complementary to each other.</p>
<p class="paragraph">Therefore, when the aptamers in the Receptor heterounits bind to the binding sites of thrombin, the heterounits join together to form a dimer. We should keep in mind that we have to optimize the thrombin concentration; an excess of thrombin causes errors because of the Receptor E and the Receptor A binding to the separate thrombin.</p>
<br>
<br>
<p class="paragraph">3. Release of the Polymerization Initiator.</p>
<p class="paragraph">
5. Releasing the Activator in the liposome
</p>
<p class="paragraph">
For efficient interactions between the Activator and the Motor-Monomers, the Activator has to be released inside the liposome because the Motor-Monomers are less likely to access the membrane anchored Activator because of steric hindrance.</p>


<p class="paragraph">We designed the Polymerization Initiator as a single-stranded DNA which is partially complementary to a specific domain of the Receptor E. When the Receptor heterounits form a dimer, the strand displacement occurs to form a more stable DNA duplex and the initially-bound ssDNA is released, which works as the Polymerization Initiator. This strategy, however, has an important flaw; even if there is no thrombin, the Receptors can be close to each other because of lateral diffusion of lipids in the liposome. To overcome this flaw, we are planning to</p>
<p class="paragraph">To release the Activator, we put another restriction enzyme in the liposome. The restriction enzyme cuts the double-stranded DNA chain that links the Activator and the Anchor, and the Activator is therefore released.</p>
<a name="1">&nbsp;</a>
</p>


<a name="2">&nbsp;</a>
<br>
<br>
<br>
<br>
<h1 class="title"><a name="motormonomer">&nbsp;The Motor-Monomer</a></h1>
<h1 class="title"><a name="motormonomer">&nbsp;The Motor-Monomer</a></h1>


<p class="paragraph">The Motor-Monomer was developed to begin the polymerization when it caught the Polymerization Initiator. For this, we designed the Motor-Monomer that has closed rings; when the Polymerization Initiator is caught, the Motor-Monomers join to form the Motor-Polymer by opening their ring structures.</p>
<p class="paragraph">
The Motor-Monomers are developed to start polymerization to form the Motor-Polymer when the Receptor recognizes the outside signal, and consequently deform the liposome. </p>
<div class="imagebox">
<p class="image"><img src="http://openwetware.org/images/4/40/Monomer.png" width="200px" height="110px"></p>
<p class="caption">Fig.5. A rough figure of the Motor-Monomer.<br>Staples with biotin and streptavidin are shown.</p>
</div>


<h1 class="sub">Requirements</h1>
<p class="paragraph">
<p class="paragraph">To develop the Motor-Monomer, the following two requirements have to be considered:</p>
For this, we design a Motor-Monomer with both biotin and streptavidin; biotin at the top part and streptavidin at the middle part. The streptavidin is inactivated and unable to bind biotin before being activated by the Activator. The. The Activator of the Receptor restores the binding capacity and the Motor-Monomers therefore start polymerizing after activation by the Activator.
</p>
<br clear="right">


<p class="paragraph">1. Controllability: the Motor-Monomers should not polymerize without the Initiator.
<br>
<h1 class="big">Requirements</h1>
<p class="paragraph">To develop the Motor-Monomer, the following four requirements have to be considered:</p>


<br>2. Bending stiffness: the Motor-Polymer should be stiff enough to deform the liposome.</p>
<p class="paragraph">
<p class="menu">1. Encapsulating the Motor-Monomers into the liposome</p>
<p class="menu">2. Deactivating the polymerizing activity of the Motor-Monomer</p>
<p class="menu">3. Restoring the polymerizing activity of the Motor-Monomer with the Activator</p>
<p class="menu">4. Forming the Motor-Polymer stiff enough to deform the liposome</p>
</p>


<h1 class="sub">Structure</h1>
<br>
<h1 class="big">Structure</h1>
<img src="http://openwetware.org/images/0/05/Monomer_rot_500.gif" width="500px" height="210px" align="right">
<p class="paragraph">
The Motor-Monomer is constructed using DNA origami building block of six honeycomb structures. It is shaped like fingers pointing, and two honeycomb structures are longer than the others for 65 nm. There is a hole in the middle part of the Motor-Monomer to embed streptavidin. The length of each part is shown in the picture. The ends of the honeycomb structures are rough to prevent aggregation.
</p>
<p class="paragraph">The ends of the honeycomb structures are rough to prevent from cohesion.</p>
<br clear="align">
<br>
<h1 class="big">Strategy</h1>
<p class="paragraph">The details of our strategy to fulfill the requirements are described below.</p>
<br>
<p class="paragraph">1. Encapsulating the Motor-Monomers into the liposome</p>
<p class="paragraph">
If the Motor-Monomers and the Activator are put into the liposome at the same time, the Motor-Monomers are activated before the signal induction. We therefore planned to connect the Activator and the Wall in the liposome first, and then fuse that liposome to another liposome containing the Motor-Monomers.
</p>


<h1 class="sub">Strategy</h1>
<p class="paragraph">The details of our strategy to fulfill the requirements are described below.</p>
<br>
<br>
<p class="paragraph">1.Controllability</p>
<p class="paragraph">
2. Deactivating the polymerizing activity of the Motor-Monomer
</p>
<div class="imagebox">
  <p class="image"><img src="http://openwetware.org/images/3/3e/BiotinKashiwa.png" width="150px" height="100px"><img src="http://openwetware.org/images/5/5e/DesthiobiotinKashiwa.png" width="150px" height="100px"></p>
  <p class="caption">Fig.6. Structural formulas of biotin and desthiobiotin.</p>
</div>
 
<p class="paragraph">
To prevent the Motor-Monomers from polymerizing in inactivated state, despite allowing polymerization in activated state, we used the following approach.
</p>
<p class="paragraph">
We used the difference of the binding strength between the biotin and desthiobiotin to streptavidin. Desthiobiotin is a biotin analogue that binds less tightly to biotin-binding proteins and is easily displaced by biotin*.
</p>
<br clear="right">
<div class="imagebox">
  <p class="image"><img src="http://openwetware.org/images/4/43/Streptavidin-monomer.png" width="300px" height="214px"></p>
  <p class="caption">Fig.7. Connecting deactivated streptavidin <br>to the Motor-Monomer.</p>
</div>
 
<p class="paragraph">
For the inactivation process, two complementally oligonucleotides having biotin or desthiobiotin were hybridized and the resulting duplex were bound with streptavidin. Supported by biotin strand, the desthiobiotin are stably tethered to the streptavidin, giving resistance to biotin displacement.
</p>
<p class="paragraph">
Finally, this inactivated streptavidins were connected to the Motor-Monomers preventing the polymerization before activation. We used alkyne-azide huisgen cycloaddition (known as "click reaction") for connection.
</p>
<br clear="right">


<p class="paragraph">We designed the Motor-Monomer as a ring structure, ssDNAs at both ends of the Monomer are partially complementary to each other. Furthermore, the Motor-Monomers comprise two different constructs which have completely complementary ssDNAs at their ends. In other words, the Motor-Polymer is the heteropolymer. It helps us prevent the Motor-Monomers from polymerizing before building ring structures. </p>
<p class="paragraph">Therefore, when the ssDNA of Initiator is released, the Monomer opens its ring via strand displacement. Then, the free end of the Monomer forms a DNA duplex by a displacement with one end of another Monomer and the other end becomes free. In this way, the Monomers polymerize one by one.</p>
<br>
<br>
<p class="paragraph">2.Bending stiffness</p>
<p class="paragraph">3. Restoring the polymerizing activity of streptavidin with the Activator</p>
<div class="imagebox">
  <p class="image"><img src="http://openwetware.org/images/3/3b/Design_SA-inactivation_ver3_000001.png" width="300px" height="200px"></p>
  <p class="caption">Fig.8. Cutting the DNA duplex by Hind&#8546;.</p>
</div>
<p class="paragraph">
As mentioned above, desthiobiotin-streptavidin binding is supported by stable biotin-avidin binding through the hybridization of DNA duplex. We supposed that by cutting the DNA duplex, desthiobiotin loses its supporter and is easily dissociated from the streptavidn, allowing the binding of biotin on the different Motor-Monomer.
</p>
<p class="paragraph">
Hind&#8546;, a restriction enzyme cleaves the specific DNA sequence, was chosen as the Activator to cut the DNA duplex. After cleavage by Hind&#8546;, the length of the remaining sequence is short, therefore dehybridization of the duplex are supposed to be occurred easily.
</p>
<br clear="right">


<p class="paragraph">Our initial approach was to design three closed rings in the Motor-Monomer to develop enough stiffness by limiting flexibility. Subsequently, we found that three-rings-Monomers are likely to form the branched Polymer, based on probability theory. Therefore, we are trying new approach to develop stiffness with one-ring-Monomers by following strategies:</p>
<br>
<p class="paragraph">4. Forming the Motor-polymer enough to deform the liposome</p>
<div class="imagebox">
  <p class="image"><img src="http://openwetware.org/images/f/fa/Polymer_2patterns.png" width="250px" height="150px"></p>
  <p class="caption">Fig.9. Two different forms of the Polymer.</p>
</div>
<p class="paragraph">
<br>
As described in Structure, the Motor-Monomer has the pointing-finger shape and the hole to embed streptavidin. These features enable the Motor-Monomers to connect with others like Lego blocks, which makes the Motor-Polymer stiffer. There are two types of structures the Motor-Polymer would take as shown in fig.9; both are supposed to be stiff enough.
</p>
<br clear="right">
<div class="imagebox">
  <p class="image"><img src="http://openwetware.org/images/0/0b/Monomerstaples.png" width="175px" height="88px"></p>
  <p class="caption">Fig.10. Single-stranded DNA adhesives<br> on the Motor-Monomer.</p>
</div>
<p class="paragraph">
<br>
Moreover, two single-stranded DNA “adhesives” complementary to each other are bound to different parts on the Motor-Monomer (showed as yellow lines in fig.10). Those adhesives form duplexes with the adhesives on other Motor-Monomers, which strengthen the connection of the Monomers.
</p>
<br clear="right">


<p class="paragraph">  
<br>
<ul><li>By designing the binding site rough, the flexibility should be limited.(参考文献) It also improves the controllability because the binding site becomes more specific.</li>
<br>
<li>By inserting a second strand connector, the binding power should be increased. (図解?参考文献)</li></ul>
<h2 class="reference">Reference</h2>
</p></body>
<p class="reference">1.M. Douglas et al, “A logic-gated nanorobot for targeted transport of molecular payloads”, Science, 2012 Feb 17; 335(6070): 831-4.
<br>2.Science. 2014;344(6179):65-9.Polyhedra self-assembled from DNA tripods and characterized with 3D DNA-PAINT.Iinuma R, Ke Y, Jungmann R, Schlichthaerle T, Woehrstein JB, Yin P.</p>
</body>


<footer style="position:relative; left:600px;">
<footer style="position:relative; left:600px;">

Latest revision as of 02:01, 26 October 2014

<html> <head> <div id="#top"></div>

<script type="text/javascript" src="http://code.jquery.com/jquery-1.9.1.min.js"></script>

<script type="text/javascript"> new Image(120,80) = "http://openwetware.org/images/7/7a/Logo2Kashiwa.png";

new image(120,80) = "http://openwetware.org/images/1/1d/Logo2.5.png";

new image(71,143) = "http://openwetware.org/images/2/2a/Top1.gif";

new image(71,143) = "http://openwetware.org/images/f/f0/Top2.gif";

new image() = "http://openwetware.org/images/3/38/BackgroundKashiwa.png";

new image() = "http://openwetware.org/images/9/9c/BackgroundKashiwa2.png"; </script>


<script type="text/javascript"> 

  $(function(){
     $("#menu li").hover(function(){
        $("ul",this).show();
     },
     function(){
        $("ul",this).hide();
     });
  });

</script>


<script type="text/javascript"> <!-- if (document.images) {

// 設定開始（使用する画像を設定してください）

// 通常の画像 var img0 = new Image(120,80); img0.src = "http://openwetware.org/images/e/ec/LogoKashiwa.png";

// ポイント時の画像1 var img1 = new Image(120,80); img1.src = "http://openwetware.org/images/2/26/Logo3Kashiwa.png";


// 設定終了

} // ポイント時の処理 function On(name) { if (document.images) { document.images['def'].src = eval(name + '.src'); } } // 放した時の処理 function Off() { if (document.images) { document.images['def'].src = img0.src; } } // --> </script>

<META http-equiv="Content-Style-Type" content="text/css">

<style type="text/css">


.page-back { position: fixed; bottom: 25px; right: 25px;}

p.paragraph { 

 font-size :100%;
 line-height: 135%;
 margin: 10px 25px;

}

p.reference { 

 font-size :90%;
 line-height: 135%;
 margin: 10px 20px;

}

p.indent-paragraph { 

 font-size :110%;
 line-height:1.5;
 margin:0 30px;
 text-indent: 1em;
 }

h1.title a{ 

 font-size :100%;
 display: block;
 text-decoration: none;
 color: #000000;

　font-weight:bolder; 

 border-left: solid 5px #e00000;
 }

h2.reference a{ 

 font-size :90%;
 display: block;
 text-decoration: none;
 border-left: solid 5px;
 }

body { 

 font-size: 12px;
 font-family: Frutiger, Helvetica, Arial;
 background-color: #B5D3FF;
 overflow-y:scroll;
 overflow-x:hidden;

} article { 

 background-color: #ffffff

} .container { 

  background-color: #ffffff;
  margin-top:0px

}

.OWWNBcpCurrentDateFilled {display: none;}

}

  1. column-content

{ 

  width: 0px;
  margin: 0;
  padding: 0;
  float:left;

} .firstHeading { 

  display:none;
  width:0px;

}

  1. globalWrapper

{ 

  width:900px;
  background-color: #ffffff;
  margin-left: auto;
  margin-right: auto;

}

  1. column-one

{ 

  display:none;
  width:0px;
  background-color: #ffffff;

}

  1. content

{ 

  margin: 0px;
  align: center;
  padding: 0px 12px;
  width:876px;
  border: 0;

}

  1. bodyContent

{ 

  width: 800px;
  padding: 0px 38px;
  align: center;
  background-color: #ffffff;
  position:relative;

}

  1. column-content

{ 

  width: 900px;
  background-color: #ffffff;

}


  1. footer

{ 

  position: center;
  width: 900px;

}

div.menubar { 

  position: fixed;
  background-color: none; /* バーの背景色 */
  opacity: 0.95;
  border-top: 0px double white; /* バーの上端線 */
  border-bottom: 0px double white; /* バーの下端線 */
  min-width: 900px; /* メインメニュー全部が収まる最低横幅 */
  z-index:3

}

div.menubar ul#menu { 

  margin: -33px 0px 0px -50px; /* メニューバー外側の余白 */
  padding: 0px; /* メニューバー内側の余白 */
  height: 80px; /* メニューバーの高さ */
  list-style-type: none;

}

div.menubar ul#menu li { 

  min-width: 110px; /* メニュー項目の横幅 */
  height: 80px; /* メニュー項目の高さ(「メニューバーの高さ」と一致させる) */ 
  float: left;
  list-style-type: none;
  list-style-image: none;
  position: relative;

}

div.menubar ul#menu a { 

  text-decoration: none; /* メニュー項目の装飾(下線を消す) */
  display: block; 
  background-color: #1F003E;/* メニュー項目の背景色 */
  color: white; /* メニュー項目の文字色 */
  line-height: 80px; /* メニュー項目のリンクの高さ(「メニュー項目の高さ」と一致させる) */
  text-align: center; /* メインメニューの文字列の配置(中央寄せ) */　
  width: 100%;
  height: 100%;

}

div.menubar ul#menu a:hover { 

  background-image: url("http://openwetware.org/images/3/38/BackgroundKashiwa.png"); /* メニュー項目にマウスが載ったときの背景色 */
  color: #8B008B; /* メニュー項目にマウスが載ったときの文字色 */

}

div.menubar ul#menu a:active { 

  background-image: url("http://openwetware.org/images/3/33/Background2.png"); /* メニュー項目にマウスが載ったときの背景色 */
  color: #8B008B; /* メニュー項目をクリックした時の文字色 */

}

/* メニューバー直後のClearfix */ div.menubar ul#menu { 

  zoom:1;

} div.menubar ul#menu:after { 

  height: 0; visibility: hidden; content: "."; display: block; clear: both;

}

div.menubar ul#menu ul.sub { 

  background-color: #1F003E; /* サブメニュー全体の背景色 */
  border-width: 0px 0px 0px 0px; /* サブメニュー全体の枠線の太さ */
  border-style: solid; /* サブメニュー全体の枠線の線種 */
  border-color: #191970; /* サブメニュー全体の枠線の色 */
  margin: 0px;
  padding: 0px;
  display: none;
  position: absolute;

}

div.menubar ul#menu ul.sub li { 

  width: 110px;
  height: 50px; /* サブメニュー1項目の高さ */
  border-width: 0px 0px 0px 0px; /* サブメニュー1項目の枠線の太さ */
  border-style: solid; /* サブメニュー1項目の枠線の線種 */
  border-color: #191970; /* サブメニュー1項目の枠線の色 */
  z-index: 3

}

div.menubar ul#menu ul.sub li a { 

  line-height: 50px; /* サブメニュー1項目の行の高さ(「サブメニュー1項目の高さ」と合わせる) */
  text-align: center;  /* サブメニュー1項目の項目名の配置(中央寄せ) */
  text-indent: 0px;  /* サブメニュー1項目の項目名前方の余白 */

}

div.menubar ul#menu ul.sub li a:hover { 

  background-image: ('back');
  background-size: 100% auto;
  background-color: #FF00FF; /* サブメニュー項目にマウスが載ったときの背景色 */
  color:  #8B008B; /* サブメニュー項目にマウスが載ったときの文字色 */

}

div.menubar ul#menu ul.sub li a:active { 

  background-image: ('backclick') /* メニュー項目にマウスが載ったときの背景色 */
  backround-size: 100% auto;
  color: #8B008B; /* メニュー項目をクリックした時の文字色 */

}

</style>

<div class="menubar"> 

 <ul id="menu">
 <font face="Frutiger,Helvetica,Arial">
   <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa"><img src="http://openwetware.org/images/e/ec/LogoKashiwa.png" onmouseover="this.src='http://openwetware.org/images/7/7a/Logo2Kashiwa.png'" onclick="this.src='http://openwetware.org/images/1/1d/Logo2.5.png'" onmouseout="this.src='http://openwetware.org/images/e/ec/LogoKashiwa.png'" height="80px" width="120px" name="def"></a>
   </li>
   <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Project" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">PROJECT</span></a>
     <ul class="sub">
       <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Project#1" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">Background</span></a></li>
       <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Project#2" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">Motivation</span></a></li>
       <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Project#3" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">Project Goals</span></a></li>
     </ul>
   </li>
   <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Trial" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">&nbsp;EARLY TRIAL&nbsp;</span></a>
     <ul class="sub">
       <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Trial#1" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">Design</span></a></li>
       <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Trial#2" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">Approaches</span></a></li>
     </ul>
   </li>
   <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Design" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">DESIGN</span></a>
     <ul class="sub">
       <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Design#2" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">The Receptor</span></a></li>
       <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Design#1" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">The Motor</span></a></li>
     </ul>
   </li>
   <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Highlights" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">&nbsp;EXPERIMENT&nbsp;&nbsp;</span></a>
     <ul class="sub">
       <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Highlights" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">Highlights</span></a></li>
       <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Receptor" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">The Receptor</span></a></li>
       <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Motor" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">The Motor</span></a></li>
     </ul>
   <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Discussion" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">&nbsp;&nbsp;DISCUSSION&nbsp;</span></a>
     <ul class="sub">
       <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Discussion#1" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">Achievements</span></a></li>
       <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Discussion#2" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">Future</span></a></li>
     </ul>
   </li>
   <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Protocols" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">PROTOCOL</span></a>
   </li>
   <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Team" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">TEAM</span></a>
     <ul class="sub">
       <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Team#1" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">Members</span></a></li>
       <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Team#2" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">Sponsors</span></a></li>
     </ul>
   </li>
 </font>
 </ul>

</div> <br> <br> <br> <div class="page-back"><a href="#top"> <img src="http://openwetware.org/images/2/2a/Top1.gif" onmouseover="this.src='http://openwetware.org/images/f/f0/Top2.gif'" onmouseout="this.src='http://openwetware.org/images/2/2a/Top1.gif'" height="150px"/></a></div> </head>


</html>

<html>

<head> <style>

h1.big{ 

 font-size :24px;
 display: block;
 text-decoration: none;
 color: #000000;

　font-weight:bolder; 

 margin: 25px 15px;
 }

p.menu { 

 font-size :100%;
 line-height: 135%;
 margin: 0px 40px;

}

div.imagebox { 

  border: 1.7px dashed #2E2EFE; /* 1.枠線 */
  background-color: #F0F8FF;  /* 2.背景色 */
  width: auto; /* 横幅 */
  float: right; /* 右に配置 */
  margin: 0px 10px;

} p.image { 

  text-align: center;
  width: auto;
  height: auto;
  margin: 5px;

} p.caption { 

  margin: 5px 10px;
  font-size: 90%;  /* 5.文字サイズ */
  color: black; /* 6.文字色 */

}


</style> </head>

<body> <font face="Futura,Arial,Frutiger" font size="24px">DESIGN</font> <br> <br> <p class="paragraph"> In order to achieve the project goal, we designed two constructs; the Receptor for the sensing system and the Motor-Monomer for the moving system. The Receptor and Motor models were based on the DNA origami structure from the earlier studies (see <a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Trials">Early Trial</a> section). We especially focused to improve the DNA origami structure to create motility upon a signal reception. <a name="2">&nbsp;</a></p>

<h1 class="title"><a name="receptor">&nbsp;The Receptor</a></h1>

<p class="paragraph"> The Receptor is developed to be inserted in the liposome with two functions: recognition of an outside signal and activation of the Motor-Monomers inside to start polymerization. </p>

<table align="right"> <td> <img src="http://openwetware.org/images/c/c5/Project1Kashiwa.png" width="200px" height="225px"> </td> <td> <img src="http://openwetware.org/images/c/c9/Project2Kashiwa.png" width="200px" height="225px"> </td> </table>

<p class="paragraph"> For this, we designed the Receptor that consists of the Activator and the Wall surrounding the Activator. The Activator stimulates the Motor-Monomers to polymerize. In the absence of the outside signal, the Wall component, which is connected to the Activator with a linker, physically blocks the Activator to associate with Motor-Monomers. Upon outside signal recognition, the Wall separates from the Activator and the Activator is released into the liposome, resulting the activation of Motor-Monomers (the activation mechanism is described in <a href="#1">the Motor-Monomer</a> part). </p>

<br clear="right"> <br> <h1 class="big">Requirements</h1> <p class="paragraph">To develop the Receptor, the following five mechanisms have to be implemented:</p>

<p class="paragraph"> <p class="menu">1. Embedding the Wall in the liposome <p class="menu">2. Linking the Activator to the liposome</p> <p class="menu">3. Surrounding the Activator with the Wall</p> <p class="menu">4. Separating the Wall from the Activator upon signal reception</p> <p class="menu">5. Releasing the Activator into the liposome</p> </p>

<br> <h1 class="big">Structure (The Wall)</h1> <img src="http://openwetware.org/images/1/15/WallKashiwa.gif" width="325px" height="309px" align="right">


<br> <p class="paragraph">The Wall is constructed using DNA origami building block of 18 honeycomb structures. It is composed of the main body and the extension bar. The body part is U-shaped to surround the receptor, and the extension bar part is 16 nm long to penetrate into the liposomal membrane. </p>

<p class="paragraph"> The upper ends of the honeycomb in the main body are smooth in order to attach to the liposomal membrane, while the other ends are rough to self- aggregation of the Wall. </p> <br clear="right">

<br> <h1 class="big" clear="both">Strategy</h1> <p class="paragraph">The details of our strategy for the Receptor to fulfill the five aforementioned requirements are described below.</p>

<br> <p class="paragraph">1. Embedding the Wall in the liposome</p> <div class="imagebox"> 

  <p class="image"><img src="http://openwetware.org/images/9/96/Wall.png" width="100px" height="110px"></p>
  <p class="caption">Fig.1. Cholesterol modification<br>of the Wall. Four yellow ellipses<br>show cholesterol-modified staples.</p>

</div> <p class="paragraph"> The whole Wall structure is so big that it cannot penetrate into the phospholipid bilayers of the liposome. We therefore insert only one honeycomb structure extended from the body part of the Wall into the membrane of liposome. Moreover, the upper side of the Wall is modified with cholesterol to decrease the polarity of the inserted part, supporting the embedding into the liposome. </p> <br clear="right">

<br> <p class="paragraph">2. Linking the Activator to the liposome</p> <div class="imagebox"> 

  <p class="image"><img src="http://openwetware.org/images/7/7a/Raft_for_wiki.png" width="200px" height="200px"></p>
  <p class="caption">Fig.2. Structure of the Activator and the Anchor.</p>

</div> <p class="paragraph"> The Activator is a restriction enzyme Hind&#8546;. Hind&#8546; is an restriction endonuclease that recognizes and cleaves base sequence 5’-AAGCTT-3’. The Activator is associated with the liposome via linkage to the following Anchor embedded in the liposomal membrane. </p> <p class="paragraph"> The Anchor is composed of a double-stranded DNA chain and MISTIC protein (Membrane Integrating Sequence for Translation of Integral Membrane Protein Constructs). The reason we chose MISTIC was that it folds into the liposomal membrane easily compared to other membrane proteins<sup>1</sup>. The double-stranded DNA chain links the MISTIC and the Activator. </p> <br clear="right"> <br> <p class="paragraph">3. Connecting the Activator and the Wall</p> <div class="imagebox"> 

  <p class="image"><img src="http://openwetware.org/images/f/f1/Bivalent_SA.png" width="150px" height="100px"></p>
  <p class="caption">Fig.3. simplified model of divalent streptavidin.</p>

</div> <p class="paragraph"> To prevent the Motor-Monomers from associating the Activator before the signal induction, the Activator has to be inactivated by the Wall.</p> <p class="paragraph"> For this, we bind biotin-modified staple strands to the Activator and the Wall. When divalent streptavidin (mutant of wild type tetravalent streptavidin) is added, the Activator and the Wall are combined by the biotin-streptavidin binding. </p> <br clear="right">

<br> <p class="paragraph">4. Separating the Wall from the Activator</p> <div class="imagebox"> 

  <p class="image"><img src="http://openwetware.org/images/1/1d/Receptormechanism.png" width="400px" height="100px"></p>
  <p class="caption">Fig.4. Separating the Wall from the Activator upon recognition<br> of the outside signal.</p>

</div> <p class="paragraph"> To activate the polymerization of Motor-Monomers, the Wall has to separate from the Activator upon recognition of the outside signal. Our ultimate goal is to start PoLICe patrolling when it recognizes the cancer markers. However, it is complicated and difficult to use a native cancer marker as a signal. We therefore used a simplified model signal: the restriction enzyme.</p>

<p class="paragraph"> When the restriction enzyme is added, it cuts the recognition site on the DNA staple strand that connects the Wall and the Anchor. The Wall consequently separates from the Anchor and the Motor-Monomers are able to associate with the Activator. </p> <br clear="right">

<br> <p class="paragraph"> 5. Releasing the Activator in the liposome </p> <p class="paragraph"> For efficient interactions between the Activator and the Motor-Monomers, the Activator has to be released inside the liposome because the Motor-Monomers are less likely to access the membrane anchored Activator because of steric hindrance.</p>

<p class="paragraph">To release the Activator, we put another restriction enzyme in the liposome. The restriction enzyme cuts the double-stranded DNA chain that links the Activator and the Anchor, and the Activator is therefore released.</p> <a name="1">&nbsp;</a> </p>

<br> <br>

<h1 class="title"><a name="motormonomer">&nbsp;The Motor-Monomer</a></h1>

<p class="paragraph"> The Motor-Monomers are developed to start polymerization to form the Motor-Polymer when the Receptor recognizes the outside signal, and consequently deform the liposome. </p> <div class="imagebox"> <p class="image"><img src="http://openwetware.org/images/4/40/Monomer.png" width="200px" height="110px"></p> <p class="caption">Fig.5. A rough figure of the Motor-Monomer.<br>Staples with biotin and streptavidin are shown.</p> </div>

<p class="paragraph"> For this, we design a Motor-Monomer with both biotin and streptavidin; biotin at the top part and streptavidin at the middle part. The streptavidin is inactivated and unable to bind biotin before being activated by the Activator. The. The Activator of the Receptor restores the binding capacity and the Motor-Monomers therefore start polymerizing after activation by the Activator. </p> <br clear="right">

<br> <h1 class="big">Requirements</h1> <p class="paragraph">To develop the Motor-Monomer, the following four requirements have to be considered:</p>

<p class="paragraph"> <p class="menu">1. Encapsulating the Motor-Monomers into the liposome</p> <p class="menu">2. Deactivating the polymerizing activity of the Motor-Monomer</p> <p class="menu">3. Restoring the polymerizing activity of the Motor-Monomer with the Activator</p> <p class="menu">4. Forming the Motor-Polymer stiff enough to deform the liposome</p> </p>

<br> <h1 class="big">Structure</h1> <img src="http://openwetware.org/images/0/05/Monomer_rot_500.gif" width="500px" height="210px" align="right"> <p class="paragraph"> The Motor-Monomer is constructed using DNA origami building block of six honeycomb structures. It is shaped like fingers pointing, and two honeycomb structures are longer than the others for 65 nm. There is a hole in the middle part of the Motor-Monomer to embed streptavidin. The length of each part is shown in the picture. The ends of the honeycomb structures are rough to prevent aggregation. </p> <p class="paragraph">The ends of the honeycomb structures are rough to prevent from cohesion.</p> <br clear="align"> <br> <h1 class="big">Strategy</h1> <p class="paragraph">The details of our strategy to fulfill the requirements are described below.</p> <br> <p class="paragraph">1. Encapsulating the Motor-Monomers into the liposome</p> <p class="paragraph"> If the Motor-Monomers and the Activator are put into the liposome at the same time, the Motor-Monomers are activated before the signal induction. We therefore planned to connect the Activator and the Wall in the liposome first, and then fuse that liposome to another liposome containing the Motor-Monomers. </p>

<br> <p class="paragraph"> 2. Deactivating the polymerizing activity of the Motor-Monomer </p> <div class="imagebox"> 

  <p class="image"><img src="http://openwetware.org/images/3/3e/BiotinKashiwa.png" width="150px" height="100px"><img src="http://openwetware.org/images/5/5e/DesthiobiotinKashiwa.png" width="150px" height="100px"></p>
  <p class="caption">Fig.6. Structural formulas of biotin and desthiobiotin.</p>

</div>

<p class="paragraph"> To prevent the Motor-Monomers from polymerizing in inactivated state, despite allowing polymerization in activated state, we used the following approach. </p> <p class="paragraph"> We used the difference of the binding strength between the biotin and desthiobiotin to streptavidin. Desthiobiotin is a biotin analogue that binds less tightly to biotin-binding proteins and is easily displaced by biotin*. </p> <br clear="right"> <div class="imagebox"> 

  <p class="image"><img src="http://openwetware.org/images/4/43/Streptavidin-monomer.png" width="300px" height="214px"></p>
  <p class="caption">Fig.7. Connecting deactivated streptavidin <br>to the Motor-Monomer.</p>

</div>

<p class="paragraph"> For the inactivation process, two complementally oligonucleotides having biotin or desthiobiotin were hybridized and the resulting duplex were bound with streptavidin. Supported by biotin strand, the desthiobiotin are stably tethered to the streptavidin, giving resistance to biotin displacement. </p> <p class="paragraph"> Finally, this inactivated streptavidins were connected to the Motor-Monomers preventing the polymerization before activation. We used alkyne-azide huisgen cycloaddition (known as "click reaction") for connection. </p> <br clear="right">

<br> <p class="paragraph">3. Restoring the polymerizing activity of streptavidin with the Activator</p> <div class="imagebox"> 

  <p class="image"><img src="http://openwetware.org/images/3/3b/Design_SA-inactivation_ver3_000001.png" width="300px" height="200px"></p>
  <p class="caption">Fig.8. Cutting the DNA duplex by Hind&#8546;.</p>

</div> <p class="paragraph"> As mentioned above, desthiobiotin-streptavidin binding is supported by stable biotin-avidin binding through the hybridization of DNA duplex. We supposed that by cutting the DNA duplex, desthiobiotin loses its supporter and is easily dissociated from the streptavidn, allowing the binding of biotin on the different Motor-Monomer. </p> <p class="paragraph"> Hind&#8546;, a restriction enzyme cleaves the specific DNA sequence, was chosen as the Activator to cut the DNA duplex. After cleavage by Hind&#8546;, the length of the remaining sequence is short, therefore dehybridization of the duplex are supposed to be occurred easily. </p> <br clear="right">

<br> <p class="paragraph">4. Forming the Motor-polymer enough to deform the liposome</p> <div class="imagebox"> 

  <p class="image"><img src="http://openwetware.org/images/f/fa/Polymer_2patterns.png" width="250px" height="150px"></p>
  <p class="caption">Fig.9. Two different forms of the Polymer.</p>

</div> <p class="paragraph"> <br> As described in Structure, the Motor-Monomer has the pointing-finger shape and the hole to embed streptavidin. These features enable the Motor-Monomers to connect with others like Lego blocks, which makes the Motor-Polymer stiffer. There are two types of structures the Motor-Polymer would take as shown in fig.9; both are supposed to be stiff enough. </p> <br clear="right"> <div class="imagebox"> 

  <p class="image"><img src="http://openwetware.org/images/0/0b/Monomerstaples.png" width="175px" height="88px"></p>
  <p class="caption">Fig.10. Single-stranded DNA adhesives<br> on the Motor-Monomer.</p>

</div> <p class="paragraph"> <br> 

Moreover, two single-stranded DNA “adhesives” complementary to each other are bound to different parts on the Motor-Monomer (showed as yellow lines in fig.10). Those adhesives form duplexes with the adhesives on other Motor-Monomers, which strengthen the connection of the Monomers.

</p> <br clear="right">

<br> <br> <h2 class="reference">Reference</h2> <p class="reference">1.M. Douglas et al, “A logic-gated nanorobot for targeted transport of molecular payloads”, Science, 2012 Feb 17; 335(6070): 831-4. <br>2.Science. 2014;344(6179):65-9.Polyhedra self-assembled from DNA tripods and characterized with 3D DNA-PAINT.Iinuma R, Ke Y, Jungmann R, Schlichthaerle T, Woehrstein JB, Yin P.</p> </body>

<footer style="position:relative; left:600px;"> <small> &copy; 2014 UTokyo Chem & Bio </small> </footer>


</html>

Retrieved from "https://openwetware.org/mediawiki/index.php?title=Biomod/2014/Kashiwa/Design&oldid=837722"