Biomod/2014/Kashiwa/Design

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   <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>
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   <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Project" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">PROJECT</span></a>
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       <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>
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   <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>
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       <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>
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   <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Design" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">DESIGN</span></a>
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       <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>
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       <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>
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   <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>
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   <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Protocols" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">PROTOCOL</span></a>
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<body> <a name="1">&nbsp;</a> <font face="Futura,Arial,Frutiger" font size="24px">DESIGN</font> <br> <br> <p class="paragraph">In order to achieve the <a href=Biomod/2014/Kashiwa/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> <br> <h1 class="title"><a name="receptor">&nbsp;The Receptor</a></h1> <p class="paragraph">The Receptor was developed to recognize an outside signal on the liposome so that it emits the Polymerization Imitator inside. For this, we designed a pair of heterounits of the Receptor, the Receptor E and the Receptor A. They dimerize when an outside signal is recognized and the Polymerization Initiator (In this case, the single-stranded DNA) emits due to the emission mechanism.</p>

<h1 class="sub">Requirements</h1> <p class="paragraph">To develop the Receptor, the following three mechanisms have to be considered :</p>

<br>1. Penetration to the liposome <br>2. Recognization of the outside signal <br>3. Emittion of the Polymerization Initiator</p>

<h1 class="sub">Structure</h1>

<h1 class="sub">Strategy</h1> <p class="paragraph">The details of our strategy to fulfill the requirements are described below.</p> 1. Penetration to the liposome <p class="paragraph">The natural Receptor often ends up sticking to the liposome and rarely penetrates it. Therefore, we modified the Receptor with cholesterol so that it penetrates the liposome.</p>

2. Recognization of the outside signal. <p class="paragraph">We used Thrombin as an outside 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. 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>

3. Emittion of the Polymerization Initiator. <p class="paragraph">We designed the Polymerization Initiator as a single-stranded DNA which is partially complementary to a specific domain of the Receoter 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> <a name="2">&nbsp;</a> <br> <br> <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>

<h1 class="sub">Requirements</h1> <p class="paragraph">To develop the Motor-Monomer, the following two requirements have to be considered:</p> <p class="paragraph">1. Controllability: the Motor-Monomers should not polymerize without the Initiator. <br>2. Bending stiffness: the Motor-Polymer should be stiff enough to deform the liposome.</p>

<h1 class="sub">Structure</h1>

<h1 class="sub">Strategy</h1> <p class="paragraph">The details of our strategy to fulfill the requirements are described below.</p> 1. Controllability <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 help us to prevent the Motor-Monomers from polymerizing before building ring structures.</p> <p class="paragraph">Therefore, when the ssDNA of Initiator is released, the Monomer open its ring by a 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>

2. Bending stiffness <p class="paragraph">Our initial approach was to design three closed rings in the Motor-Monomer to develop enough stiffness by limiting flexibility. Later on, however, we found that three-rings-Monomers are likely to form the brached Polymer, based on probability theory. Therefore, we are trying new approach to develop stiffness with one-ring-Monomers by following strategies:</p>

<p class="paragraph"> <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> <li>By inserting a second strand connector, the binding power should be increased. (図解？参考文献)</li> </p></body>

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