Biomod/2014/Kashiwa/Trial

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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>
     <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>
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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>
     <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>
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   <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>
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       <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>
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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/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>
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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>
   </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>
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       <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>
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 </font>
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</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>

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<body> <font face="Futura,Arial,Frutiger" font size="24pt">EARLY TRIAL</font> <br> <br> <p class="paragraph"> In the early stage of our research, we tried a different approach to achieve the project goal. This section shows a summary of the early approach; initial designs and experiments. </p>

<p class="paragraph"> In our early approach, the Receptor consists of a pair of hetero-units and emits a single-stranded DNA by dimerization. The single-stranded DNA works as the Polymerization Initiator, causing the Motor-Monomers to form the Motor-Polymer by ring-opening polymerization. </p>

<p class="paragraph"> The main reasons why we changed our approach were following: <p class="menu"> 1. The stiffness of the Motor-Polymer would not be enough. </p> <p class="menu"> 2. Only one Polymerization Initiator is emitted, which is not enough for certain polymerization. </p> <p class="menu"> 3. Controlling the Polymerization (making a ring structure of the Motor-Monomer) was rather difficult. <a name="1"> </a> </p> </p>

<p class="paragraph"> Please <a href="#1">read more about the design</a>, or <a href="#2">skip to the experiments</a>. </p>

<br> <h1 class="title"><a name="Abstract"> Design</a></h1>

<h1 class="big">The Receptor</h1>

<p class="paragraph"> The Receptor was designed to consist of a pair of hetero-units. They dimerize when an outside signal is recognized and release the Polymerization Initiator (In this case, a single-stranded DNA). </p>

<p class="paragraph"> The Receptor’s structure is shown at right. The Receptor has the following three features. </p> <p class="paragraph"> 1. The Receptor penetrates a liposome by its yellow part. Underside of the blue part is modified with cholesterol to float on the liposome membrane. </p> <p class="paragraph"> 2. The red staple on the top is DNA aptamer sequence which binds to thrombin. Thrombin has two specific binding sites for DNA aptamers. The Receptor hetero-units bind to different sites of thrombin, and therefore they join together to form a dimer via thrombin. </p> <img src="http://openwetware.org/images/3/32/Kashiwareceptor2.jpg" align="right" width="280px" height="120px" style="margin: 5px 25px 5px 10px;"> <p class="paragraph"> 3. The orange staple at the bottom is a single-stranded DNA, which works as the Polymerization Initiator. The green staple is also a single-stranded DNA to bind the Initiator. When the Receptor hetero-units dimerized, the green staple of two hetero-units form a DNA duplex by the strand displacement, the Initiator is consequently released. </p>

<br clear="right"> <h1 class="big">The Motor-Monomer</h1> <p class="paragraph"> The Motor-Monomers were designed to form the Motor-Polymer via ring-opening polymerization. We made two hetero-units of Motor-Monomers which have different DNA staple strands to form stiffer Polymer. (As shown in under figure, the Polymer made of hetero-Monomers get closer to each others.) </p> <center> <img src="http://openwetware.org/images/b/bf/Homoheterokashiwa.png" width="700px" height="100px"> </center>

<br>

<p class="paragraph"> The Motor-Monomer’s structure is shown at right. The Motor-Monomer has the following three features. </p> <p class="paragraph"> 1. The Motor-Monomer is designed to form a ring structure with the brown staple and the green staple, because of partially complementary DNA sequences. </p> <p class="paragraph"> 2. Similar color staples of different Motor-Monomers (green and yellowish green, brown and orange) are completely complementary to each others. The green staple is also completely complementary to the Polymerization Initiator. When the Polymerization Initiator is released, the strand displacement occurs successively and therefore the Polymer forms. </p> <img src="http://openwetware.org/images/5/5f/Coverstaplekashiwa.png" width="200px" height="150px" align="right" style="margin: 5px 25px 5px 10px;"> <p class="paragraph"> 3. There is the difficulty in forming the ring structure; the persistence length of the single-stranded DNA is only a few nano-meters while the length of the Motor-Monomer is 34 nano-meters. To solve the problem, we decided to connect the brown staple and the green staple before they are bound to the Motor-Monomer. Short single-stranded DNAs then cover the staples to make the staples double-stranded, increasing the persistence length to about 50 nano-meters. Finally, the double-stranded DNA is bound to the Motor-Monomer and the cover staples are dissociated by raising temperature to their melting temperature. <a name="2"> </a></p>

<br> <br> <h1 class="title"><a name="Experiments"> Experiments</a></h1> <p class="paragraph"> In the research for one month, we succeeded in folding the Receptor and the Motor-Monomer, confirming the binding ability of thrombin-aptamers, emitting the Polymerization Initiator by chain displacement, embedding the Receptor in a liposome, and forming the short Motor-Polymer. </p> <p class="paragraph"> This page shows some highlights selected from the whole experiments. </p>

<h1 class="big"> Folding the Receptor and the Motor-Monomer </h1>

<div class="right"><img src="http://openwetware.org/images/2/22/OCKtem.png"></div> <p class="paragraph"> The Receptor’s structure was clearly appeared in the TEM image. </p> <br clear="right"> <div class="right"><img src="http://openwetware.org/images/a/a4/Koala_001.jpg"></div> <p class="paragraph"> As for the Motor-Monomer, the folding was verified by agarose gel electrophoresis analysis but not by the TEM. We considered it is because the hollow structure of the Monomer cannot be seen with the TEM. </p>

<br clear="right"> <h1 class="big"> Forming the Motor-Polymer </h1>

<p class="paragraph"> In this experiment, two different Motor-Monomers were prepared; one has one arm and the other has three. They were used to examine an effect of the number of the arms on polymerization. </p>

<br clear="left"> <p class="name"> MoX and MoY: two hetero-units of the Motor-Monomers that make the Polymer together. </p> <p class="name"> MoN: the Motor-Monomer with no arm. </p>

<p class="paragraph"> Formation of the Motor-Polymer was checked by agarose gel analysis and the ratio of the Motor-Dimers was shown in the bar chart. </p> <p class="paragraph"> The Motor-Monomers with three arms show a positive correlation between the ratio of Monomer-dimers and the Monomers concentration while the Motor-Monomers with one arm don’t show such a correlation. It indicates that the polymerization occurs more surely in the Motor-Monomers with three arms. </p> <p class="paragraph"> This analysis, however, cannot rule out the possibility that the bands caused by cohesion of the Monomers. It is necessary to corroborate the formation of the Motor-Polymer with a TEM by changing its hollow structure. </p> <br> <br> </body>

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Biomod/2014/Kashiwa/Trial

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