Biomod/2013/Todai/Result: Difference between revisions

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<!--Method-->
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     <div class="zairyou-heading">[Method]</div>
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       <p class="paragraph>
       <p class="paragraph">
We first measured the Cu-free click reaction in solution (without no
We first measured the Cu-free click reaction in solution (without no
catalyst nor accelerator). The association time at 2 uM oligonucleotide
catalyst nor accelerator). The association time at 2 uM oligonucleotide
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8.1 [1/M/s].
8.1 [1/M/s].
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</p>
</p>
               <div class="res-conclusion">
               <div class="res-conclusion">
               --> Optimum concentration of CuSO<sub>4</sub>: 625 uM
               --> Cu-free click reaction has suitable character for specific oligomerization.
     </div>
     </div>
      
      

Revision as of 22:46, 26 October 2013

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<div class="sidebar"> 

 <ul>
    <li><a href="http://openwetware.org/wiki/Biomod/2013/Todai">Home</a>
    </li>
    <li><a href="http://openwetware.org/wiki/Biomod/2013/Todai/Project">Project</a>
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    <li><a href="http://openwetware.org/wiki/Biomod/2013/Todai/Design">Design</a>
    </li>
    <li><a href="http://openwetware.org/wiki/Biomod/2013/Todai/Result">Result</a>
    </li>
    <li><a href="http://openwetware.org/wiki/Biomod/2013/Todai/Experiment">Experiment</a>
       <ul style="list-style-type: none;">

<li> 

          <a href="http://openwetware.org/wiki/Biomod/2013/Todai/Experiment#Contents">
          Contents</a></li>
          <li>
          <a href="http://openwetware.org/wiki/Biomod/2013/Todai/Experiment#PilotStudy">
          Pilot Study</a></li>
          <li>
          <a href="http://openwetware.org/wiki/Biomod/2013/Todai/Experiment#Protocols">
          Protocols</a></li>
       </ul>
    </li>
    <li><a href="http://openwetware.org/wiki/Biomod/2013/Todai/Team">Team</a>
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<html> <head> <title>Result-Todai nanORFEVRE-</title> <style>

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<body>

<!--◆◆Result◆◆--> 

  <h1 class="big-title"><a name="Result">&nbsp;Result</a></h1>
      <figure>
       <center>
        <img src="

http://openwetware.org/images/1/15/Todai_Projectsteps_map.png" width=720px height=480px> 

       </center>
      </figure>

  <h1 class="heading"><a name="Contents">&nbsp;Contents</a></h1>

<ul> <li><h3><a href="#STEP1">STEP 1: DNA strands assemble to form designed structures</a></h3> <ul style="list-style: none;"> <li><a href="#Optimize_the_condition_to_assemble_OCK">1)Optimize the condition to assemble OCK</a></li> <li><a href="#Conformation_of_the_3D_structure_of_OCK_by_TEM">2)TEM imaging of the 3D structure of OCK</a></li></ul> </li> <li><h3><a href="#STEP2">STEP 2: Penetration into the membrane</a></h3> <ul style="list-style: none;"> <li><a href="#Flotation_assay">1)Flotation asssay</a></li> <li><a href="#Preparation_of_GUVs">2)Preparation of GUVs</a></li> </ul> </li> <li><h3><a href="#STEP3">STEP 3: Recognition of cancer-specific proteins</a></h3> <ul style="list-style:none;"> <li><a href="Optimization_of_aptamer_lock_system">1) Optimization of aptamer-lock system</a> </li> <li><a href="Embedding_of_recognition_system_to_OCK">2) Embedding of recognition system to OCK</a></li> </ul> </li> <li><h3><a href="#STEP4">STEP 4: Oligomerization in solution</a></h3> <ul style="list-style: none;"> <li><a href="#Oligomerization_by_streptavidin-biotin_complex">1)Oligomerization by streptavidin-biotin complex</a></li> <li><a href="#Tem_imaging_of_OCK_dimers_by_streptavidin-biotin_complex">2)TEM imaging of OCK dimers connected by streptavidin-biotin interaction</a></li> <li><a href="#Oligomerization_by_Click_reaction">3)Oligomerization by Click reaction</a></li> </ul> </li> 

  <h1 class="heading"><a name="Oligomeric_Cell_Killer_(OCK)">&nbsp;Oligomeric Cell Killer (OCK)</a></h1>

<!--◆◆STEP 1: DNA strands assemble to form designed structures◆◆--> 

    <article>
     <h2 class="small-title"><a name="STEP1">&nbsp;STEP 1: DNA strands assemble to form designed structures</a></h2>

<!--◆◆Optimize the condition to assemble OCK◆◆--> <h3><a name ="Optimize_the_condition_to_assemble_OCK"></a>1)Optimize the condition to assemble OCK</h3> <!--Method--> 

    <div class="zairyou-heading">[Method]</div>
    <figure>
        <iframe style="float:left; margin:0;margin-right:-10px;margin-bottom:10px; position:relative;left:-20px;" width="420" height="315" src="//www.youtube.com/embed/1ci93_QI6QA" frameborder="0" allowfullscreen></iframe>

</figure> 

    <div id ="step0_1)">
      <p class="paragraph">
      The DNA nanostructure,
      <a target="_bramk" href="http://openwetware.org/wiki/Biomod/2013/Todai/Design#Oligomeric_Cell_Killer " style="color:#e00000">
      "Oligomeric Cell Killer"
      </a>
      , was designed to achieve our goal(--><a href="http://openwetware.org/wiki/Biomod/2013/Todai/Project">Project</a>). 
      </p>

     <p class ="paragraph">
     The result of simulation by "CanDo<sup>[1]</sup>" showed the shape and flexibility of OCK.

To know the optimum condition of the structure assembly, we did experiments in three conditions as follows. <ul style="position:relative;left:30px;"> <li>At different concentration of MgCl<sub>2</sub></li> <li>At different incubate temperature</li> <li>At different length of incubate time</li> 

       </ul>
       (<a target="_blank" href="http://openwetware.org/wiki/Biomod/2013/Todai/Experiment#Protocols" style="color:#e00000;">
       protocols
       </a>
       )
       </p>
   </div>
   <br>
   <br>

<br> <br> <br> <br> <!--Result&Discussion1--> 

     <div class="zairyou-heading">[Result & Discussion]</div>

    <div class="res-conclusion">
   Optimum concentration of MgCl<sub>2</sub>
    </div>

      <figure>
       <center>
        <img src="http://openwetware.org/images/a/af/OOCK_Optimize_MgConc.png" width=640px height=360px>
           <figcaption> <b>Agarose-gel electrophoresis to research the optimum concentration of MgCl<sub>2</sub>

</b> 

           </figcaption>
       </center>
      </figure>

    <p class="paragraph">

Fast-migrating species upon agarose-gel electrophoresis was yielded at 10~20mM MgCl<sub>2</sub> condition. At higher MgCl<sub>2</sub> concentration, a sub-band, which might be a dimer, appeared. 

    </p>
    <div class="res-conclusion">
    --->> Optimum concentration of MgCl<sub>2</sub>: 10mM   
    </div>

    <br>
    <br>

<!--Result&Discussion2--> 

    <div class="res-conclusion">
   Optimum incubate temparature
    </div>
  
    <figure>
       <center>
          <img src="http://openwetware.org/images/9/95/OOCK_Optimize_Temp-Todai.png" width=640px height=360px >
          <figcaption>
          <b>Agarose-gel electrophoresis to research the optimum temperature</b>
          </figcaption>
       </center>
    </figure>

   <p class="paragraph">

Fast-migrating species upon agarose-gel electrophoresis was yielded at 52.0 °C. 

    </p>
    <div class="res-conclusion">
    --->> Optimum temparature : 52.0 °C  
    </div>
    <br>
    <br>

<!--Result&Discussion2--> 

    <div class="res-conclusion">
    To decide optimum length of incubate time
    </div>

      <figure>
        <center>
          <img src="http://openwetware.org/images/9/94/OOCK_Optimize_Time-Todai.png" width=640px height=360px >
          <figcaption>
          <b>Agarose-gel electrophoresis to research the optimum time</b>
          </figcaption>
       </center>
      </figure>

   <p class="paragraph">

The band for 3h is fast migrated and sharp. 

    </p>

    <div class="res-conclusion">
    --->> Optimum incubate time : 3h 
    </div>

    <br>

<!--◆◆1.2 Conformation of the 3D structure of OCK by TEM◆◆--> 

    <h3><a name="Conformation_of_the_3D_structure_of_OCK_by_TEM"></a>2) TEM imaging of the 3D structure of OCK</h3>
    <!--Method-->
    
    <div class="zairyou-heading">[Method]</div>

    <div id ="step0_2)">
      <p class="paragraph">

Gel electrophoresis cannot visualize the 3D structure of OCK, so it was confirmed by Transmission electron microscopy (TEM). 

       (<a target="_blank" href="http://openwetware.org/wiki/Biomod/2013/Todai/Experiment#Protocols" style="color:#e00000;">
       protocols
       </a>
       )
       </p>
   </div>
   <br>
   <br>

    </article>
    <br>

<!--Result&Discussion1--> 

     <div class="zairyou-heading">[Result & Discussion]</div>

    <div class="res-conclusion">
    TEM imaging of OCK
    </div>

      <figure>
       <center>
        <img src="http://openwetware.org/images/e/ea/Monomer-Todai.png" width=480px height=360px>
           <figcaption> <b>TEM image of OCK</b>
           Three monomers of OCK were observed in this figure. 
        
           </figcaption>
       </center>
      </figure>

    <p class="paragraph">
    TEM images confirm that our OCK has two domains. Comparing the observed structure to our design, one domain match the shape and size to plane-like domain. And the other domain matches to stick-like domain. Furthermore, in close watching the images, DNA well, which exists one side of plane-like domain, could be detected.
    </p>
    <div class="res-conclusion">
    </div>
    </article>

<!--◆◆2. Penetration◆◆--> <!--editting--> 

     <h2 class="small-title"><a name="STEP2">&nbsp;STEP 2: Penetration into the membrane</a></h2>
     <article>

<!--◆◆2.1 Flotation assay◆◆--> 

    <h3><a name=" Flotation_assay"></a>1) Flotation assay</h3>
    <!--Method-->
    <div class="zairyou-heading">[Method]</div>

    <div id ="step3_1)">
      <p class="paragraph">
      OCK was designed to penetrate lipid bilayer. However, it is difficult to conclude the penetration of OCK. Therefore, we first did flotation assay to detect the interaction of OCK with lipid. 
       (<a target="_blank" href="http://openwetware.org/wiki/Biomod/2013/Todai/Experiment#Protocols" style="color:#e00000;">
       protocols
       </a>
       )
       </p>
   </div>
   <br>
   <br>

    <br>

<!--Result&Discussion1--> 

     <div class="zairyou-heading">[Result & Discussion]</div>

    <div class="res-conclusion">
    
    </div>

      <figure>
       <center>
        <img src="http://openwetware.org/images/c/ca/Todai_result_step2_fluolescence.JPG" width=450px height=350px>
           <figcaption> <b>The fluorescence intensity of NIL (in liposome) in each fraction </b><br>
           The result of fluorescence spectrophotometer (JASCO, FP-6500) showed that liposome distributed mostly in fraction 3(lower layer).
           </figcaption>
       </center>
      </figure>
          <figure>
       <center>
        <img src="http://openwetware.org/images/f/fe/FlotationOCK12gel-Todai.png" width=480px height=360px>
           <figcaption> <b>1% Agarose gel electrophoresis of each fraction in sample 1, 2</b>
           </figcaption>
       </center>
      </figure>
          <figure>
       <center>
        <img src="http://openwetware.org/images/0/0d/FlotationOCK34gel-Todai.png" width=480px height=360px>
           <figcaption> <b>1% Agarose gel electrophoresis of each fraction in sample 3, 4</b>
           </figcaption>
       </center>
      </figure>
          <figure>
       <center>
        <img src="http://openwetware.org/images/f/ff/450px_FloatingOCKprofile-Todai.jpg" width=450px height=300px>

       </center>
      </figure>

    <p class="paragraph">
    With the condition of cholesterol +/ liposome+, the peak fraction was No.3, which coinced with peak fraction of liposome. In contrast, lacking of cholesterol or liposome, OCK exist mainly in fraction No.2.

As the peak fraction of OCK shifted from fraction No.2 to No.3, with the attachment of cholesterol and existence of liposome, we concluded that OCK stack in liposome. 

    </p>
    <div class="res-conclusion">
    --> OCK stack in liposome.
    </div>
    </article>

<!--◆◆2.2 Preparation of GUVs◆◆--> 

     <article>
    <h3><a name="Preparation_of_GUVs"></a>2) Preparation of GUVs</h3>
    <!--Method-->
    <div class="zairyou-heading">[Method]</div>

    <div id ="step2_2)">
      <p class="paragraph">
     	GUV, Giant Unilamellar Vesicle, was prepared to visualize the sticking of OCK in membrane. The comparation between the fluorescence of OCK (Cy5) and GUV(NIL, Nile Red) was expected to suggest the sticking.
       (<a target="_blank" href="http://openwetware.org/wiki/Biomod/2013/Todai/Experiment#Protocols" style="color:#e00000;">
       protocols
       </a>
       )
       </p>
   </div>
   <br>
   <br>

    <br>

<!--Result&Discussion1--> 

     <div class="zairyou-heading">[Result & Discussion]</div>

    <div class="res-conclusion">
    
    </div>

          <figure>
       <center>
        <img src="http://openwetware.org/images/b/be/GUVconfocal_scale-Todai.png" width=450px height=350px>

       </center>
      </figure>

    <p class="paragraph">

GUVs were observed with confocal laser scanning microscope (Carl Zeiss, LSM 5 Exciter). As the tracer of GUVs, 0.1 mol% Nile Red (Ex 553 nm, Em 637 nm) was used. 

About 10 um of GUVs were observed.

    </p>



    </article>

<!--◆◆STEP 3: Recognition of cancer-specific proteins◆◆--> 

    <article>
     <h2 class="small-title"><a name="STEP3">&nbsp;STEP3: Subunits recognize cancer-specific proteins. </a></h2>
    <h3><a name="Optimization_of_aptamer_lock_system"></a>1) Optimization of aptamer-lock system</h3>

<!--Method--> 

    <div class="zairyou-heading">[Method]</div>
           <figure>
       <center>
        <img src="http://openwetware.org/images/5/50/Todai_Recognition_ideal_mod.png" width=480px height=270px>
       </center>
      </figure>



    <div id ="step0_1)">
      <p class="paragraph">
      
      We did pilot study of oligomerization process triggered by membrane
      protein recognition. We used cholesterol modified PDGF as model membrane
      protein, as DNA origami embedded aptamer system recognizing PDGF was
      reported (Douglass et al. (2012)).
      
      </p>

     <p class ="paragraph">
     Our simplified model lock system is consisted with two steps: 1)

> Blocking of streptavidin binding to biotin by steric hindrance. Our lock > system consists of two strands: biotin attached strands (biotin strands) > and aptamer attached strands (aptamer strands). These two strands > hybridize each other in inactive form and hide biotin moiety from the > streptavidin by steric hindrance effect. 2) Upon binding of ligands > (PDGF in this study) to aptamer strands, the complementary strand(biotin > strands) is released from the DNA aptamer, because ligands take over the > DNA strands of DNA aptamer from the complementally strands, and biotin > can now bind to streptavidin. Therefore, the cancer cell recognition and > OCK oligomerization are achieved simultaneously in the future study. 

     </p>

   </div>
   <br>
   <br>

<br> 

    </article>
    <br>

<!--Result&Discussion1--> 

     <div class="zairyou-heading">[Result & Discussion]</div>

    <div class="res-conclusion">
    Integration of aptamer strands into DNA origami tile
    </div>
                    <figure>
       <center>
        <img src="http://openwetware.org/images/7/7f/Tile-ins.png" width=480px height=360px>
           <figcaption> <b></b>

           </figcaption>
       </center>
      </figure>
      <p class="paragraph">                               We confirmed the integration of aptamer attached strands (aptamer

strands) into rectangle DNA origami tile (rect-tile). </p> <br> 

    <div class="res-conclusion">
    Responsibility of aptamer
    </div>
      <figure>
       <center>
        <img src="http://openwetware.org/images/f/f5/Figure9_10-Todai.png" width=480px height=270px>
           <figcaption> <b></b>



           </figcaption>
       </center>
      </figure>
      <p class ="paragraph">
           We confirmed the responsibility of aptamer sequence embedded in

rect-tile (shown above). The position of Cy5-PDGF band coincided with that of DNA tile, showing that the aptamers work also on rect-tile. Furthermore, the linker length between aptamer sequence and staple sequence, the latter staple sequence is embedded into rect-tile, does not affect the binding ability of aptamer to PDGF. </p> <br> 

    <div class="res-conclusion">
    Blocking capabbility of lock system  by aptamer
    </div>
      <table cellpadding="0" style="position:relative;left:-50px;">
       <tbody><tr>
       <td>
       <figure>
         <img src="http://openwetware.org/images/3/30/Figure11_12-Todai.png" width="360px" height="240px">

       </figure>
       </td>



       <td>
       <figure  style="position:relative;left:-50px;">
         <img src="http://openwetware.org/images/2/24/Koyama_131027_1-Todai.JPG" width="240px" height="240px">

       </figure>
       </td>
       </tr>
     </tbody></table>



    <p class="paragraph">

We confirmed the blocking capability of our lock system for streptavidin binding (left figure, the image of gel electrophoresis). Our lock system consists of two strands: biotin attached strands (biotin strands) and aptamer attached strands (aptamer strands). These two strands hybridize each other in inactive form and hide biotin moiety from the streptavidin by steric hindrance effect. We confirmed this blocking capability by mixing Cy3 labeled streptavidin with lock system embedded rect-tile. Data indicates that the slight blocking capability upon shorten the polyT linker between aptamer sequence and staple sequence. Recently, we tried other sequence and have better results, which may be presented in the Jamboree in Boston. <br> 

    <div class="res-conclusion">

Optimum embedding condition of our lock system into rect-tile 

    </div>
      <figure>
       <center>
        <img src="http://openwetware.org/images/c/c8/Tile_double_insertion-Todai.png" width=480px height=360px>
           <figcaption> <b></b>



           </figcaption>
       </center>
      </figure>

Next, we optimize the embedding condition of our lock system into rect-tile. This time full length of biotin strands were used instead of truncated ones used in above figure. Data indicate that the integrate efficiency of both biotin strands and aptamer strands into rect-tile is independent on the incubate temperature.

We improve our lock system everyday. Don't miss our presentation in Jaboree in Boston ! 

    </p>
    <div class="res-conclusion">
    </div>

<!--◆◆3.2Embedding_of_recognition_system_to_OCK◆◆--> 

    <h3><a name="Embedding_of_recognition_system_to_OCK"></a>2) Embedding of recognition system to OCK</h3>
   <!--Method-->
    <div class="zairyou-heading">[Method]</div>

    <div id ="step3_1)">
      <p class="paragraph">

To embed recognition system to OCK, we equiped PDGF aptamer used in rect-tile to OCK and confirmed the association of aptamer and PDGF . 

       (<a target="_blank" href="http://openwetware.org/wiki/Biomod/2013/Todai/Experiment#Protocols" style="color:#e00000;">
       protocols
       </a>
       )
       </p>
   </div>

<!--Result&Discussion1--> 

     <div class="zairyou-heading">[Result & Discussion]</div>

    <div class="res-conclusion">
    Recognition of PDGF by DNA aptamer on OCK
    </div>
    <figure>
       <center>
        <img src="http://openwetware.org/images/c/c3/OCK_PDGFWeb-Todai.png" width=320px height=180px>

       </center>
      </figure>
      
         <div class="res-conclusion">
    -->PDGF was recognized by the aptamer of OCK
    </div>

<!--◆◆STEP 4: Oligomerization in solution◆◆--> 

    <article>
     <h2 class="small-title"><a name="STEP4">&nbsp;STEP 4: Oligomerization in solution</a></h2>

<!--◆◆4.1 Oligomerization by streptavidin-biotin complex◆◆--> 

    <h3><a name="Oligomerization_by_streptavidin-biotin_complex"></a>1) Oligomerization by streptavidin-biotin complex</h3>
    <!--Method-->
    <div class="zairyou-heading">[Method]</div>

    <div id ="step3_1)">
      <p class="paragraph">
      Biotins are equipped to OCK for oligomerization. The experiment which confirmed that streptavidins induced oligomeriation. 
       (<a target="_blank" href="http://openwetware.org/wiki/Biomod/2013/Todai/Experiment#Protocols" style="color:#e00000;">
       protocols
       </a>
       )
       </p>
   </div>
   <br>
   <br>

    </article>
    <br>

<!--Result&Discussion1--> 

     <div class="zairyou-heading">[Result & Discussion]</div>

    <div class="res-conclusion">
    Streptavidins induced oligomerization
    </div>

      <figure>
       <center>
        <img src="http://openwetware.org/images/5/5b/Streptavidin_dimer-Todai.png" width=600px height=450px>

<figcaption>The mixing ratio of streptavidin to OCK was equal to 5:3, which means the mixing ratio of streptavidin to biotin was equal to 5:6 in the condition (L+R).</figcaption> 

       </center>
      </figure>
    </article>

<!--◆◆4.2 EMm imaging of dimers by streptavidin-biotin complex◆◆--> 

    <article>
         <h3><a name="Tem_imaging_of_OCK_dimers_by_streptavidin-biotin_complex"></a>2) TEM imaging of OCK dimers connected by streptavidin-biotin interaction</h3>
    <!--Method-->
    <div class="zairyou-heading">[Method]</div>

    <div id ="step4_2)">
      <p class="paragraph">
      Dimers of OCKs were also imaged by TEM to confirm the bands observed in the experiment 3.1) originated from the dimers.
       (<a target="_blank" href="http://openwetware.org/wiki/Biomod/2013/Todai/Experiment#Protocols" style="color:#e00000;">
       protocols
       </a>
       )
       </p>
   </div>
   <br>
   <br>

    </article>
    <br>

<!--Result&Discussion1--> 

     <div class="zairyou-heading">[Result & Discussion]</div>

    <div class="res-conclusion">
    </div>
    <center>
     <table cellpadding="0" style ="position:relative;left:-30px;">
       <tr>
       <td>
       <figure>
         <img src="http://openwetware.org/images/c/c1/Dimerv2-Todai.png" width="300px" height="300px" >
       </figure>
       </td>



       <td>
       <figure>
         <img src="http://openwetware.org/images/5/53/Dimer_2v2-Todai.png" width="300px" height="300px" >
       </figure>
       </td>
       </tr>
     </table>
     </center>
    <p class="paragraph">
    	Dimers of OCKs  were observed in this experiment and two of them were shown above. 
    </p>
    <div class="res-conclusion">
    -->The dimerization by streptavidin-biotin complex was confirmed.
    </div>

<!--◆◆4.3 Optimum concentration of CuSO4◆◆--> 

    <article>
    <h3><a name="Oligomerization_by_Click_reaction"></a>3) Oligomerization by Click reaction</h3>
    <!--Method-->
    <div class="zairyou-heading">[Method]</div>

    <div id ="step4_3)">
      <p class="paragraph">
      Azide and alkyne, which function as a reactive group of click reaction, are also equiped to OCK. It demands Cu<sup>+</sup> as catalyst, but too high concentration of Cu<sup>+</sup> (cation) might denaturate OCK like Mg<sup>2+</sup>. Therefore, we optimized the concentration of Cu<sup>+</sup> to OCK first, and then the optimum Cu<sup>+</sup> concentration to click reaction was investigated.
       (<a target="_blank" href="http://openwetware.org/wiki/Biomod/2013/Todai/Experiment#Protocols" style="color:#e00000;">
       protocols
       </a>
       )
       </p>
   </div>
   
     <div class="zairyou-heading">[Result & Discussion]</div>
      <div class="res-conclusion">
      a) Optimum concentration of CuSO<sub>4</sub> to OCK
    </div>
          <figure>
       <center>
        <img src="http://openwetware.org/images/5/5e/CuAlive-Todai.png" width=600px height=450px>

       </center>
      </figure>
             <div class="res-conclusion">
      -->Optimum concentration of CuSO<sub>4</sub>: 625 uM or less
    </div>
   <br>
   <br>
   </div>
      <div class="res-conclusion">
      b) Optimum concentration of CuSO<sub>4</sub> to click reaction
    </div>
          <figure>
       <center>
        <img src="http://openwetware.org/images/9/94/ClickResult-Todai.png" width=600px height=450px>

       </center>
      </figure>
      <p class ="paragraph">
      Product of click reaction appeared over 375 uM CuSO<sub>4</sub> concentration. Combining with the stability data, we decided to use 625 uM CuSO<sub>4</sub> condition.
      </p>
             <div class="res-conclusion">
             --> Optimum concentration of CuSO<sub>4</sub>: 625 uM
    </div>
    </article>
    <!--◆◆4.4 Cupper-free click chemistry◆◆-->

    <article>
    <h3><a name="Cupper-free_click_reaction"></a>4) Cupper-free click reaction</h3>
    <!--Method-->
    <div class="zairyou-heading">[Method]</div>

    <div id ="step4_3)">
      <p class="paragraph">

Click reaction demands cupper catalyst, which works as a toxine in human body. Therefore, we studied about cupper-free click reaction for the application to human body. 

       (<a target="_blank" href="http://openwetware.org/wiki/Biomod/2013/Todai/Experiment#Protocols" style="color:#e00000;">
       protocols
       </a>
       )
       </p>
   </div>
   
     <div class="zairyou-heading">[Result & Discussion]</div>
      <div class="res-conclusion">
      a) Optimum concentration of CuSO<sub>4</sub> to OCK
    </div>

<center> 

      <table cellpadding="0" style="position:relative;left:-25px;">
       <tbody><tr>
       <td>
       <figure>
         <img src="http://openwetware.org/images/2/25/Gelphoto1-Todai.png" width="400px" height="225px">
       </figure>
       </td>
       <td>
       <figure  style="position:relative;left:-50px;">
         <img src="http://openwetware.org/images/2/2f/Yatagai_131027_1.JPG" width="225px" height="225px">

<figcaption> <b>The reaction rate of cupper-free click reaction with no accelerator</b> </figcaption> 

       </figure>
       </td>
       
       </tr>
     </tbody></table>

</center> 

      <p class="paragraph">

We first measured the Cu-free click reaction in solution (without no catalyst nor accelerator). The association time at 2 uM oligonucleotide condition was 17.1 h, and appearent association time was estimated as 8.1 [1/M/s]. </p> <center> 

       <table cellpadding="0" style="position:relative;left:-25px;">
       <tbody><tr>
     
       <td>
       <figure>
         <img src="http://openwetware.org/images/4/45/Gelphoto2-Todai.png" width="400px" height="225px">
       </figure>
       </td>
      
       <td>
       <figure  style="position:relative;left:-50px;">
         <img src="http://openwetware.org/images/2/23/YatagaiclickStA-Todai.png" width="225px" height="225px">
       </figure>
       </td>
       </tr>
     </tbody></table>

</center> <center> 

        <table cellpadding="0" style="position:relative;left:-25px;">
       <tbody><tr>
       <td>
       <figure>
         <img src="http://openwetware.org/images/1/12/Gelphoto3-Todai.png" width="360px" height="270px">
       </figure>
       </td>



       <td>
       <figure  style="position:relative;left:-50px;">
         <img src="http://openwetware.org/images/c/c6/Yatagaiclickhybri-Todai.png" width="270px" height="270px">

       </figure>
       </td>
       </tr>
     </tbody></table>

</center> 

               <figure  style="position:relative;left:-50px;">
         <img src="http://openwetware.org/images/a/a2/Yatagai_131027_2.JPG" width="270px" height="270px">

       </figure>
       
       	<p class="paragraph">

We add accelerator, which can work as a scaffold and make alkyne and azide reactive group close. Acceleration of the click reaction was observed. In other words, azide and alkyne reactive groups do not react each other in solution, but easy to react each other after proximally-positioned. This character is very suitable to prevent non-specific oligomerization, while accelerating the specific oligomerization in OCK. </p> <p class ="paragraph"> Note: Streptavidin has 4 identical subunits. So we can not control the binding order of alkyne and azide oligo with streptavidin method. Therefore, vicinity subunit may have identical reactive groups (e.g. alkyne-alkyne or azide-azide, instead of alkyne-azide or azide-alkyne), and may reduce the yield. </p> 

             <div class="res-conclusion">
             --> Cu-free click reaction has suitable character for specific oligomerization.
    </div>
    
    </article>
    
       <h1 class="title"><a name="Reference">&nbsp;Reference</a></h1>

    <div>     
       <div class="reference-title">
       <a name="proref-1">
       [1] CanDo(<a href="http://cando-dna-origami.org/usersguide">http://cando-dna-origami.org/usersguide</a>)
       </a>
       </div>
    </div>
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