Difference between revisions of "Biomod/2013/Todai/Experiment"

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<title>Experiment-Todai nanORFEVRE-</title>
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  <h1 class="big-title"><a name="Result">&nbsp;Result</a></h1>
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http://openwetware.org/images/1/15/Todai_Projectsteps_map.png" width=720px height=480px >
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<!--Experiment-->
 +
    <h1 class="big-title"><a name="Experiment">&nbsp;Experiment</a></h1>
 +
    <div id="Explist">
 +
    <ul>
 +
      <li><div class="mokuji"><a href="#Contents">Contents of pilot study</a></div></li>
 +
      <li><div class="mokuji"><a href="#Contents">Contents of Protocols</a></div></li>
 +
      <li><div class="mokuji"><a href="#PilotStudy">Pilot Study</a></div></li>
 +
      <li><div class="mokuji"><a href="#Protocols">Protocols</a></div></li>
 +
    </ul>
 +
    </div>
 +
    <br>
 +
 
 +
<!--Contents-->
 +
 
 +
  <article>
 +
    <h1 class="title"><a name="Contents">&nbsp;Contents of pilot study</a></h1>
  
  <h1 class="heading"><a name="Contents">&nbsp;Contents</a></h1>
+
      <article>
<ul>
+
        <ul>
<li><h3><a href="#STEP1">STEP 1: DNA strands assemble to form designed structures</a></h3>
+
<li><div class="mini-title" style="color:#BBBBBB;">STEP 1: DNA strands assemble to form designed structures.</div>
 +
</li>
 +
<li><div class="mini-title"><a href="#STEP2">STEP 2: Subunits penetrate into the membrane.</a></div>
 +
<ul style="list-style: none;">
 +
<li><a href="#hybridization_of_Cholesterol_Oligo_with_OCK">1) hybridization of cholesterol oligo with OCK</a></li>
 +
<li><a href="#Preparation_of_liposome">2) Preparation of liposome</a></li>
 +
<li><a href="#Flotation_assay_of_liposome_and_DNA_origami">3) Floatation assay of liposome and Rectangular tile(DNA origami)</a></li>
 +
</ul>
 +
</li>
 +
<br>
 +
<li><div class="mini-title"><a href="#STEP3">STEP 3: Subunits recognize cancer-specific proteins.</a></div>
 +
</li>
 +
<li><div class="mini-title"><a href="#STEP4">STEP 4: The formed subunits oligomerize in solution.</a></div>
 
<ul style="list-style: none;">
 
<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="#Click_reaction_via_(3+2)_cycloaddition">1) Optimum time of click reaction via (3+2) cycloaddition</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><a href="#OptimumConc_SA">2) OptimumConc SA</a></li>
 +
</ul>
 +
</li>
 +
</ul>
 +
  </article>
 +
</article>
 +
 +
 +
  <article>
 +
    <h1 class="title"><a name="Contents">&nbsp;Contents of protocols</a></h1>
 +
      <article>
 +
        <ul>
 +
<li><div class="mini-title"><a href="#STEP1">STEP 1: DNA strands assemble to form designed structures.</a></div> <ul style="list-style: none;">
 +
<li><a href="#Assembling_of_OCK">1) Assembly of OCK</a></li>
 +
<li><a href="#Transmission_electron_microscopy">2) Transmission electron microscopy(TEM)</a></li>
 +
</ul>
 
</li>
 
</li>
<li><h3><a href="#STEP2">STEP 2: Penetration into the membrane</a></h3>
+
<br>
 +
<li><div class="mini-title"><a href="#STEP2">STEP 2: Subunits stick in the membrane.</a></div>
 
<ul style="list-style: none;">
 
<ul style="list-style: none;">
<li><a href="#Flotation_assay">1)Flotation assay</a></li>
+
<li><a href="#Flotation_assay_[OCK]">1) Flotation assay [OCK]</a></li>
<li><a href="#Preparation_of_GUVs">2)Preparation of GUVs</a></li>
+
<li><a href="#Preparation_of_GUVs">2) Preparation of GUVs</a></li>
 +
<li><a href="Preparation_of_SUVs">3) Preparation of SUVs</a>
 +
<li><a href="#hybridization_of_Cholesterol_Oligo_with_OCK">4) hybridization of cholesterol oligo with OCK</a></li>
 
</ul>
 
</ul>
 
</li>
 
</li>
<li><h3><a href="#STEP3">STEP 3: Recognition of cancer-specific proteins</a></h3>
+
<br>
<ul style="list-style:none;">
+
<li><div class="mini-title"><a href="#STEP3">STEP 3: Recognition of target cells</a></div>
<li><a href="#Optimization_of_aptamer_lock_system">1) Optimization of aptamer-lock system</a>
+
<ul style="list-style: none;">
</li>
+
<li><a href="#Reaction_of_a_biotinized_oligo_to_streptavidin">1) Reaction of a biotinized oligo to streptavidin</a></li>
<li><a href="#Embedding_of_recognition_system_to_OCK">2) Embedding of recognition system to OCK</a></li>
+
<li>      <a href="Reaction_between_aptamer_embedded_in_rect_tile_and_PDGF10">2) Reaction_between_aptamer_embedded_in_rect_tile_and_PDGF</a>
 +
<li><a href="#efficient_hybridization_(changing_mixture_ratio)">3) Efficient hybridization (changing mixture ratio)</a></li>
 +
<li><a href="#efficient_hybridization_(incubation_time)">4) efficient hybridization (incubation time)</a></li>
 +
<li><a href="#insertion_of__hybridized_double-stranded_DNA_into_tile">5) Insertion of hybridized double-stranded DNA into tile</a></li>
 +
<li> <a href="#Reaction_between_aptamer_(3ap-M5t10f-T0)_and_PDGF">6) Reaction_between_aptamer_(3ap-M5t10f-T0)_and_PDGF</a></li>
 +
<li><a href="Double insertion_of__hybridized_double-stranded_DNA_into_tile">7) Double insertion of hybridized double-stranded DNA into tile</a></li>
 
</ul>
 
</ul>
 
</li>
 
</li>
<li><h3><a href="#STEP4">STEP 4: Oligomerization in solution</a></h3>
+
<br>
 +
<li><div class="mini-title"><a href="#STEP4">STEP 4: The formed subunits oligomerize in solution.</a></div>
 
<ul style="list-style: none;">
 
<ul style="list-style: none;">
<li><a href="#Oligomerization_by_streptavidin-biotin_complex">1)Oligomerization by streptavidin-biotin complex</a></li>
+
<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="#Click_reaction_via_(3+2)_cycloaddition"> 2) Click reaction via (3+2) cycloaddition
<li><a href="#Oligomerization_by_Click_reaction">3)Oligomerization by Click reaction</a></li>
+
      </a>
 +
      </li>
 +
<li>
 +
      <a href="#Accelerated_Click_reaction">3) Accelerated Click reaction (using streptavidin to make the aklyne and azide reactive groups close) </a>
 +
      </li>
 +
<li><a href="#Click_reaction_(using_hybridization_to_make_the_aklyne_and_azide_reactive_groups close)">4) Click reaction (using hybridization to make the aklyne and azide reactive groups close) </a>
 +
      </li>
 +
<li><a href="#Click_reaction_(copper_catalyst-free)">5) Click reaction (copper catalyst-free)</a></li>
 +
<li><a href="#Synthesis_of_streptavidin_mutants">6) Synthesis of streptavidin mutants</a>
 
</ul>
 
</ul>
 
</li>
 
</li>
+
</ul>
  <h1 class="heading"><a name="Oligomeric_Cell_Killer_(OCK)">&nbsp;Oligomeric Cell Killer (OCK)</a></h1>
+
  </article>
 +
</article>
  
<!--◆◆STEP 1: DNA strands assemble to form designed structures◆◆-->
+
<!--◆◆Pilot Study◆◆-->
 +
    <h1 class="title"><a name="PilotStudy">&nbsp;Pilot Study</a></h1>
 +
   
 +
<!--◆◆STEP2◆◆-->
 +
<h2 class="PS_title"><a name="STEP2">&nbsp;STEP 2: Subunits penetrate into the membrane </a></h2>
 +
<!--◆◆hybridizing of Cholesterol Oligo with OCK◆◆-->
 +
  <article>
 +
  <div class="mini-title">
 +
      <a name="hybridization_of_Cholesterol_Oligo_with_OCK">1) Hybridization of cholesterol Oligo with OCK</a>
 +
</div>
 +
      <figure>
 +
        <center>
 +
        <img src="http://openwetware.org/images/d/d7/480px_OCKchol-Todai.png" width=480px height=300px >
  
    <article>
+
        </center>
      <h2 class="small-title"><a name="STEP1">&nbsp;STEP 1: DNA strands assemble to form designed structures</a></h2>
+
      </figure>
     
 
<!--◆◆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>
 
<br>
<!--Result&Discussion1-->
 
  
      <div class="zairyou-heading">[Result & Discussion]</div>
+
  <div class="zairyou-heading">[Discussion]</div>
 +
    <p class="paragraph">
 +
The result of 1 % agarose gel electrophoresis showed that the band of
 +
sample 5 and 7 were smeared, showing the successful of hybridization of
 +
cholesterol oligo with OCK (Langecker et al. (2012)). We concluded that
 +
the optimized condition for hybridization is: 1 hour incubation at room
 +
temperature with 5 times excess cholesterol oligo to OCK.
 +
 +
    </p>
 +
  <br>
 +
</article>
 +
 +
<!--◆◆Preparation_of_liposome◆◆-->
 +
<article
 +
  <div class="mini-title">
 +
      <a name="Preparation_of_liposome">2) Preparation of liposome</a>
 +
  </div>
  
    <div class="res-conclusion">
+
      <figure>
    Optimum concentration of MgCl<sub>2</sub>
+
        <center>
    </div>
+
        <img src="http://openwetware.org/images/7/71/640px_suv_dls_popg50r60-Todai.png" width=640px height=360px >
 +
<figcaption> <b>The result of DLS (Viscotek, 802 DLS)</b>
 +
      </figcaption>
 +
        </center>
 +
      </figure>
 +
<div class="zairyou-heading">[Discussion]</div>
 +
    <p class="paragraph">For floating assay, uniformly-sized liposome were prepared. DLS data shows sharp peak with the mean radius of 60 nm, indicating the homogenity of liposomes.
 +
    </p>
 +
  </article>
 +
  <br>
 +
<!--◆◆Flotation assay(Rect-tile)◆◆-->
  
 +
  <article>
 +
  <div class="mini-title">
 +
      <a name="Flotation_assay_of_liposome_and_DNA_origami">3) Floatation assay of liposome and Rectangular tile(DNA origami)</a>
 +
      </div>
 +
      <figure>
 +
        <center>
 +
        <img src="http://openwetware.org/images/1/1d/640pxflotationassay-Todai.jpg" width=300px height=300px >
 +
<figcaption> <b>Result of agarose gel electrophoresis of the sample of flotation assay</b> <br>
 +
The result of 1% agarose gel electrophoresis(100V,30min). In this measurement, the fluorescence of Cy5, which is
 +
integrated into DNA origami(Rect tile<sup>[1]</sup>) ,is observed. Fraction1 is the liquid in the top layer, and fra ction 5 is in the bottom layer. Fraction 6 is the sample retrieved from precipitation. DNA origami solely was also l oaded on the extreme right lane.
 +
</figcaption>
 +
        </center>
 +
      </figure>
 
       <figure>
 
       <figure>
 
         <center>
 
         <center>
         <img src="http://openwetware.org/images/a/af/OOCK_Optimize_MgConc.png" width=640px height=360px>
+
         <img src="http://openwetware.org/images/0/0d/300pxNILGraph-Todai.PNG" width=350px height=350px >
            <figcaption> <b>Agarose-gel electrophoresis to research the optimum concentration of MgCl<sub>2</sub>
+
<figcaption> <b>Fluorescence intensity of the samples of flotation assay(DNA Rect tile +liposome)</b><br>
</b>
+
Although the size of liposome might change during the flotation assay(data not shown), the intensity of the fluoresc ence of NIL(Nile Red, ex 500nm, em 550~700nm )
            </figcaption>
+
suggests the amount of lipid membrane,liposome. The fluorescence spectrum of water was subtracted as background
 +
      </figcaption>
 
         </center>
 
         </center>
 
       </figure>
 
       </figure>
 +
<br>
  
 +
  <div class="zairyou-heading">[Discussion]</div>
 
     <p class="paragraph">
 
     <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.
+
To confirm the flotation assay, mixed tiles(DNA origami) and liposomes were assayed. Five samples (fraction 1,2,..., 5, from the top) were
 +
retrieved from supermetant liquid and a sample(fraction 6) from precipitation by the addition of buffer used in
 +
assay. When the sample, tile mixed with liposomes, were assayed, tiles were observed in the top layer. The distribut ion of liposomes is observed by the fluorescence of NIL(Nile Red).
 +
    </p>
 +
  <br>
 +
</article>
  
    </p>
 
    <div class="res-conclusion">
 
    --->> Optimum concentration of MgCl<sub>2</sub>: 10mM 
 
    </div>
 
  
    <br>
+
<!--◆◆STEP3◆◆-->
    <br>
+
<h2 class="PS_title"><a name="STEP3">&nbsp;STEP 3: Subunits recognize cancer-specific proteins</a></h2>
 +
<article>
 +
</article>
  
<!--Result&Discussion2-->
+
<!--◆◆STEP4◆◆-->
 +
<h2 class="PS_title"><a name="STEP4">&nbsp;STEP 4: The formed subunits oligomerize in solution</a></h2>
  
    <div class="res-conclusion">
+
<!--◆◆Click reaction ◆◆-->
    Optimum incubate temparature
+
  <div class="mini-title">
    </div>
+
      <a name="Click_reaction_via_(3+2)_cycloaddition">
 
+
        1)Optimum time of Click reaction via (3+2) cycloaddition<sup>[4]</sup>
    <figure>
+
      </a>
 +
  </div>
 +
 
 +
  <article>
 +
      <figure>
 
         <center>
 
         <center>
          <img src="http://openwetware.org/images/9/95/OOCK_Optimize_Temp-Todai.png" width=640px height=360px >
+
        <img src="http://openwetware.org/images/a/ab/450pxclick0828-Todai.jpg" width=480px height=360px >
          <figcaption>
+
<figcaption> <b>Result of urea gel electrophoresis of the sample of click reaction</b><br>
          <b>Agarose-gel electrophoresis to research the optimum temperature</b>
+
 
          </figcaption>
+
      </figcaption>
 
         </center>
 
         </center>
     </figure>
+
      </figure>
 +
<div class="zairyou-heading">[Discussion]</div>
 +
     <p class="paragraph">Copper(Ⅰ) catalyzed click reaction was used to dimerize of oligo DNA(length of 20bp and 14bp) . The time cause of the reaction indicate that the click reaction is so quick(<5min).</p>
  
    <p class="paragraph">
+
<!--◆◆Optimum_Conc_SA◆◆-->
Fast-migrating species upon agarose-gel electrophoresis was yielded at 52.0 °C.  
+
  <article>
 +
  <div class="mini-title">
 +
      <a name="OptimumConc_SA">2) Optimum concentration of SA</a>
 +
  </div>
 +
          <figure>
 +
        <center>
 +
        <img src="http://openwetware.org/images/1/1b/OptimumConc_SA-Todai.png" width=480px height=360px >
 +
<figcaption> <b>Optimization of the mixing ratio between </b><br>
 +
The density of the band of dimers was the highest when the mixing ratio of streptavidin to SA was 3/1, so
 +
the optimum ratio of streptavidin to OCK was 3/1.
  
    </p>
+
      </figcaption>
    <div class="res-conclusion">
+
        </center>
    --->> Optimum temparature : 52.0 °C 
+
      </figure>
    </div>
+
  <br>
    <br>
 
    <br>
 
  
<!--Result&Discussion2-->
+
  <br>
 
+
</article>
    <div class="res-conclusion">
+
<!--◆◆Evaluation of streptavidin mutants◆◆-->
    To decide optimum length of incubate time
+
  <article>
    </div>
+
  <div class="mini-title">
 +
      <a name="Evaluation_of_streptavidin_mutants">3) Evaluation of streptavidin mutants</a>
 +
  </div>
 +
          <figure>
 +
        <center>
 +
        <img src="http://openwetware.org/images/1/1e/SA_result-Todai.png" width=400px height=400px >
  
      <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>
 
         </center>
 
       </figure>
 
       </figure>
 +
  <br>
 +
<p class="paragraph">
 +
We added a 6His tag to the active wild-type subunit ("alive" (A)
 +
subunit), hence no 6His tag was added to inactive subunit ("dead" (D)
 +
subunit). We mixed A and D subunits at a molar ratio of 1:1 in GuHCl and
 +
refolded in PBS. Then, refolded streptavidins were purified by Ni-NTA
 +
column and the tetramers were distinguished by non-denatured SDS-PAGE.
 +
</p>
 +
  <br>
 +
<!--◆◆Protocols◆◆-->
 +
 +
    <h1 class="title"><a name="Protocols">&nbsp;Protocols</a></h1>
 +
<!--◆◆STEP1◆◆-->
 +
<h2 class="PS_title"><a name="STEP1">&nbsp;STEP 1:DNA strands assemble to form designed structures.</a></h2>
 +
 +
<!--◆◆Assembly of OCK◆◆-->
 +
 +
  <div class="mini-title">
 +
      <a name="Assembling_of_OCK">1) Assembly of OCK<sup>[2]</sup></a>
 +
      </div>
 +
    <br>
 +
  <article>
 +
  
 +
<!--Reagent-->
 +
      <div class="zairyou-heading">[Reagent]</div>
 +
      <br>
 +
 +
    <table>
 +
    <tr>
 +
    <th>M13mp18ss</th>
 +
    <td>4.5 ul</td>
 +
    </tr>
 +
 +
    <tr>
 +
    <th>Staple mix</th>
 +
    <td>4.5 µL</td>
 +
    </tr>
 +
 +
    <tr>
 +
    <th>10x OCK buffer<sup>*</sup></th>
 +
    <td>1 µL</td>
 +
    </tr>
 +
 +
 +
    </table>
 +
    <br>
 +
 +
&nbsp;*...10x OCKbuffer(f.100 ul)
 +
    <table>
 +
    <tr>
 +
    <th>Tris-HCl(ph 7.5)</th>
 +
    <td>f.50 mM</td>
 +
    <td>1 M</td>
 +
    <td>5 µL</td>
 +
    </tr>
 +
    <tr>
 +
    <th>EDTA-Na(pH 8)</th>
 +
    <td>f.10 mM</td>
 +
    <td>0.5 M</td>
 +
    <td>2 µL</td>
 +
    </tr>
 +
        <tr>
 +
    <th>MgCl<sub>2</sub></th>
 +
    <td>f.200 mM</td>
 +
    <td>1 M</td>
 +
    <td>20 µL</td>
 +
    </tr>
 +
    <tr>
 +
    <th>NaCl</th>
 +
    <td>f.500 mM</td>
 +
    <td>5 M</td>
 +
    <td>1 µL</td>
 +
    </tr>
 +
        <tr>
 +
    <th>MQ</th>
 +
    <td>-</td>
 +
    <td>-</td>
 +
    <td>72 µL</td>
 +
    </tr>
 +
    </table>
 +
 +
<!--Procedure-->
 +
  <div class="zairyou-heading">[Procedure]</div>
 +
    <ul class="procedure-list">
 +
      <li>mix the solutions.</li>
 +
      <li>It was annealed at 85 °C for 25 min and then at 52 °C for 3 or 4 hours.</li>
 +
    </ul>
 +
 +
    </article>
 +
    <br>
 +
 +
 +
<!--◆◆TEM◆◆-->
 +
 +
  <article>
 +
  <div class="mini-title">
 +
      <a name="Transmission_electron_microscopy">
 +
      2) Transmission electron microscopy(TEM)
 +
      </a>
 +
  </div>
 +
<!--Procedure-->
 +
  <div class="zairyou-heading">[Procedure]<sup>[6]</sup></div>
 
     <p class="paragraph">
 
     <p class="paragraph">
The band for 3h is fast migrated and sharp.
+
    The procedure of TEM was refered to previous researches<sup>[6]</sup>.  
 
+
    </p>
    </p>
+
    </article>
 +
<!--◆◆STEP2◆◆-->
 +
<h2 class="PS_title"><a name="STEP2">&nbsp;STEP 2: Subunits penetrate into the membrane.</a></h2>
 +
 
 +
<!--◆◆flotation asssay of OCK◆◆-->
 +
 
 +
  <div class="mini-title">
 +
      <a name="Flotation_assay_[OCK]">1) Flotation assay [OCK]</a>
 +
  </div>
 +
  <article>
 +
<!--Reagent-->
 +
      <div class="zairyou-heading">[Reagent]</div>
 +
      <br>
 +
 
 +
    <table>
 +
    <tr>
 +
    <th>OCK</th>
 +
    <td>100 µL</td>
 +
    </tr>
 +
 
 +
    <tr>
 +
    <th>Cholesterol hybridized OCK</th>
 +
    <td>100 µL</td>
 +
    </tr>
 +
 
 +
    <tr>
 +
    <th>Liposome (1 mg/mL SUVs)</th>
 +
    <td>100 µL</td>
 +
    </tr>
 +
 
 +
    <tr>
 +
    <th>2.25 M Sucrose buffer<sup>*</sup></th>
 +
    <td>500 µL</td>
 +
    </tr>
 +
 
 +
    <tr>
 +
    <th>1.6 M Sucrose buffer<sup>**</sup></th>
 +
    <td>900 µL</td>
 +
    </tr>
  
    <div class="res-conclusion">
+
    <tr>
    --->> Optimum incubate time : 3h
+
    <th>150 mM KCl solution</th>
    </div>
+
    <td>100 µL</td>
 +
    </tr>
  
    <br>
+
    <tr>
 +
    <th>1×Flotation buffer<sup>***</sup></th>
 +
    <td>600 µL</td>
 +
    </tr>
  
<!--◆◆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)">
+
     </table>
      <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>
 +
&nbsp;*...2.25 M Sucrose buffer
 +
    <table>
 +
    <tr>
 +
    <th>HEPES-KOH (pH 7.6)</th>
 +
    <td>50 mM</td>
 +
    </tr>
 +
    <tr>
 +
    <th>KCl</th>
 +
    <td>100 mM</td>
 +
    </tr>
 +
        <tr>
 +
    <th>MgCl<sub>2</sub></th>
 +
    <td>20 mM</td>
 +
    </tr>
 +
    <tr>
 +
    <th>Sucrose</th>
 +
    <td>2.25 M</td>
 +
    </tr>       
 +
    </table>
 +
 +
 +
    </table>
 
     <br>
 
     <br>
 +
&nbsp;**...1.6 M Sucrose buffer
 +
    <table>
 +
    <tr>
 +
    <th>HEPES-KOH (pH 7.6)</th>
 +
    <td>50 mM</td>
 +
    </tr>
 +
    <tr>
 +
    <th>KCl</th>
 +
    <td>100 mM</td>
 +
    </tr>
 +
        <tr>
 +
    <th>MgCl<sub>2</sub></th>
 +
    <td>20 mM</td>
 +
    </tr>
 +
    <tr>
 +
    <th>Sucrose</th>
 +
    <td>1.6 M</td>
 +
    </tr>       
 +
    </table>
 +
  
    </article>
+
    </table>
    <br>
+
    <br>
<!--Result&Discussion1-->
+
&nbsp;***...1×Flotation buffer
 +
    <table>
 +
    <tr>
 +
    <th>HEPES-KOH (pH 7.6)</th>
 +
    <td>50 mM</td>
 +
    </tr>
 +
    <tr>
 +
    <th>KCl</th>
 +
    <td>100 mM</td>
 +
    </tr>
 +
        <tr>
 +
    <th>MgCl<sub>2</sub></th>
 +
    <td>20 mM</td>
 +
    </tr>   
 +
    </table>
  
      <div class="zairyou-heading">[Result & Discussion]</div>
 
  
    <div class="res-conclusion">
+
<!--Procedure-->
    TEM imaging of OCK
+
  <div class="zairyou-heading">[Procedure]</div>
    </div>
+
    <ul class="procedure-list">
 +
      <li>Each sample was mixed as shown below:<sup>****</sup></li>
 +
    </table>
 +
    <br>
 +
&nbsp;****...Table1. Breakdown of Samples
 +
    <table>
 +
    <tr>
 +
    <th>Sample No.</th>
 +
    <td>1</td>
 +
    <td>2</td>
 +
    <td>3</td>
 +
    <td>4</td>
 +
    </tr>
 +
    <tr>
 +
    <th>Cholesterol hybridized OCK</th>
 +
    <td>50 µL</td>
 +
    <td>50 µL</td>
 +
    <td>-</td>
 +
    <td>-</td>
 +
    </tr>
 +
        <tr>
 +
    <th>OCK</th>
 +
    <td>-</td>
 +
    <td>-</td>
 +
    <td>50 µL</td>
 +
    <td>50 µL</td>
 +
    </tr>
 +
    <tr>
 +
    <th>Liposome</th>
 +
    <td>50 µL</td>
 +
    <td>-</td>
 +
    <td>50 µL</td>
 +
    <td>-</td>
 +
    </tr>
 +
    <tr>
 +
    <th>150 mM aqueous KCl solution</th>
 +
    <td>-</td>
 +
    <td>50 µL</td>
 +
    <td>-</td>
 +
    <td>50 µL</td>
 +
    </tr>
 +
        <tr>
 +
    <th>2.25 M Sucrose buffer</th>
 +
    <td>125 µL</td>
 +
    <td>125 µL</td>
 +
    <td>125 µL</td>
 +
    <td>125 µL</td>
 +
    </tr>
 +
    </table>
  
      <figure>
+
      <li>225 µL of 1.6 M sucrose buffer was overlaid with 225 µL of sample
        <center>
+
mixture in centrifuge tubes (Beckman, cat#343778, 11 x 34 mm).
        <img src="http://openwetware.org/images/e/ea/Monomer-Todai.png" width=480px height=360px>
+
</li>
            <figcaption> <b>TEM image of OCK</b>
+
      <li>Centrifuge for 16 minutes at 100 krpm at 4 ℃ using TLA 100.2 rotor (BECKMAN COULTER) with Ultracentrifuge (BECKMAN COULTER, Optima MAX-XP).</li>
            Three monomers of OCK were observed in this figure.  
+
      <li>150 µL of supernatant was extracted from top to bottom for 3 times (Fraction 1 to 3) and the pellet was retrieved with 150 µL of 1×Flotation buffer (Fraction 4).</li>
       
+
      <li>Fraction 1-4 of each sample were analyzed by 1 % agaraose gel
            </figcaption>
+
electrophoresis (100V, 1 hour).
        </center>
+
</li>
      </figure>
+
      <li>The Intensity of fluorescence of NileRed (Liposome) was measured with fluorescence spectrophotometer (JASCO, FP-6500) to investigate the existence of liposome in each Fraction.</li>
 +
      <li>The radiuses of liposome of each fraction were measured with DLS (Viscotek, 802 DLS).</li>
 +
    </ul>
  
    <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◆◆-->
+
    </article>
<!--editting-->
+
    <br>
  
      <h2 class="small-title"><a name="STEP2">&nbsp;STEP 2: Penetration into the membrane</a></h2>
+
    <br>
      <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>
 +
 +
<!--◆◆Preparation of GUVs◆◆-->
 +
 +
  <div class="mini-title">
 +
      <a name="Preparation_of_GUVs">2) Preparation of GUVs</a>
 +
  </div>
 +
  <article>
 +
<!--Reagent-->
 +
      <div class="zairyou-heading">[Reagent]</div>
 +
      <br>
 +
 +
    <table>
 +
    <tr>
 +
    <th>Lipid mix<sup>*</sup></th>
 +
    <td>3 ml</td>
 +
    </tr>
 +
 +
    <tr>
 +
    <th>150 mM KCl solution</th>
 +
    <td>1 µL</td>
 +
    </tr>
 +
 +
 +
    </table>
 
     <br>
 
     <br>
 +
&nbsp;*...Lipid mix
 +
    <table>
 +
    <tr>
 +
    <th>5 mg/mL POPC</th>
 +
    <td>0.1 mL</td>
 +
    </tr>
 +
    <tr>
 +
    <th>5 mg/mL POPG</th>
 +
    <td>0.1 mL</td>
 +
    </tr>
 +
        <tr>
 +
    <th>10 uM NileRed solution</th>
 +
    <td>0.13 mL</td>
 +
    </tr>
 +
    <tr>
 +
    <th>Chloroform</th>
 +
    <td>2.67 mL</td>
 +
    </tr>       
 +
    </table>
  
    <br>
+
<!--Procedure-->
<!--Result&Discussion1-->
+
  <div class="zairyou-heading">[Procedure]</div>
 +
    <ul class="procedure-list">
 +
      <li>A lipid film was formed by evaporating 3 ml of lipid mix in a 50 ml
 +
eggplant flask, using a rotational evaporator (EYELA, model#N1110) for
 +
10 mins.
 +
</li>
 +
      <li>The flask was kept under vacuum overnight to evaporate remaining chloroform.</li>
 +
      <li>The lipid film was resuspended in 1 mL of 150 mM KCl solution.</li>
 +
    </ul>
  
      <div class="zairyou-heading">[Result & Discussion]</div>
+
    </article>
 +
    <br>
  
    <div class="res-conclusion">
 
   
 
    </div>
 
  
      <figure>
+
<!--◆◆Preparation of SUVs◆◆-->
        <center>
+
 
        <img src="http://openwetware.org/images/c/ca/Todai_result_step2_fluolescence.JPG" width=450px height=350px>
+
  <div class="mini-title">
            <figcaption> <b>The fluorescence intensity of NIL (in liposome) in each fraction </b><br>
+
       <a name="Preparation_of_SUVs">3) Preparation of SUVs</a>
            The result of fluorescence spectrophotometer (JASCO, FP-6500) showed that liposome distributed mostly in fraction 3(lower layer).
+
  </div>
            </figcaption>
+
  <article>
        </center>
+
<b>Type 1: POPC 100%</b>
       </figure>
+
<!--Reagent-->
          <figure>
+
      <div class="zairyou-heading">[Reagent]</div>
        <center>
+
      <br>
        <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>
 
<figcaption>The ratios of OCK in each fraction were analyzed by the density of band.</figcaption>
 
        </center>
 
      </figure>
 
  
    <p class="paragraph">
+
    <table>
    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.
+
    <tr>
 +
    <th>150mM KCl solution</th>
 +
    <td>3mL</td>
 +
    </tr>
  
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.
+
    <tr>
 +
    <th>POPC</th>
 +
    <td>3mg</td>
 +
    </tr>
  
    </p>
+
    <tr>
    <div class="res-conclusion">
+
    <th>Chloroform (99.0%)</th>
    --> OCK stack in liposome.
+
    <td>3mL</td>
    </div>
+
    </tr>
    </article>
 
<!--◆◆2.2 Preparation of GUVs◆◆-->
 
  
      <article>
+
    <tr>
    <h3><a name="Preparation_of_GUVs"></a>2) Preparation of GUVs</h3>
+
    <th>40μM Nile Red solution</th>
    <!--Method-->
+
    <td>0.1mL</td>
    <div class="zairyou-heading">[Method]</div>
+
    </tr>
  
    <div id ="step2_2)">
+
     </table>
      <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>
 +
 +
<!--Procedure-->
 +
  <div class="zairyou-heading">[Procedure]</div>
 +
    <ul class="procedure-list">
 +
      <li>POPC were dissolved in 3mL of Chloroform.</li>
 +
      <li>A lipid film was formed by evaporating 3mL of POPC solution in a 50mL eggplant flask, using a rotational
 +
 +
evaporator for 5 minutes.</li>
 +
      <li>The flask was kept under vacuum overnight to evaporate remaining chloroform.</li>
 +
      <li>The lipid film was resuspended in 3mL of a 150mM KCl solution.</li>
 +
      <li>The solution was filtered through 200nm polar filter with extruder to even the size of liposome.</li>
 +
      <li>The size of liposome was measured with DLS (Viscotek 802 DLS).</li>
 +
      <li>The solution was kept at 3 degree C until usage.</li>
 +
    </ul>
 +
 +
    </article>
 
     <br>
 
     <br>
 +
<!--Preparation of SUVs_Added-->
 +
  <article>
 +
<b>Type 2: POPC 50%, POPG 50%</b>
 +
 +
<!--Reagent-->
 +
      <div class="zairyou-heading">[Reagent]</div>
 +
      <br>
  
    <br>
+
    <table>
<!--Result&Discussion1-->
+
    <tr>
 +
    <th>Lipid mix<sup>*</sup></th>
 +
    <td>3 ml</td>
 +
    </tr>
  
      <div class="zairyou-heading">[Result & Discussion]</div>
+
    <tr>
 +
    <th>150 mM KCl solution</th>
 +
    <td>1 µL</td>
 +
    </tr>
  
    <div class="res-conclusion">
 
   
 
    </div>
 
  
          <figure>
+
    </table>
        <center>
+
    <br>
        <img src="http://openwetware.org/images/b/be/GUVconfocal_scale-Todai.png" width=450px height=350px>
+
&nbsp;*...Lipid mix
 +
    <table>
 +
    <tr>
 +
    <th>5 mg/mL POPC</th>
 +
    <td>0.1 mL</td>
 +
    </tr>
 +
    <tr>
 +
    <th>5 mg/mL POPG</th>
 +
    <td>0.1 mL</td>
 +
    </tr>
 +
        <tr>
 +
    <th>10 uM NileRed solution</th>
 +
    <td>0.13 mL</td>
 +
    </tr>
 +
    <tr>
 +
    <th>Chloroform</th>
 +
    <td>2.67 mL</td>
 +
    </tr>       
 +
    </table>
  
        </center>
+
<!--Procedure-->
      </figure>
+
  <div class="zairyou-heading">[Procedure]</div>
 +
    <ul class="procedure-list">
 +
      <li>A lipid film was formed by evaporating 3 ml of lipid mix in a 50 ml
 +
eggplant flask, using a rotational evaporator (EYELA, model#N1110) for
 +
10 mins.
 +
</li>
 +
      <li>The flask was kept under vacuum overnight to evaporate remaining chloroform.</li>
 +
      <li>The lipid film was resuspended in 1 mL of 150 mM KCl solution.</li>
 +
      <li>Lipid suspended solution was filtered through 100nm polar filter using extruder (Avanti) to prepare uniformly-sized liposome.</li>
 +
      <li>The size of liposome was measured with DLS (Viscotek 802 DLS).</li>
 +
      <li>The solution was kept at 3℃ until usage.</li>
 +
    </ul>
  
    <p class="paragraph">
+
    </article>
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.
+
<!--◆◆Hybridization of cholesterol modified oligo◆◆-->
 +
  <article>
 +
  <div class="mini-title">
 +
      <a name="Protocol_Hybridization_of_cholesterol_oligo_with_OCK">4) Hybridization of cholesterol oligo with OCK</a>
 +
  </div>
  
    </p>
+
<!--Reagent-->
 +
      <div class="zairyou-heading">[Reagent]</div>
 +
      <br>
  
 +
    <table>
 +
    <tr>
 +
    <th>OCK</th>
 +
    <td>48 µL</td>
 +
    </tr>
  
    </article>
+
    <tr>
 +
    <th>Cholesterol oligo (0.32, 0.64, 3.2, 6.4 µM)</th>
 +
    <td>100 µL</td>
 +
    </tr>
  
<!--◆◆STEP 3: Recognition of cancer-specific proteins◆◆-->
+
    </table>
 +
<center>
 +
Marker; GeneRuler DNA Ladder Mix (Fermentas, GeneRuler DNA Ladder Mix #SM0331)
 +
</center>
  
    <article>
+
<!--Procedure-->
      <h2 class="small-title"><a name="STEP3">&nbsp;STEP3: Subunits recognize cancer-specific proteins. </a></h2>
+
  <div class="zairyou-heading">[Procedure]</div>
    <h3><a name="Optimization_of_aptamer_lock_system"></a>1) Optimization of aptamer-lock system</h3>
+
    <ul class="procedure-list">
<!--Method-->
+
      <li>Centrifuge for 16 minutes at 100 krpm at 4 ℃ using TLA 100.2 rotor (BECKMAN COULTER) with Ultracentrifuge (BECKMAN COULTER, Optima MAX-XP).</li>
    <div class="zairyou-heading">[Method]</div>
+
      <li>Each sample was mixed and incubated as shown below:<sup>*</sup></li>
            <figure>
+
    </table>
 +
    <br>
 +
&nbsp;*...Table1.
 +
    <table>
 +
<center>
 +
    <tr>
 +
    <th>Sample No.</th>
 +
    <td>1</td>
 +
    <td>2</td>
 +
    <td>3</td>
 +
    <td>4</td>
 +
    <td>5</td>
 +
    <td>6</td>
 +
    <td>7</td>
 +
    <td>8</td>
 +
    </tr>
 +
    <tr>
 +
    <th>Purified OCK (40 µM)</th>
 +
    <td>6 µL</td>
 +
    <td>6 µL</td>
 +
    <td>6 µL</td>
 +
    <td>6 µL</td>
 +
    <td>6 µL</td>
 +
    <td>6 µL</td>
 +
    <td>6 µL</td>
 +
    <td>6 µL</td>
 +
    </tr>
 +
        <tr>
 +
    <th>0.32 µM Cholesterol oligo</th>
 +
    <td>1.5 µL</td>
 +
    <td>1.5 µL</td>
 +
    <td>-</td>
 +
    <td>-</td>
 +
    <td>-</td>
 +
    <td>-</td>
 +
    <td>-</td>
 +
    <td>-</td>
 +
    </tr>
 +
    <tr>
 +
    <th>0.64 µM Cholesterol oligo</th>
 +
    <td>-</td>
 +
    <td>-</td>
 +
    <td>1.5 µL</td>
 +
    <td>1.5 µL</td>
 +
    <td>-</td>
 +
    <td>-</td>
 +
    <td>-</td>
 +
    <td>-</td>
 +
    </tr>
 +
    <tr>
 +
    <th>3.2 µM Cholesterol oligo</th>
 +
    <td>-</td>
 +
    <td>-</td>
 +
    <td>-</td>
 +
    <td>-</td>
 +
    <td>1.5 µL</td>
 +
    <td>1.5 µL</td>
 +
    <td>-</td>
 +
    <td>-</td>
 +
    </tr>
 +
        <tr>
 +
    <th>6.4 µM Cholesterol oligo</th>
 +
    <td>-</td>
 +
    <td>-</td>
 +
    <td>-</td>
 +
    <td>-</td>
 +
    <td>-</td>
 +
    <td>-</td>
 +
    <td>1.5 µL</td>
 +
    <td>1.5 µL</td>
 +
    </tr>
 +
        <tr>
 +
    <th>[Cholesterol oligo]/[OCK] (see #Note)</th>
 +
    <td>1/2</td>
 +
    <td>1/2</td>
 +
    <td>1</td>
 +
    <td>1</td>
 +
    <td>5</td>
 +
    <td>5</td>
 +
    <td>10</td>
 +
    <td>10</td>
 +
    </tr>
 +
<tr>
 +
    <th>Incubation time [min]</th>
 +
    <td>60</td>
 +
    <td>30</td>
 +
    <td>60</td>
 +
    <td>30</td>
 +
    <td>60</td>
 +
    <td>30</td>
 +
    <td>60</td>
 +
    <td>30</td>
 +
    </tr>
 +
</center>
 +
    </table>
 +
#Note; OCK has 4 cholesterol oligo binding sites. Therefore, we devided the molar ratio of cholesterol oligo to OCK with 4.
 +
                <figure>
 
         <center>
 
         <center>
         <img src="http://openwetware.org/images/5/50/Todai_Recognition_ideal_mod.png" width=480px height=270px>
+
         <img src="http://openwetware.org/images/6/66/OCK_Cholesterol-Todai.png" width=300px height=300px>
 +
       
 
         </center>
 
         </center>
 
       </figure>
 
       </figure>
 +
 +
      <li>Each sample was analyzed by 1% agarose gel electrophoresis (100V, 1 hour).</li>
 +
 +
    </ul>
 +
 +
 +
    </article>
 +
    <br>
  
  
    <div id ="step0_1)">
+
 
      <p class="paragraph">
+
 
     
+
<!--◆◆STEP3◆◆-->
      We did pilot study of oligomerization process triggered by membrane
+
<h2 class="PS_title"><a name="STEP3">&nbsp;STEP 3: Subunits recognize cancer-specific proteins.</a></h2>
      protein recognition. We used cholesterol modified PDGF as model membrane
+
<article>
      protein, as DNA origami embedded aptamer system recognizing PDGF was
+
<!--◆◆Reaction_of_a_biotinized_oligo_to_streptavidin◆◆-->
       reported (Douglass et al. (2012)).
+
  <article>
     
+
  <div class="mini-title">
      </p>
+
       <a name="Reaction_of_a_biotinized_oligo_to_streptavidin">1) Reaction of a biotinized oligo to streptavidin</a>
 +
  </div>
  
       <p class ="paragraph">
+
<!--Reagent-->
       Our simplified model lock system is consisted with two steps: 1)
+
       <div class="zairyou-heading">[Reagent]</div>
Blocking of streptavidin binding to biotin by steric hindrance. Our lock
+
       <br>
system consists of two strands: biotin attached strands (biotin strands)
+
<li>materials for hybridization</li>
and aptamer attached strands (aptamer strands). These two strands
+
        <li>5ap_M-3t4e_T0 (1uM) (oligo): 5’ to 3’</li>
hybridize each other in inactive form and hide biotin moiety from the
+
TACTCAGGGCACTGCAAGCAATTGTGGTCCCAATGGGCTGAGTACGCGACCTCATCTTTGACCCCCAGGCAGGGAG
streptavidin by steric hindrance effect. 2) Upon binding of ligands
+
        <li>5ap_5B_16 (10uM) (oligo, which has biotin in 5’ end):</li>
(PDGF in this study) to aptamer strands, the complementary strand(biotin
+
Biotin-TACTCAGCCCATTGGG
strands) is released from the DNA aptamer, because ligands take over the
+
        <li>10x tile buffer<sup>*</sup></li>
DNA strands of DNA aptamer from the complementally strands, and biotin
+
        <li>MilliQ</li>
can now bind to streptavidin. Therefore, the cancer cell recognition and
 
OCK oligomerization are achieved simultaneously in the future study.
 
      </p>
 
  
     </div>
+
     </table>
 
     <br>
 
     <br>
 +
&nbsp;*...10x tile buffer(f.100 µl)
 +
    <table>
 +
    <tr>
 +
    <th>Mg(OAc)<sub>2</sub></th>
 +
    <td>f.100 mM</td>
 +
    </tr>
 +
    <tr>
 +
    <th>Tris-HCl (pH7.5)</th>
 +
    <td>f.200 mM</td>
 +
    </tr>
 +
        <tr>
 +
    <th>EDTA</th>
 +
    <td>f.10 mM</td>
 +
    </tr>
 +
 +
    </table>
 +
 +
<li>5ap_tile</li>
 +
        <li>M13mp18 (scaffold)</li>
 +
        <li>Cy5_Rmix (staples)</li>
 +
        <li>10x tile buffer</li>
 +
        <li>Cy3 streptavidin (800nM)</li>
 +
 +
<!--Procedure-->
 +
  <div class="zairyou-heading">[Procedure]</div>
 +
    <ul class="procedure-list">
 +
      <li>Hybridization</li>
 +
<li>Mix materials (mentioned above in [hybridization]) in 0.2 ml PCR-tubes.</li>
 +
<li>Incubate the mixture at the room temperature (25 ℃) for 1 hour.</li>
 +
 +
      <li>Making 5ap_tile</li>
 +
<li>Mix materials (mentioned above in [5ap_tile]) in 0.2 ml PCR-tubes.</li>
 +
<li>Anneal the mixture using PCR machine (from 85 ℃ to 25 ℃, -2 ℃/min).</li>
 +
 +
      <li>Insertion of hybridized double-stranded DNA into 5ap_tile</li>
 +
<li>Mix hybridized double-stranded DNA and 5ap_tile in 0.2 ml PCR-tubes.</li>
 +
<li>Incubate the mixture at 48 ℃ for 1 hour.</li>
 +
<li>Mix the mixture and Cy3 streptavidin.</li>
 +
 +
      <li>1wt% Agarose-gel Electrophoresis</li>
 +
<li>Electrophoresis the inserted 5ap_tile for 50 minutes at 100 V at 4 ℃.</li>
 +
<li>Take photographs of the electrophoresed gel by LAS-4000.</li>
 +
 +
 +
    </ul>
 +
 +
    </article>
 
     <br>
 
     <br>
<br>
 
    </article>
 
    <br>
 
<!--Result&Discussion1-->
 
  
       <div class="zairyou-heading">[Result & Discussion]</div>
+
<!--◆◆Reaction_between_aptamer_embedded_in_rect_tile_and_PDGF10◆◆-->
 +
  <article>
 +
  <div class="mini-title">
 +
      <a name="Reaction_between_aptamer_embedded_in_rect_tile_and_PDGF10">2) Reaction between aptamer embedded in rect tile and PDGF</a>
 +
  </div>
 +
 
 +
<!--Reagent-->
 +
       <div class="zairyou-heading">[Reagent]</div>
 +
      <br>
 +
<li>materials for hybridization</li>
 +
        <li>5ap_M-3t4e_T0 (1uM) (oligo): 5’ to 3’</li>
 +
TACTCAGGGCACTGCAAGCAATTGTGGTCCCAATGGGCTGAGTACGCGACCTCATCTTTGACCCCCAGGCAGGGAG
 +
  <li>5ap_M-3t4e_T-3(1µM) (oligo):</li> 
 +
TACTCAGGGCACTGCAAGCAATTGTGGTCCCAATGGGCTGAGTAGACCTCATCTTTGACCCCCAGGCAGGGAG
 +
 
 +
<li>5ap_M-3t4e_T-1(1µM) (oligo):</li>
 +
TACTCAGGGCACTGCAAGCAATTGTGGTCCCAATGGGCTGAGTAGCGACCTCATCTTTGACCCCCAGGCAGGGAG
 +
  <li>5ap_M-3t4e_T7(1µM) (oligo):</li>
 +
TACTCAGGGCACTGCAAGCAATTGTGGTCCCAATGGGCTGAGTAtttttttCGCGACCTCATCTTTGACCCCCAGGCAGGGAG
 +
<li>5ap_5B_16 (10uM) (oligo, which has biotin in 5’ end):</li>
 +
Biotin-TACTCAGCCCATTGGG
 +
        <li>10x tile buffer<sup>*</sup></li>
 +
        <li>MilliQ</li>
 +
 
 +
    </table>
 +
    <br>
 +
&nbsp;*...10x tile buffer(f.100 µl)
 +
    <table>
 +
    <tr>
 +
    <th>Mg(OAc)<sub>2</sub></th>
 +
    <td>f.100 mM</td>
 +
    </tr>
 +
    <tr>
 +
    <th>Tris-HCl (pH7.5)</th>
 +
    <td>f.200 mM</td>
 +
    </tr>
 +
        <tr>
 +
    <th>EDTA</th>
 +
    <td>f.10 mM</td>
 +
    </tr>
 +
 
 +
    </table>
 +
 
 +
<li>5ap_tile</li>
 +
        <li>M13mp18 (scaffold)</li>
 +
        <li>5ap_Rmix (staples)</li>
 +
        <li>10x tile buffer</li>
 +
        <li>PDGF(dye 45nM)</li>
 +
<!--Procedure-->
 +
  <div class="zairyou-heading">[Procedure]</div>
 +
    <ul class="procedure-list">
 +
      <li>Hybridization</li>
 +
<li>Mix materials (mentioned above in [hybridization]) in 0.2 ml PCR-tubes.</li>
 +
<li>Incubate the mixture at the room temperature (25 ℃) for 1 hour.</li>
 +
 
 +
      <li>Making 5ap_tile</li>
 +
<li>Mix materials (mentioned above in [5ap_tile]) in 0.2 ml PCR-tubes.</li>
 +
<li>Anneal the mixture using PCR machine (from 85 ℃ to 25 ℃, -2 ℃/min).</li>
 +
 
 +
      <li>Insertion of hybridized double-stranded DNA into 5ap_tile</li>
 +
<li>Mix hybridized double-stranded DNA and 5ap_tile in 0.2 ml PCR-tubes.</li>
 +
<li>Incubate the mixture at 48 ℃ for 1 hour.</li>
 +
<li>Mix the mixture and PDGF.</li>
 +
        <li>Incubate at 37 ℃ for 30 min and then at 4 ℃ for 25 hours.</li>
 +
 
 +
      <li>1wt% Agarose-gel Electrophoresis</li>
 +
<li>Electrophoresis the inserted 5ap_tile for 50 minutes at 100 V at 4 ℃.</li>
 +
        <li>Stain the gel by SYBR Gold in TBE.</li>
 +
<li>Take photographs of the electrophoresed gel by LAS-4000.</li>
 +
 
 +
 
 +
    </ul>
 +
 
 +
    </article>
 +
    <br>
 +
 
 +
 
 +
 
 +
<!--◆◆efficient_hybridization_(changing_mixture_ratio)_Added◆◆-->
 +
  <article>
 +
  <div class="mini-title">
 +
      <a name="efficient_hybridization_(changing_mixture_ratio)">3) Efficient hybridization (changing mixture ratio)</a>
 +
  </div>
  
    <div class="res-conclusion">
+
<!--Reagent-->
    Integration of aptamer strands into DNA origami tile
+
      <div class="zairyou-heading">[Reagent]</div>
    </div>
+
      <br>
                          <figure>
+
    <li>5ap_M-3t4e_T0 (1 µM): 5’ to 3’</li>
        <center>
+
TACTCAGGGCACTGCAAGCAATTGTGGTCCCAATGGGCTGAGTACGCGACCTCATCTTTGACCCCCAGGCAGGGAG
        <img src="http://openwetware.org/images/d/d3/Todai_intofapt_1.png" width=600px height=450px>
+
    <li>5ap_5B_16 (1 or 10 µM): Biotin in 5’ end</li>
        </center>
+
Biotin - TACTCAGCCCATTGGG
        </figure>
+
    <li>10x tile buffer<sup>*</sup></li>
                                  <figure>
+
    <li>MilliQ</li>
        <center>
 
        <img src="http://openwetware.org/images/6/6c/Todai_intofapt_2.png" width=600px height=450px>
 
        </center>
 
        </figure>
 
                    <figure>
 
        <center>
 
        <img src="http://openwetware.org/images/7/7f/Tile-ins.png" width=480px height=360px>
 
            <figcaption> <b></b>
 
  
            </figcaption>
+
    </table>
        </center>
+
    <br>
      </figure>
+
&nbsp;*...10x tile buffer
      <p class="paragraph">                               We confirmed the integration of aptamer attached strands (aptamer
+
    <table>
strands) into rectangle DNA origami tile (rect-tile).
+
    <tr>
</p>
+
    <th>Mg(OAc)<sub>2</sub></th>
<br>
+
    <td>f.100 mM</td>
    <div class="res-conclusion">
+
    </tr>
    Responsibility of aptamer
+
    <tr>
    </div>
+
    <th>Tris-HCl (pH7.5)</th>
                <figure>
+
    <td>f.200 mM</td>
         <center>
+
    </tr>
        <img src="http://openwetware.org/images/4/42/Todai_Recognition_tile_B_PDGF_mod.png" width=600px height=450px>
+
         <tr>
 +
    <th>EDTA</th>
 +
    <td>f.10 mM</td>
 +
    </tr>
  
        </center>
+
    </table>
      </figure>
 
      <figure>
 
        <center>
 
        <img src="http://openwetware.org/images/f/f5/Figure9_10-Todai.png" width=480px height=270px>
 
            <figcaption> <b></b>
 
  
  
            </figcaption>
+
<!--Procedure-->
        </center>
+
  <div class="zairyou-heading">[Procedure]</div>
      </figure>
+
    <ul class="procedure-list">
      <p class ="paragraph">
+
<li>Mix materials to make samples, following the ratio written in Table.3.<sup>*</sup></li>
            We confirmed the responsibility of aptamer sequence embedded in
+
    </table>
rect-tile (shown above). The position of Cy5-PDGF band coincided
+
    <br>
with that of DNA tile, showing that the aptamers work also on rect-tile.
+
&nbsp;*...Table.3
Furthermore, the linker length between aptamer sequence and staple
+
    <table>
sequence, the latter staple sequence is embedded into rect-tile, does
+
<center>
not affect the binding ability of aptamer to PDGF.
+
    <tr>
</p>
+
    <th>Sample No.</th>
<br>
+
    <td>1</td>
    <div class="res-conclusion">
+
    <td>2</td>
    Blocking capability of lock system  by aptamer
+
    <td>3</td>
    </div>
+
    <td>4</td>
                    <figure>
+
    <td>5</td>
        <center>
+
    <td>6</td>
        <img src="http://openwetware.org/images/9/96/Todai_Recognition_tile_B_SA_mod.png" width=600px height=450px>
+
    <td>7</td>
 +
    <td>8</td>
 +
    </tr>
  
        </center>
+
    <tr>
      </figure>
+
    <th>5ap_M-3t4e_T0 (1 µM)</th>
      <table cellpadding="0" style="position:relative;left:-50px;">
+
    <td>3 µL</td>
        <tbody><tr>
+
    <td>-</td>
        <td>
+
    <td>-</td>
        <figure>
+
    <td>3 µL</td>
          <img src="http://openwetware.org/images/3/30/Figure11_12-Todai.png" width="360px" height="240px">
+
    <td>3 µL</td>
 +
    <td>3 µL</td>
 +
    <td>3 µL</td>
 +
    <td>3 µL</td>
 +
    </tr>
  
         </figure>
+
         <tr>
        </td>
+
    <th>5ap_5B_16 (10 µM)</th>
 +
    <td>-</td>
 +
    <td>-</td>
 +
    <td>-</td>
 +
    <td>-</td>
 +
    <td>-</td>
 +
    <td>1.5 µL</td>
 +
    <td>3 µL</td>
 +
    <td>6 µL</td>
 +
    </tr>
  
        <td>
+
    <tr>
        <figure  style="position:relative;left:-50px;">
+
    <th>5ap_5B_16 (1 µM)</th>
          <img src="http://openwetware.org/images/2/24/Koyama_131027_1-Todai.JPG" width="240px" height="240px">
+
    <td>-</td>
 +
    <td>3 µL</td>
 +
    <td>-</td>
 +
    <td>3 µL</td>
 +
    <td>6 µL</td>
 +
    <td>-</td>
 +
    <td>-</td>
 +
    <td>-</td>
 +
    </tr>
  
         </figure>
+
    <tr>
         </td>
+
    <th>10x tile buffer</th>
        </tr>
+
    <td>1 µL</td>
      </tbody></table>
+
    <td>1 µL</td>
 +
    <td>1 µL</td>
 +
    <td>1 µL</td>
 +
    <td>1 µL</td>
 +
    <td>1 µL</td>
 +
    <td>1 µL</td>
 +
    <td>1 µL</td>
 +
    </tr>
 +
         <tr>
 +
    <th>MilliQ</th>
 +
    <td>6 µL</td>
 +
    <td>6 µL</td>
 +
    <td>9 µL</td>
 +
    <td>3 µL</td>
 +
    <td>-</td>
 +
    <td>4.5 µL</td>
 +
    <td>3 µL</td>
 +
    <td>-</td>
 +
    </tr>
 +
         <tr>
 +
    <th>Ratio of concentration of 5ap_5B_16 to 5ap_M-3t4e_T0</th>
 +
    <td>-</td>
 +
    <td>-</td>
 +
    <td>-</td>
 +
    <td>1 %</td>
 +
    <td>2 %</td>
 +
    <td>5 %</td>
 +
    <td>10 %</td>
 +
    <td>20 %</td>  
 +
    </tr>
 +
</center>
 +
    </table>
  
 +
<li>Apply the samples to 10 % Native-PAGE for 85 minutes at 100 V at 4 ℃.</li>
 +
<li>Stain the gel by SYBR Gold in TBE.</li>
 +
<li>Take a photograph of the gel by LAS-4000.</li>
  
    <p class="paragraph">
+
    </ul>
  
We confirmed the blocking capability of our lock system for streptavidin
+
    </article>
binding (left figure, the image of gel electrophoresis). Our lock system consists of two strands:
+
    <br>
biotin attached strands (biotin strands) and aptamer attached strands
+
    <!--◆◆efficient hybridization (incubation time)◆◆-->
(aptamer strands). These two strands hybridize each other in inactive
+
  <article>
form and hide biotin moiety from the streptavidin by steric hindrance
+
  <div class="mini-title">
effect. We confirmed this blocking capability by mixing Cy3 labeled
+
      <a name="efficient_hybridization_(incubation_time)">4) efficient hybridization (incubation time)</a>
streptavidin with lock system embedded rect-tile. Data indicates that
+
  </div>
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/e/ec/Todai_intofapt_3.png" width=600px height=450px>
 
        </center>
 
        </figure>
 
      <figure>
 
        <center>
 
        <img src="http://openwetware.org/images/c/c8/Tile_double_insertion-Todai.png" width=480px height=360px>
 
            <figcaption> <b></b>
 
  
 +
<!--Reagent-->
 +
      <div class="zairyou-heading">[Reagent]</div>
 +
      <br>
 +
    <li>5ap_M-3t4e_T0 (1 µM): 5’ to 3’</li>
 +
TACTCAGGGCACTGCAAGCAATTGTGGTCCCAATGGGCTGAGTACGCGACCTCATCTTTGACCCCCAGGCAGGGAG
 +
    <li>5ap_5B_16 (10 µM): (biotin in 5’ end)</li>
 +
Biotin - TACTCAGCCCATTGGG
 +
    <li>10x tile buffer<sup>*</sup></li>
 +
    <li>MilliQ</li>
  
            </figcaption>
+
    </table>
        </center>
+
    <br>
      </figure>
+
&nbsp;*...10x tile buffer(f.100 µl)
Next, we optimize the embedding condition of our lock system into
+
    <table>
rect-tile. This time full length of biotin strands were used instead of
+
    <tr>
truncated ones used in above figure. Data indicate that the integrate
+
    <th>Mg(OAc)<sub>2</sub></th>
efficiency of both biotin strands and aptamer strands into rect-tile is
+
    <td>f.100 mM</td>
independent on the incubate temperature.
+
    </tr>
 +
    <tr>
 +
    <th>Tris-HCl (pH7.5)</th>
 +
    <td>f.200 mM</td>
 +
    </tr>
 +
        <tr>
 +
    <th>EDTA</th>
 +
    <td>f.10 mM</td>
 +
    </tr>
  
We improve our lock system everyday. Don't miss our presentation in
+
     </table>
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>
+
<!--Procedure-->
 +
  <div class="zairyou-heading">[Procedure]</div>
 +
    <ul class="procedure-list">
 +
<li>Mix the materials in 0.2 ml PCR-tubes.</li>
 +
<li>Denature the oligos at 95 ℃ for 30 seconds.</li>
 +
<li>Incubate the mixture at room temperature (25 ℃).</li>
 +
<li>Freeze the samples into nitrogen liquid at planned incubation time.</li>
 +
<li>Pick up the samples out from nitrogen liquid immediately before applying into gel.</li>
 +
<li>Apply the samples to 10 % Native PAGE for 85 minutes at 100 V at 4 ℃.</li>
 +
<li>Take a photograph of the electrophoresed gel by LAS-4000.</li>
  
    <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>
+
    </ul>
      </figure>
 
     
 
          <div class="res-conclusion">
 
    -->PDGF was recognized by the aptamer of OCK
 
    </div>
 
<!--◆◆STEP 4: Oligomerization in solution◆◆-->
 
  
    <article>
+
    </article>
      <h2 class="small-title"><a name="STEP4">&nbsp;STEP 4: Oligomerization in solution</a></h2>
+
    <br>
     
+
<!-- ◆◆Insertion of hybridized double-stranded DNA into tile◆◆-->
<!--◆◆4.1 Oligomerization by streptavidin-biotin complex◆◆-->
+
  <article>
 +
  <div class="mini-title">
 +
      <a name="insertion_of__hybridized_double-stranded_DNA_into_tile">5) Insertion of hybridized double-stranded DNA into tile</a>
 +
  </div>
  
    <h3><a name="Oligomerization_by_streptavidin-biotin_complex"></a>1) Oligomerization by streptavidin-biotin complex</h3>
+
<!--Reagent-->
    <!--Method-->
+
      <div class="zairyou-heading">[Reagent]</div>
    <div class="zairyou-heading">[Method]</div>
+
      <br>
 +
hybridization
 +
    <li>5ap_M-3t4e_T0 (1 µM) (oligo): 5’ to 3’</li>
 +
TACTCAGGGCACTGCAAGCAATTGTGGTCCCAATGGGCTGAGTACGCGACCTCATCTTTGACCCCCAGGCAGGGAG
 +
    <li>5ap_5B3G_16 (1 µM) (oligo, which has biotin in 5’ end and Cy3 in 3’ end):</li>
 +
Biotin–TACTCAGCCCATTGGG–Cy3
 +
    <li>10x tile buffer<sup>*</sup></li>
 +
    <li>MilliQ</li>
  
    <div id ="step3_1)">
+
     </table>
      <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>
 +
&nbsp;*...10x tile buffer(f.100 µl)
 +
    <table>
 +
    <tr>
 +
    <th>Mg(OAc)<sub>2</sub></th>
 +
    <td>f.100 mM</td>
 +
    </tr>
 +
    <tr>
 +
    <th>Tris-HCl (pH7.5)</th>
 +
    <td>f.200 mM</td>
 +
    </tr>
 +
        <tr>
 +
    <th>EDTA</th>
 +
    <td>f.10 mM</td>
 +
    </tr>
 +
 +
    </table>
 +
 +
5ap_tile
 +
    <li>M13mp18 (scaffold)</li>
 +
    <li>5ap_Rmix (staples)</li>
 +
    <li>10x tile buffer</li>
 +
 +
<!--Procedure-->
 +
  <div class="zairyou-heading">[Procedure]</div>
 +
    <ul class="procedure-list">
 +
Hybridization
 +
<li>Mix materials (mentioned above in [hybridization]) in 0.2 ml PCR-tubes.</li>
 +
<li>Incubating the mixture at the room temperature (25 ℃) for 1 hour.</li>
 +
 +
Making 5ap_tile
 +
<li>Mix materials (mentioned above in [5ap_tile]) in 0.2 ml PCR-tubes.</li>
 +
<li>Anneal the mixture using PCR machine (from 85 ℃ to 25 ℃, -2 ℃/min).</li>
 +
 +
Insertion of hybridized double-stranded DNA into 5ap_tile
 +
<li>Mix hybridized double-stranded DNA and 5ap_tile in 0.2 ml PCR-tubes.</li>
 +
<li>Incubate the mixture at 48 ℃ for 1 hour.</li>
 +
 +
1 wt% Agarose-gel Electrophoresis
 +
<li>Electrophoresis the inserted 5ap_tile for 50 minutes at 100 V at 4 ℃.</li>
 +
        <li>Take photographs of the electrophoresed gel by LAS-4000.</li>
 +
 +
    </ul>
 +
 +
    </article>
 
     <br>
 
     <br>
 +
<!--Reaction_between_aptamer_(3ap-M5t10f-T0)_and_PDGF_Added-->
 +
  <article>
 +
  <div class="mini-title">
 +
      <a name="Reaction_between_aptamer_(3ap-M5t10f-T0)_and_PDGF">6) Reaction between aptamer (3ap-M5t10f-T0) and PDGF</a>
 +
  </div>
 +
 +
<!--Reagent-->
 +
      <div class="zairyou-heading">[Reagent]</div>
 +
      <br>
 +
<li>materials for the reaction</li>
 +
    <table>
 +
    <tr>
 +
    <th>10x tile buffer </th>
 +
    <td>f. 1x tile buffer</td>
 +
    </tr>
 +
 +
    <tr>
 +
    <th>1 µM aptamer</th>
 +
    <td>f. 0.3µM</td>
 +
    </tr>
 +
 +
    <tr>
 +
    <th>1 µM Ladder151515_1</th>
 +
    <td>f. 0.3µM</td>
 +
    </tr>
  
    </article>
+
    <tr>
    <br>
+
    <th>PDGF (dye 45nM)</th>
<!--Result&Discussion1-->
+
    <td>f. 20mM</td>
 +
    </tr>
  
      <div class="zairyou-heading">[Result & Discussion]</div>
+
    </table>
  
    <div class="res-conclusion">
+
<li>materials for the electrophoresis</li>
    Streptavidins induced oligomerization
+
    <gel for 10% Native-PAGE>
    </div>
+
    <table>
 +
    <tr>
 +
    <th>MilliQ</th>
 +
    <td>7.9 mL</td>
 +
    </tr>
 +
 
 +
    <tr>
 +
    <th>30% Acrylamide mix</th>
 +
    <td>6.7 mL</td>
 +
    </tr>
 +
 
 +
    <tr>
 +
    <th>1.5 M Tris-HCl (pH 8.8)</th>
 +
    <td>5 mL</td>
 +
    </tr>
 +
 
 +
    <tr>
 +
    <th>1 M MgCl2</th>
 +
    <td>200 µL</td>
 +
    </tr>
 +
 
 +
    <tr>
 +
    <th>10 % APS</th>
 +
    <td>80 µL</td>
 +
    </tr>
 +
 
 +
    <tr>
 +
    <th>TEMED</th>
 +
    <td>80 µL</td>
 +
    </tr>
 +
 
 +
    </table>
 +
 
 +
    <Electrophoresis buffer for 10 % Native-PAGE>
 +
      1x TBE
 +
 
 +
    <materials for stain>
 +
    <table>
 +
    <tr>
 +
    <th>Electrophoresis buffer for 10 % Native-PAGE</th>
 +
    <td>50 mL</td>
 +
    </tr>
 +
 
 +
    <tr>
 +
    <th>SYBR Gold</th>
 +
    <td>5 µL</td>
 +
    </tr>
 +
 
 +
    </table>
 +
 
 +
    <others>
 +
    <table>
 +
    <tr>
 +
    <th>Loading buffer</th>
 +
    <th>20 % glycerol (as used 6x)</th>
 +
    </tr>
 +
 
 +
    <tr>
 +
    <th>Marker</th>
 +
    <th>Cy5 38 mer</th>
 +
    </tr>
 +
 
 +
    </table>
  
      <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)">
+
<!--Procedure-->
      <p class="paragraph">
+
  <div class="zairyou-heading">[Procedure]</div>
      Dimers of OCKs were also imaged by TEM to confirm the bands observed in the experiment 3.1) originated from the dimers.
+
    <ul class="procedure-list">
        (<a target="_blank" href="http://openwetware.org/wiki/Biomod/2013/Todai/Experiment#Protocols" style="color:#e00000;">
+
      <li>Mix the solutions as shown below:<sup>*</sup></li>
        protocols
+
     </table>
        </a>
 
        )
 
        </p>
 
     </div>
 
 
     <br>
 
     <br>
 +
&nbsp;*...Table.2
 +
    <table>
 +
<center>
 +
    <tr>
 +
    <th>Sample No.</th>
 +
    <td>1</td>
 +
    <td>2</td>
 +
    <td>3</td>
 +
    <td>4</td>
 +
    <td>5</td>
 +
    <td>6</td>
 +
    </tr>
 +
 +
    <tr>
 +
    <th>10x tile buffer</th>
 +
    <td>1 µL</td>
 +
    <td>1 µL</td>
 +
    <td>1 µL</td>
 +
    <td>1 µL</td>
 +
    <td>1 µL</td>
 +
    <td>1 µL</td>
 +
    </tr>
 +
 +
        <tr>
 +
    <th>1 µM aptamer</th>
 +
    <td>3 µL</td>
 +
    <td>-</td>
 +
    <td>-</td>
 +
    <td>3 µL</td>
 +
    <td>-</td>
 +
    <td>-</td>
 +
    </tr>
 +
 +
    <tr>
 +
    <th>1 µM Ladder151515_1</th>
 +
    <td>-</td>
 +
    <td>-</td>
 +
    <td>-</td>
 +
    <td>-</td>
 +
    <td>3 µL</td>
 +
    <td>3 µL</td>
 +
    </tr>
 +
 +
    <tr>
 +
    <th>PDGF (dye 45 nM)</th>
 +
    <td>-</td>
 +
    <td>6 µL</td>
 +
    <td>-</td>
 +
    <td>6 µL</td>
 +
    <td>-</td>
 +
    <td>6 µL</td>
 +
    </tr>
 +
        <tr>
 +
    <th>MilliQ</th>
 +
    <td>6 µL</td>
 +
    <td>3 µL</td>
 +
    <td>9 µL</td>
 +
    <td>-</td>
 +
    <td>6 µL</td>
 +
    <td>-</td>
 +
    </tr>
 +
 +
</center>
 +
    </table>
 +
 +
      <li>Incubate at 37 ℃ for 30 min and then at 4 ℃ for 30 min.</li>
 +
      <li>Make 10 % Native PAGE gel as mentioned above.</li>
 +
      <li>Add loading buffer into each samples.</li>
 +
      <li>Apply the samples to 10 % Native-PAGE for 85 minutes at 100V at 4℃.</li>
 +
      <li>Take a photograph of the electrophoresed gel by LAS-4000 for Cy5.</li>
 +
      <li>Stain the gel by SYBR Gold for 20 min.</li>
 +
      <li>Take a photograph by LAS-4000.</li>
 +
 +
    </ul>
 +
 +
    </article>
 
     <br>
 
     <br>
  
    </article>
 
    <br>
 
<!--Result&Discussion1-->
 
  
      <div class="zairyou-heading">[Result & Discussion]</div>
+
</article>
  
    <div class="res-conclusion">
+
<!-- ◆◆Double insertion of hybridized double-stranded DNA into tile◆◆-->
    </div>
+
  <article>
    <center>
+
  <div class="mini-title">
       <table cellpadding="0" style ="position:relative;left:-30px;">
+
      <a name="Double insertion_of__hybridized_double-stranded_DNA_into_tile">7) Double insertion of hybridized double-stranded DNA into tile</a>
 +
  </div>
 +
 
 +
<!--Reagent-->
 +
       <div class="zairyou-heading">[Reagent]</div>
 +
      <br>
 +
hybridization
 +
    <li>5ap_R125_T-3_Cy5 (1 µM) (oligo):</li>
 +
TACTCAGGGCACTGCAAGCAATTGTGGTCCCAATGGGCTGAGTAGACCTCATCTTTGACCCCCAGGCAGGGAG
 +
    <li>5ap_5B3T30_16_Cy3 (1 µM) (oligo, which has Cy3 in 5’ end):</li>
 +
TACTCAGCCCATTGGGttttttttttttttttttttttttttttttAAAACACTGCTCCATGTTACTTAACAAAGCT 
 +
    <li>10x tile buffer<sup>*</sup></li>
 +
    <li>MilliQ</li>
 +
 
 +
    </table>
 +
    <br>
 +
&nbsp;*...10x tile buffer(f.100 µl)
 +
    <table>
 +
    <tr>
 +
    <th>Mg(OAc)<sub>2</sub></th>
 +
    <td>f.100 mM</td>
 +
    </tr>
 +
    <tr>
 +
    <th>Tris-HCl (pH7.5)</th>
 +
    <td>f.200 mM</td>
 +
    </tr>
 
         <tr>
 
         <tr>
         <td>
+
    <th>EDTA</th>
         <figure>
+
    <td>f.10 mM</td>
          <img src="http://openwetware.org/images/c/c1/Dimerv2-Todai.png" width="300px" height="300px" >
+
    </tr>
        </figure>
+
 
        </td>
+
    </table>
 +
 
 +
5ap_tile
 +
    <li>M13mp18 (scaffold)</li>
 +
    <li>5ap_Rmix (staples)</li>
 +
    <li>10x tile buffer</li>
 +
 
 +
<!--Procedure-->
 +
  <div class="zairyou-heading">[Procedure]</div>
 +
    <ul class="procedure-list">
 +
Hybridization
 +
<li>Mix materials (mentioned above in [hybridization]) in 0.2 ml PCR-tubes.</li>
 +
<li>Incubating the mixture at the room temperature (25 ℃) for 1 hour.</li>
 +
 
 +
Making 5ap_tile
 +
<li>Mix materials (mentioned above in [5ap_tile]) in 0.2 ml PCR-tubes.</li>
 +
<li>Anneal the mixture using PCR machine (from 85 ℃ to 25 ℃, -2 ℃/min).</li>
 +
 
 +
Insertion of hybridized double-stranded DNA into 5ap_tile
 +
<li>Mix hybridized double-stranded DNA and 5ap_tile in 0.2 ml PCR-tubes.</li>
 +
<li>Incubate the mixture at 48 ℃, 46℃, 44℃, 42℃,or 40℃ for 1 hour.</li>
 +
 
 +
1 wt% Agarose-gel Electrophoresis
 +
<li>Electrophoresis the inserted 5ap_tile for 50 minutes at 100 V at 4 ℃.</li>
 +
         <li>Stain the gel by SYBR Gold in TBE.</li>
 +
         <li>Take photographs of the electrophoresed gel by LAS-4000.</li>
 +
 
 +
    </ul>
 +
 
 +
    </article>
 +
    <br>
 +
 
 +
 
 +
<!--◆◆STEP4◆◆-->
 +
<h2 class="PS_title"><a name="STEP4">&nbsp;STEP 4: The formed subunits oligomerize in solution.</a></h2>
 +
 
 +
<!--◆◆SA dimer◆◆-->
 +
  <article>
 +
  <div class="mini-title">
 +
      <a name="Dimerization_of_OCK--using_biotin,_streptavidin_and_click_ reaction">1) Dimerization of OCK--using biotin, streptavidin and click reaction</a>
 +
  </div>
 +
 
 +
<!--Reagent-->
 +
      <div class="zairyou-heading">[Reagent]</div>
 +
      <br>
 +
 
 +
    <table>
 +
    <tr>
 +
    <th>OCK (90 nM)</th>
 +
    <td>8 µL</td>
 +
    </tr>
 +
 
 +
    <tr>
 +
    <th>Streptavidin (190 nM)</th>
 +
    <td>2 µL</td>
 +
    </tr>
 +
 
 +
    <tr>
 +
    <th>CuSO4 aq (8 mM)</th>
 +
    <td>1 µL</td>
 +
    </tr>
 +
 
 +
    <tr>
 +
    <th>THTA (32.5 mM)</th>
 +
    <td>1 µL</td>
 +
    </tr>
 +
 
 +
    <tr>
 +
    <th>Sodium ascorbate (3.25 mM)</th>
 +
    <td>1 µL</td>
 +
    </tr>
 +
 
 +
 
 +
    </table>
 +
    <br>
 +
 
 +
 
 +
 
 +
<!--Procedure-->
 +
  <div class="zairyou-heading">[Procedure]</div>
 +
    <ul class="procedure-list">
 +
      <li>7.4 µL of OCK and 1 µL Streptavidin (190 nM) were mixed and kept at room temperature (27 ℃) for an hour. (Mix1)</li>
 +
      <li>10 µL of Mix1 and 1 µL of Sodium ascorbate (3.25 mM) were mixed and then 1 µL of CuSO4 aq (8 mM) was added into that solution.</li>
 +
      <li>The solution was mixed and 1µL of THTA (20 mM) was added in it and mixed.</li>
 +
      <li>That solution was kept at room temperature (27 ℃) for a day.</li>
 +
    </ul>
 +
 
 +
    </article>
 +
    <br>
 +
 
 +
 
 +
<!--◆◆Click_reaction◆◆-->
 +
      <div class="mini-title">
 +
      <a name="Click_reaction_via_(3+2)_cycloaddition">
 +
      2) Click reaction via (3+2) cycloaddition
 +
      </a>
 +
  </div>
 +
  <article>
 +
 
 +
<!--Reagent-->
 +
      <div class="zairyou-heading">[Reagent]</div>
 +
      <br>
 +
 
 +
    <table>
 +
 
 +
    <tr>
 +
    <th>azide solution (10μM)</th>
 +
    <td>3μL</td>
 +
    </tr>
 +
 
 +
    <tr>
 +
    <th>alkyne solution (10μM)</th>
 +
    <td>3μL</td>
 +
    </tr>
 +
 
 +
    <tr>
 +
    <th>CuSO<sub>4</sub> solution (50mM)</th>
 +
    <td>1μL</td>
 +
    </tr>
 +
 
 +
    <tr>
 +
    <th>THTA solution (100mM)</th>
 +
    <td>1μL</td>
 +
    </tr>
 +
 
 +
    <tr>
 +
    <th>sodium ascorbate solution (100mM)</th>
 +
    <td>1μL</td>
 +
    </tr>
 +
 
 +
    </table>
 +
    <br>
 +
 
 +
<!--Procedure-->
 +
  <div class="zairyou-heading">[Procedure]<sup>[4]</sup></div>
 +
    <ul class="procedure-list">
 +
      <li>The above all solutions were mixed, using a vortex.</li>
 +
      <li>The solution was kept at room temperature.</li>
 +
    </ul>
 +
    </article>
 +
    <br>
 +
<!--◆◆Accelerated_Click_reaction◆◆-->
 +
  <article>
 +
  <div class="mini-title">
 +
      <a name="Accelerated_Click_reaction">3) Accelerated Click reaction (using streptavidin to make the aklyne and azide reactive groups close) </a>
 +
  </div>
 +
 
 +
<!--Reagent-->
 +
      <div class="zairyou-heading">[Reagent]</div>
 +
      <br>
 +
 
 +
    <table>
 +
    <tr>
 +
    <th>2x barrel buffer</th>
 +
    <td>6 µL</td>
 +
    </tr>
 +
 
 +
    <tr>
 +
    <th>alkyne oligo (carrying biotin) (15 µM)</th>
 +
    <td>1 µL</td>
 +
    </tr>
 +
 
 +
    <tr>
 +
    <th>azide oligo (carrying biotin) (15 µM)</th>
 +
    <td>1 µL</td>
 +
    </tr>
 +
 
 +
 
 +
    <tr>
 +
    <th>streptavidin (500 µM)</th>
 +
    <td>1 µL</td>
 +
    </tr>
  
 +
    </table>
 +
    <br>
  
        <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>
+
<!--Procedure-->
    <h3><a name="Oligomerization_by_Click_reaction"></a>3) Oligomerization by Click reaction</h3>
+
  <div class="zairyou-heading">[Procedure]</div>
    <!--Method-->
+
    <ul class="procedure-list">
    <div class="zairyou-heading">[Method]</div>
+
      <li>mix reagents</li>
 +
      <li>incubate the tube at 37 ℃ for indicated reaction time.</li>
 +
      <li>boil at 95 ℃ for 30 minutes to break down streptavidin</li>
 +
    </ul>
  
    <div id ="step4_3)">
+
    </article>
      <p class="paragraph">
+
    <br>
      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>
+
<!--◆◆Click_reaction_hybridization◆◆-->
      <div class="res-conclusion">
+
  <article>
      a) Optimum concentration of CuSO<sub>4</sub> to OCK
+
  <div class="mini-title">
    </div>
+
      <a name="Click_reaction_(using_hybridization_to_make_the_aklyne_and_azide_reactive_groups close)">4) Click reaction (using hybridization to make the aklyne and azide reactive groups close) </a>
          <figure>
+
  </div>
        <center>
+
 
        <img src="http://openwetware.org/images/5/5e/CuAlive-Todai.png" width=600px height=450px>
+
<!--Reagent-->
 +
      <div class="zairyou-heading">[Reagent]</div>
 +
      <br>
 +
 
 +
    <table>
 +
    <tr>
 +
    <th>2x barrel buffer</th>
 +
    <td>7 µL</td>
 +
    </tr>
 +
 
 +
    <tr>
 +
    <th>alkyne oligo (15 µM)</th>
 +
    <td>1 µL</td>
 +
    </tr>
 +
 
 +
    <tr>
 +
    <th>azide oligo (15 µM)</th>
 +
    <td>1 µL</td>
 +
    </tr>
 +
 
 +
 
 +
    <tr>
 +
    <th>scaffold (15 µM)</th>
 +
    <td>1 µL</td>
 +
    </tr>
  
        </center>
+
    </table>
      </figure>
 
              <div class="res-conclusion">
 
      -->Optimum concentration of CuSO<sub>4</sub>: 625 uM or less
 
    </div>
 
 
     <br>
 
     <br>
 +
 +
 +
<!--Procedure-->
 +
  <div class="zairyou-heading">[Procedure]</div>
 +
    <ul class="procedure-list">
 +
      <li>mix reagents</li>
 +
      <li>incubate the tube at 37 ℃.</li>
 +
      <li>add loading buffer into the reaction mixture and boil at 95 ℃ for 5 minutes to denature the double strand to single strand.</li>
 +
    </ul>
 +
 +
<!--◆◆Click reaction cupper free◆◆-->
 +
  <div class="mini-title">
 +
      <a name="Click_reaction_(copper_catalyst-free)">5) Click reaction (copper catalyst-free)</a>
 +
  </div>
 +
 +
<!--Reagent-->
 +
      <div class="zairyou-heading">[Reagent]</div>
 +
      <br>
 +
 +
    <table>
 +
    <tr>
 +
    <th>2x barrel buffer</th>
 +
    <td>7 µL</td>
 +
    </tr>
 +
 +
    <tr>
 +
    <th>alkyne oligo (15 µM)</th>
 +
    <td>1 µL</td>
 +
    </tr>
 +
 +
    <tr>
 +
    <th>azide oligo (15 µM)</th>
 +
    <td>1 µL</td>
 +
    </tr>
 +
 +
 +
    <tr>
 +
    <th>scaffold (15 µM)</th>
 +
    <td>1 µL</td>
 +
    </tr>
 +
 +
    </table>
 
     <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>
+
<!--※10x OCK buffer (f. 100 µl)-->
       </figure>
+
  <div class="zairyou-heading">[※※ 2x barrel buffer]</div>
      <p class ="paragraph">
+
       <br>
      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>
+
    <table>
              <div class="res-conclusion">
+
    <tr>
              --> Optimum concentration of CuSO<sub>4</sub>: 625 uM
+
    <th>1M Tris (pH 7.5)</th>
    </div>
+
    <td>5 µL</td>
    </article>
+
    </tr>
    <!--◆◆4.4 Cupper-free click chemistry◆◆-->
+
 
 +
 
 +
    <tr>
 +
    <th>0.5M EDTA</th>
 +
    <td>2 µL</td>
 +
    </tr>
 +
 
 +
 
 +
    <tr>
 +
    <th>5M NaCl</th>
 +
    <td>1 µL</td>
 +
    </tr>
 +
 
 +
 
 +
    <tr>
 +
    <th>MQ</th>
 +
    <td>32 µL</td>
 +
    </tr>
 +
 
 +
    </table>
 +
    <br>
 +
 
 +
<!--Procedure-->
 +
  <div class="zairyou-heading">[Procedure]</div>
 +
    <ul class="procedure-list">
 +
      <li>mix reagents</li>
 +
      <li>incubate the tube at 37 ℃.</li>
 +
      <li>add loading buffer into the reaction mixture and boil at 95 ℃ for 5 minutes to denature the double strand to single strand.</li>
 +
    </ul>
 +
 
 +
    </article>
 +
 
  
    <article>
+
<article>
    <h3><a name="Cupper-free_click_reaction"></a>4) Cupper-free click reaction</h3>
+
  <div class="mini-title">
    <!--Method-->
+
      <a name="Synthesis_of_streptavidin_mutants">
    <div class="zairyou-heading">[Method]</div>
+
      6) Synthesis of streptavidin mutants
 +
      </a>
 +
  </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>
+
<!--Procedure-->
      <table cellpadding="0" style="position:relative;left:-25px;">
+
  <div class="zairyou-heading">[Procedure]<sup>[7],[8]</sup></div>
        <tbody><tr>
+
    <p class="paragraph">Mono-, di-, tri-, tetra-valent streptavidin were prepared as described [7,8] with some modifications. Shortly, BL21 Star (DE3) pLysSRARE and C43 (DE3) was transformed with pET21a(+) SA-Alive-his or pET21a(+) SA-Dead plasmids and cultured in LB at 37℃. Collected cells were resuspended in B-PER (Pierce) and inclusion bodies were purified, and dissolved in 6M guanidinium hydrochloride (GuHCl; pH 1.5). After mixing the unfolded subunits in desired ratio, the unfolded subunits were refolded by rapid dilution into PBS, then concentrated by ammonium sulfate precipitation. After dialyzed 3 x against PBS, refolded streptavidin were purified by Ni-NTA column (GE 17-5248-02) using AKTA system (GE AKTAexplorer 10S). Fractionized samples were concentrated by Amicon Ultra (Millipore).
        <td>
+
    </p>
        <figure>
+
    </article>
          <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:-20px;">
 
        <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:-35px;">
 
          <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:-20px;">
 
        <tbody><tr>
 
        <td>
 
        <figure>
 
          <img src="http://openwetware.org/images/c/c5/Gelphoto3kai-Todai.png" width="360px" height="270px">
 
        </figure>
 
        </td>
 
  
 +
<!--Reference-->
  
        <td>
+
    <h1 class="title"><a name="Reference">&nbsp;Reference</a></h1>
        <figure style="position:relative;left:-35px;">
 
          <img src="http://openwetware.org/images/c/c6/Yatagaiclickhybri-Todai.png" width="270px" height="270px">
 
  
         </figure>
+
    <div>   
         </td>
+
        <div class="reference-title">
         </tr>
+
         <a name="proref-1">
      </tbody></table>
+
        [1] Folding DNA to create nanoscale shapes and patterns
</center>
+
         </a>
 +
         </div>
 +
          <div class="reference-author">
 +
          Rothemund, P. W.
 +
          </div>
 +
              <div class="reference-journal">
 +
              Nature 440, 297–302 (2006)
 +
              </div>
 +
    </div>
  
                <figure  style="position:relative;left:-45px;">
+
    <div>
          <img src="http://openwetware.org/images/a/a2/Yatagai_131027_2.JPG" width="270px" height="270px">
+
        <div class="reference-title">
 +
        <a name="proref-1">
 +
        [2] Rapid Folding of DNA into Nanoscale Shapes at Constant Temperature
 +
        </a>
 +
        </div>
 +
          <div class="reference-author">
 +
            Jean-Philippe J. Sobczak, Thomas G. Martin, Thomas Gerling, Hendrik Dietz
 +
          </div>
 +
              <div class="reference-journal">
 +
              Science, 2012, 338, 1458
 +
              </div>
 +
    </div>
  
        </figure>
+
    <div>    
          
+
         <div class="reference-title">
        <p class="paragraph">
+
        <a name="proref-1">
We add accelerator, which can work as a scaffold and make alkyne and
+
        [3] Transcription Regulation System Mediated by Mechanical Operation of a DNA &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbs p;Nanostructure
azide reactive group close. Acceleration of the click reaction was
+
        </a>
observed. In other words, azide and alkyne reactive groups do not react
+
        </div>
each other in solution, but easy to react each other after
+
          <div class="reference-author">
proximally-positioned. This character is very suitable to prevent
+
          Masayuki Endo, Ryoji Miyazaki, Tomoko Emura, Kumi Hidaka, and Hiroshi Sugiyama
non-specific oligomerization, while accelerating the specific
+
          </div>
oligomerization in OCK.
+
               <div class="reference-journal">
</p>
+
               Journal of the American Chemical Society, 2012, 134, 2852-2855
      <p class ="paragraph">
+
              </div>
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>
 
     </div>
   
 
    </article>
 
   
 
        <h1 class="title"><a name="Reference">&nbsp;Reference</a></h1>
 
  
 
     <div>     
 
     <div>     
 
         <div class="reference-title">
 
         <div class="reference-title">
 
         <a name="proref-1">
 
         <a name="proref-1">
         [1] CanDo(<a href="http://cando-dna-origami.org/usersguide">http://cando-dna-origami.org/usersguide</a>)
+
         [4] the protocol of Jena Bioscience GmbH
 +
        </a>
 +
        </div>
 +
          <div class="reference-journal">
 +
          <a target="_blank" href="http://www.jenabioscience.com" style="color:#e00000">
 +
          http://www.jenabioscience.com</a>
 +
          </div>
 +
    </div>
 +
 
 +
    <div>
 +
        <div class="reference-title">
 +
        <a name="proref-1">
 +
        [5] Substrate-Assisted Assembly of Interconnected Single-Duplex DNA Nanostructures
 
         </a>
 
         </a>
 
         </div>
 
         </div>
 +
          <div class="reference-author">
 +
          Shogo Hamada, Satoshi Murata Prof.
 +
          </div>
 +
              <div class="reference-journal">
 +
              Angewandte Chemie International Edition,2009,48(37),6820–6823
 +
              </div>
 
     </div>
 
     </div>
     <br>
+
     <div>    
 +
        <div class="reference-title">
 +
        <a name="proref-1">
 +
        [6] A primer to scaffolded DNA origami.</a>
 +
        </div>
 +
          <div class="reference-author">
 +
          Castro CE, Kilchherr F, Kim DN, Shiao EL, Wauer T, Wortmann P, Bathe M
 +
and Dietz H.
 +
          </div>
 +
              <div class="reference-journal">
 +
              Nat Methods 221-229 (2011, Mar;8(3))
 +
              </div>
 +
    </div>
 +
          <div>   
 +
        <div class="reference-title">
 +
        <a name="proref-1">
 +
        [7] A monovalent streptavidin with a single femtomolar biotin binding site.</a>
 +
        </div>
 +
          <div class="reference-author">
 +
          Howarth M, Chinnapen DJ, Gerrow K, Dorrestein PC, Grandy MR, Kelleher NL, El-Husseini A and Ting AY.
 +
          </div>
 +
              <div class="reference-journal">
 +
              Nat Methods 267-273 (2006, Apr;3(4))
 +
              </div>
 +
    </div>
 +
          <div>   
 +
        <div class="reference-title">
 +
        <a name="proref-1">
 +
        [8] Imaging proteins in live mammalian cells with biotin ligase and monovalent streptavidin.</a>
 +
        </div>
 +
          <div class="reference-author">
 +
          Howarth M and Ting AY.
 +
          </div>
 +
              <div class="reference-journal">
 +
              Nat Protoc 534-545 (2008, Mar;3(3)); doi: 10.1038/nprot.2008.20.
 +
              </div>
 +
    </div>
 +
             
 
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    Copyright &copy; Todai nanORFEVRE, all rights reserved.
Copyright &copy; Todai nanORFEVRE, all rights reserved.
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 <ul>
    <li><a href="http://openwetware.org/wiki/Biomod/2013/Todai">Home</a>
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<!--Experiment--> <h1 class="big-title"><a name="Experiment">&nbsp;Experiment</a></h1>

   	<div id="Explist">
   	<ul>
      	<li><div class="mokuji"><a href="#Contents">Contents of pilot study</a></div></li>
      	<li><div class="mokuji"><a href="#Contents">Contents of Protocols</a></div></li>
      	<li><div class="mokuji"><a href="#PilotStudy">Pilot Study</a></div></li>
      	<li><div class="mokuji"><a href="#Protocols">Protocols</a></div></li>
   	</ul>
   	</div>
   	<br>

<!--Contents-->

  	<article>
    	<h1 class="title"><a name="Contents">&nbsp;Contents of pilot study</a></h1>
      	<article>
       		<ul>

<li><div class="mini-title" style="color:#BBBBBB;">STEP 1: DNA strands assemble to form designed structures.</div> </li> <li><div class="mini-title"><a href="#STEP2">STEP 2: Subunits penetrate into the membrane.</a></div> <ul style="list-style: none;"> <li><a href="#hybridization_of_Cholesterol_Oligo_with_OCK">1) hybridization of cholesterol oligo with OCK</a></li> <li><a href="#Preparation_of_liposome">2) Preparation of liposome</a></li> <li><a href="#Flotation_assay_of_liposome_and_DNA_origami">3) Floatation assay of liposome and Rectangular tile(DNA origami)</a></li> </ul> </li> <br> <li><div class="mini-title"><a href="#STEP3">STEP 3: Subunits recognize cancer-specific proteins.</a></div> </li> <li><div class="mini-title"><a href="#STEP4">STEP 4: The formed subunits oligomerize in solution.</a></div> <ul style="list-style: none;"> <li><a href="#Click_reaction_via_(3+2)_cycloaddition">1) Optimum time of click reaction via (3+2) cycloaddition</a></li>

<li><a href="#OptimumConc_SA">2) OptimumConc SA</a></li> </ul> </li> </ul>

 		</article>
		</article>
		
		
		   	<article>
    	<h1 class="title"><a name="Contents">&nbsp;Contents of protocols</a></h1>
      	<article>
       		<ul>

<li><div class="mini-title"><a href="#STEP1">STEP 1: DNA strands assemble to form designed structures.</a></div> <ul style="list-style: none;"> <li><a href="#Assembling_of_OCK">1) Assembly of OCK</a></li> <li><a href="#Transmission_electron_microscopy">2) Transmission electron microscopy(TEM)</a></li> </ul> </li> <br> <li><div class="mini-title"><a href="#STEP2">STEP 2: Subunits stick in the membrane.</a></div> <ul style="list-style: none;"> <li><a href="#Flotation_assay_[OCK]">1) Flotation assay [OCK]</a></li> <li><a href="#Preparation_of_GUVs">2) Preparation of GUVs</a></li> <li><a href="Preparation_of_SUVs">3) Preparation of SUVs</a> <li><a href="#hybridization_of_Cholesterol_Oligo_with_OCK">4) hybridization of cholesterol oligo with OCK</a></li> </ul> </li> <br> <li><div class="mini-title"><a href="#STEP3">STEP 3: Recognition of target cells</a></div> <ul style="list-style: none;"> <li><a href="#Reaction_of_a_biotinized_oligo_to_streptavidin">1) Reaction of a biotinized oligo to streptavidin</a></li> <li> <a href="Reaction_between_aptamer_embedded_in_rect_tile_and_PDGF10">2) Reaction_between_aptamer_embedded_in_rect_tile_and_PDGF</a> <li><a href="#efficient_hybridization_(changing_mixture_ratio)">3) Efficient hybridization (changing mixture ratio)</a></li> <li><a href="#efficient_hybridization_(incubation_time)">4) efficient hybridization (incubation time)</a></li> <li><a href="#insertion_of__hybridized_double-stranded_DNA_into_tile">5) Insertion of hybridized double-stranded DNA into tile</a></li> <li> <a href="#Reaction_between_aptamer_(3ap-M5t10f-T0)_and_PDGF">6) Reaction_between_aptamer_(3ap-M5t10f-T0)_and_PDGF</a></li> <li><a href="Double insertion_of__hybridized_double-stranded_DNA_into_tile">7) Double insertion of hybridized double-stranded DNA into tile</a></li> </ul> </li> <br> <li><div class="mini-title"><a href="#STEP4">STEP 4: The formed subunits oligomerize in solution.</a></div> <ul style="list-style: none;"> <li><a href="#Oligomerization_by_streptavidin-biotin_complex">1) Oligomerization by streptavidin-biotin complex</a></li> <li> <a href="#Click_reaction_via_(3+2)_cycloaddition"> 2) Click reaction via (3+2) cycloaddition

      </a>
      </li>

<li>

      <a href="#Accelerated_Click_reaction">3) Accelerated Click reaction (using streptavidin to make the aklyne and azide 	reactive groups close) </a>
      </li>

<li><a href="#Click_reaction_(using_hybridization_to_make_the_aklyne_and_azide_reactive_groups close)">4) Click reaction (using hybridization to make the aklyne and azide reactive groups close) </a>

      </li>

<li><a href="#Click_reaction_(copper_catalyst-free)">5) Click reaction (copper catalyst-free)</a></li> <li><a href="#Synthesis_of_streptavidin_mutants">6) Synthesis of streptavidin mutants</a> </ul> </li> </ul>

 		</article>
		</article>

<!--◆◆Pilot Study◆◆-->

    <h1 class="title"><a name="PilotStudy">&nbsp;Pilot Study</a></h1>
    

<!--◆◆STEP2◆◆--> <h2 class="PS_title"><a name="STEP2">&nbsp;STEP 2: Subunits penetrate into the membrane </a></h2> <!--◆◆hybridizing of Cholesterol Oligo with OCK◆◆-->

  <article>
  <div class="mini-title">
      <a name="hybridization_of_Cholesterol_Oligo_with_OCK">1) Hybridization of cholesterol Oligo with OCK</a>

</div>

      <figure>
       <center>
        <img src="http://openwetware.org/images/d/d7/480px_OCKchol-Todai.png" width=480px height=300px >
       </center>
      </figure>

<br>

  <div class="zairyou-heading">[Discussion]</div>
    <p class="paragraph">

The result of 1 % agarose gel electrophoresis showed that the band of sample 5 and 7 were smeared, showing the successful of hybridization of cholesterol oligo with OCK (Langecker et al. (2012)). We concluded that the optimized condition for hybridization is: 1 hour incubation at room temperature with 5 times excess cholesterol oligo to OCK.

    </p>
  	<br>

</article>

<!--◆◆Preparation_of_liposome◆◆--> <article

  	<div class="mini-title">
      <a name="Preparation_of_liposome">2) Preparation of liposome</a>
  	</div>
      <figure>
       <center>
        <img src="http://openwetware.org/images/7/71/640px_suv_dls_popg50r60-Todai.png" width=640px height=360px >

<figcaption> <b>The result of DLS (Viscotek, 802 DLS)</b>

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

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

    <p class="paragraph">For floating assay, uniformly-sized liposome were prepared. DLS data shows sharp peak with the mean radius of 60 nm, indicating the homogenity of liposomes.
    </p>
  </article>
  <br>

<!--◆◆Flotation assay(Rect-tile)◆◆-->

  <article>
  <div class="mini-title">
      <a name="Flotation_assay_of_liposome_and_DNA_origami">3) Floatation assay of liposome and Rectangular tile(DNA origami)</a>
      </div>
      <figure>
       <center>
        <img src="http://openwetware.org/images/1/1d/640pxflotationassay-Todai.jpg" width=300px height=300px >
	<figcaption> <b>Result of agarose gel electrophoresis of the sample of flotation assay</b> <br>
	The result of 1% agarose gel electrophoresis(100V,30min). In this measurement, the fluorescence of Cy5, which is 

integrated into DNA origami(Rect tile<sup>[1]</sup>) ,is observed. Fraction1 is the liquid in the top layer, and fra ction 5 is in the bottom layer. Fraction 6 is the sample retrieved from precipitation. DNA origami solely was also l oaded on the extreme right lane. </figcaption>

       </center>
      </figure>
      <figure>
       <center>
        <img src="http://openwetware.org/images/0/0d/300pxNILGraph-Todai.PNG" width=350px height=350px >
	<figcaption> <b>Fluorescence intensity of the samples of flotation assay(DNA Rect tile +liposome)</b><br>

Although the size of liposome might change during the flotation assay(data not shown), the intensity of the fluoresc ence of NIL(Nile Red, ex 500nm, em 550~700nm ) suggests the amount of lipid membrane,liposome. The fluorescence spectrum of water was subtracted as background

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

<br>

  <div class="zairyou-heading">[Discussion]</div>
    <p class="paragraph">

To confirm the flotation assay, mixed tiles(DNA origami) and liposomes were assayed. Five samples (fraction 1,2,..., 5, from the top) were retrieved from supermetant liquid and a sample(fraction 6) from precipitation by the addition of buffer used in assay. When the sample, tile mixed with liposomes, were assayed, tiles were observed in the top layer. The distribut ion of liposomes is observed by the fluorescence of NIL(Nile Red).

    </p>
  	<br>

</article>


<!--◆◆STEP3◆◆--> <h2 class="PS_title"><a name="STEP3">&nbsp;STEP 3: Subunits recognize cancer-specific proteins</a></h2> <article> </article>

<!--◆◆STEP4◆◆--> <h2 class="PS_title"><a name="STEP4">&nbsp;STEP 4: The formed subunits oligomerize in solution</a></h2>

<!--◆◆Click reaction ◆◆-->

  	<div class="mini-title">
      <a name="Click_reaction_via_(3+2)_cycloaddition">
       1)Optimum time of Click reaction via (3+2) cycloaddition<sup>[4]</sup>
      </a>
  	</div>
  	
  	<article>
      <figure>
       <center>
        <img src="http://openwetware.org/images/a/ab/450pxclick0828-Todai.jpg" width=480px height=360px >
	<figcaption> <b>Result of urea gel electrophoresis of the sample of click reaction</b><br>
     </figcaption>
       </center>
      </figure>

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

    <p class="paragraph">Copper(Ⅰ) catalyzed click reaction was used to dimerize of oligo DNA(length of 20bp and 14bp)	. The time cause of the reaction indicate that the click reaction is so quick(<5min).</p>

<!--◆◆Optimum_Conc_SA◆◆-->

  <article>
  <div class="mini-title">
      <a name="OptimumConc_SA">2) Optimum concentration of SA</a>
  </div>
         <figure>
       <center>
        <img src="http://openwetware.org/images/1/1b/OptimumConc_SA-Todai.png" width=480px height=360px >
	<figcaption> <b>Optimization of the mixing ratio between </b><br>
	The density of the band of dimers was the highest when the mixing ratio of streptavidin to SA was 3/1, so
the optimum ratio of streptavidin to OCK was 3/1.
     </figcaption>
       </center>
      </figure>
  <br>
  <br>

</article> <!--◆◆Evaluation of streptavidin mutants◆◆-->

  <article>
  <div class="mini-title">
      <a name="Evaluation_of_streptavidin_mutants">3) Evaluation of streptavidin mutants</a>
  </div>
         <figure>
       <center>
        <img src="http://openwetware.org/images/1/1e/SA_result-Todai.png" width=400px height=400px >
       </center>
      </figure>
  <br>

<p class="paragraph"> We added a 6His tag to the active wild-type subunit ("alive" (A) subunit), hence no 6His tag was added to inactive subunit ("dead" (D) subunit). We mixed A and D subunits at a molar ratio of 1:1 in GuHCl and refolded in PBS. Then, refolded streptavidins were purified by Ni-NTA column and the tetramers were distinguished by non-denatured SDS-PAGE. </p>

  <br>

<!--◆◆Protocols◆◆-->

    <h1 class="title"><a name="Protocols">&nbsp;Protocols</a></h1>

<!--◆◆STEP1◆◆--> <h2 class="PS_title"><a name="STEP1">&nbsp;STEP 1:DNA strands assemble to form designed structures.</a></h2>

<!--◆◆Assembly of OCK◆◆-->

  <div class="mini-title">
      <a name="Assembling_of_OCK">1) Assembly of OCK<sup>[2]</sup></a>
      </div>
   <br>
  <article>


<!--Reagent-->

     <div class="zairyou-heading">[Reagent]</div>
     <br>
   <table>
   <tr>
   <th>M13mp18ss</th>
   <td>4.5 ul</td>
   </tr>
   <tr>
   <th>Staple mix</th>
   <td>4.5 µL</td>
   </tr>
   <tr>
   <th>10x OCK buffer<sup>*</sup></th>
   <td>1 µL</td>
   </tr>


   </table>
   <br>

&nbsp;*...10x OCKbuffer(f.100 ul) <table>

   <tr>
   <th>Tris-HCl(ph 7.5)</th>
   <td>f.50 mM</td>
   <td>1 M</td>
   <td>5 µL</td>
   </tr>
   <tr>
   <th>EDTA-Na(pH 8)</th>
   <td>f.10 mM</td>
   <td>0.5 M</td>
   <td>2 µL</td>
   </tr>
       <tr>
   <th>MgCl<sub>2</sub></th>
   <td>f.200 mM</td>
   <td>1 M</td>
   <td>20 µL</td>
   </tr>
   <tr>
   <th>NaCl</th>
   <td>f.500 mM</td>
   <td>5 M</td>
   <td>1 µL</td>
   </tr>
       <tr>
   <th>MQ</th>
   <td>-</td>
   <td>-</td>
   <td>72 µL</td>
   </tr>
   </table>

<!--Procedure-->

  <div class="zairyou-heading">[Procedure]</div>
   <ul class="procedure-list">
     <li>mix the solutions.</li>
     <li>It was annealed at 85 °C for 25 min and then at 52 °C for 3 or 4 hours.</li>
   </ul>
   </article>
   <br>


<!--◆◆TEM◆◆-->

  <article>
  <div class="mini-title">
      <a name="Transmission_electron_microscopy">
      2) Transmission electron microscopy(TEM)
      </a>
  </div>

<!--Procedure-->

  <div class="zairyou-heading">[Procedure]<sup>[6]</sup></div>
   <p class="paragraph">
   The procedure of TEM was refered to previous researches<sup>[6]</sup>. 
   </p>
   </article>

<!--◆◆STEP2◆◆--> <h2 class="PS_title"><a name="STEP2">&nbsp;STEP 2: Subunits penetrate into the membrane.</a></h2>

<!--◆◆flotation asssay of OCK◆◆-->

  <div class="mini-title">
      <a name="Flotation_assay_[OCK]">1) Flotation assay [OCK]</a>
  </div>
  <article>

<!--Reagent-->

     <div class="zairyou-heading">[Reagent]</div>
     <br>
   <table>
   <tr>
   <th>OCK</th>
   <td>100 µL</td>
   </tr>
   <tr>
   <th>Cholesterol hybridized OCK</th>
   <td>100 µL</td>
   </tr>
   <tr>
   <th>Liposome (1 mg/mL SUVs)</th>
   <td>100 µL</td>
   </tr>
   <tr>
   <th>2.25 M Sucrose buffer<sup>*</sup></th>
   <td>500 µL</td>
   </tr>
   <tr>
   <th>1.6 M Sucrose buffer<sup>**</sup></th>
   <td>900 µL</td>
   </tr>
   <tr>
   <th>150 mM KCl solution</th>
   <td>100 µL</td>
   </tr>
   <tr>
   <th>1×Flotation buffer<sup>***</sup></th>
   <td>600 µL</td>
   </tr>


   </table>
   <br>

&nbsp;*...2.25 M Sucrose buffer <table>

   <tr>
   <th>HEPES-KOH (pH 7.6)</th>
   <td>50 mM</td>
   </tr>
   <tr>
   <th>KCl</th>
   <td>100 mM</td>
   </tr>
       <tr>
   <th>MgCl<sub>2</sub></th>
   <td>20 mM</td>
   </tr>
   <tr>
   <th>Sucrose</th>
   <td>2.25 M</td>
   </tr>        
   </table>


   </table>
   <br>

&nbsp;**...1.6 M Sucrose buffer <table>

   <tr>
   <th>HEPES-KOH (pH 7.6)</th>
   <td>50 mM</td>
   </tr>
   <tr>
   <th>KCl</th>
   <td>100 mM</td>
   </tr>
       <tr>
   <th>MgCl<sub>2</sub></th>
   <td>20 mM</td>
   </tr>
   <tr>
   <th>Sucrose</th>
   <td>1.6 M</td>
   </tr>        
   </table>


   </table>
   <br>

&nbsp;***...1×Flotation buffer <table>

   <tr>
   <th>HEPES-KOH (pH 7.6)</th>
   <td>50 mM</td>
   </tr>
   <tr>
   <th>KCl</th>
   <td>100 mM</td>
   </tr>
       <tr>
   <th>MgCl<sub>2</sub></th>
   <td>20 mM</td>
   </tr>     
   </table>


<!--Procedure-->

  <div class="zairyou-heading">[Procedure]</div>
   <ul class="procedure-list">
     <li>Each sample was mixed as shown below:<sup>****</sup></li>
   </table>
   <br>

&nbsp;****...Table1. Breakdown of Samples <table>

   <tr>
   <th>Sample No.</th>
   <td>1</td>
   <td>2</td>
   <td>3</td>
   <td>4</td>
   </tr>
   <tr>
   <th>Cholesterol hybridized OCK</th>
   <td>50 µL</td>
   <td>50 µL</td>
   <td>-</td>
   <td>-</td>
   </tr>
       <tr>
   <th>OCK</th>
   <td>-</td>
   <td>-</td>
   <td>50 µL</td>
   <td>50 µL</td>
   </tr>
   <tr>
   <th>Liposome</th>
   <td>50 µL</td>
   <td>-</td>
   <td>50 µL</td>
   <td>-</td>
   </tr>
   <tr>
   <th>150 mM aqueous KCl solution</th>
   <td>-</td>
   <td>50 µL</td>
   <td>-</td>
   <td>50 µL</td>
   </tr>
       <tr>
   <th>2.25 M Sucrose buffer</th>
   <td>125 µL</td>
   <td>125 µL</td>
   <td>125 µL</td>
   <td>125 µL</td>
   </tr>
   </table>
     <li>225 µL of 1.6 M sucrose buffer was overlaid with 225 µL of sample

mixture in centrifuge tubes (Beckman, cat#343778, 11 x 34 mm). </li>

     <li>Centrifuge for 16 minutes at 100 krpm at 4 ℃ using TLA 100.2 rotor (BECKMAN COULTER) with Ultracentrifuge (BECKMAN COULTER, Optima MAX-XP).</li>
     <li>150 µL of supernatant was extracted from top to bottom for 3 times (Fraction 1 to 3) and the pellet was retrieved with 150 µL of 1×Flotation buffer (Fraction 4).</li>
     <li>Fraction 1-4 of each sample were analyzed by 1 % agaraose gel

electrophoresis (100V, 1 hour). </li>

     <li>The Intensity of fluorescence of NileRed (Liposome) was measured with fluorescence spectrophotometer (JASCO, FP-6500) to investigate the existence of liposome in each Fraction.</li>
     <li>The radiuses of liposome of each fraction were measured with DLS (Viscotek, 802 DLS).</li>
   </ul>


   </article>
   <br>
   <br>


   <br>

<!--◆◆Preparation of GUVs◆◆-->

  <div class="mini-title">
      <a name="Preparation_of_GUVs">2) Preparation of GUVs</a>
  </div>
  <article>

<!--Reagent-->

     <div class="zairyou-heading">[Reagent]</div>
     <br>
   <table>
   <tr>
   <th>Lipid mix<sup>*</sup></th>
   <td>3 ml</td>
   </tr>
   <tr>
   <th>150 mM KCl solution</th>
   <td>1 µL</td>
   </tr>


   </table>
   <br>

&nbsp;*...Lipid mix <table>

   <tr>
   <th>5 mg/mL POPC</th>
   <td>0.1 mL</td>
   </tr>
   <tr>
   <th>5 mg/mL POPG</th>
   <td>0.1 mL</td>
   </tr>
       <tr>
   <th>10 uM NileRed solution</th>
   <td>0.13 mL</td>
   </tr>
   <tr>
   <th>Chloroform</th>
   <td>2.67 mL</td>
   </tr>        
   </table>

<!--Procedure-->

  <div class="zairyou-heading">[Procedure]</div>
   <ul class="procedure-list">
     <li>A lipid film was formed by evaporating 3 ml of lipid mix in a 50 ml

eggplant flask, using a rotational evaporator (EYELA, model#N1110) for 10 mins. </li>

     <li>The flask was kept under vacuum overnight to evaporate remaining chloroform.</li>
     <li>The lipid film was resuspended in 1 mL of 150 mM KCl solution.</li>
   </ul>
   </article>
   <br>


<!--◆◆Preparation of SUVs◆◆-->

  <div class="mini-title">
      <a name="Preparation_of_SUVs">3) Preparation of SUVs</a>
  </div>
  	<article>

<b>Type 1: POPC 100%</b> <!--Reagent-->

     <div class="zairyou-heading">[Reagent]</div>
     <br>
   <table>
   <tr>
   <th>150mM KCl solution</th>
   <td>3mL</td>
   </tr>
   <tr>
   <th>POPC</th>
   <td>3mg</td>
   </tr>
   <tr>
   <th>Chloroform (99.0%)</th>
   <td>3mL</td>
   </tr>
   <tr>
   <th>40μM Nile Red solution</th>
   <td>0.1mL</td>
   </tr>
   </table>
   <br>

<!--Procedure-->

  <div class="zairyou-heading">[Procedure]</div>
   <ul class="procedure-list">
     <li>POPC were dissolved in 3mL of Chloroform.</li>
     <li>A lipid film was formed by evaporating 3mL of POPC solution in a 50mL eggplant flask, using a rotational 

evaporator for 5 minutes.</li>

     <li>The flask was kept under vacuum overnight to evaporate remaining chloroform.</li>
     <li>The lipid film was resuspended in 3mL of a 150mM KCl solution.</li>
     <li>The solution was filtered through 200nm polar filter with extruder to even the size of liposome.</li>
     <li>The size of liposome was measured with DLS (Viscotek 802 DLS).</li>
     <li>The solution was kept at 3 degree C until usage.</li>
   </ul>
   </article>
   <br>

<!--Preparation of SUVs_Added-->

  <article>

<b>Type 2: POPC 50%, POPG 50%</b>

<!--Reagent-->

     <div class="zairyou-heading">[Reagent]</div>
     <br>
   <table>
   <tr>
   <th>Lipid mix<sup>*</sup></th>
   <td>3 ml</td>
   </tr>
   <tr>
   <th>150 mM KCl solution</th>
   <td>1 µL</td>
   </tr>


   </table>
   <br>

&nbsp;*...Lipid mix <table>

   <tr>
   <th>5 mg/mL POPC</th>
   <td>0.1 mL</td>
   </tr>
   <tr>
   <th>5 mg/mL POPG</th>
   <td>0.1 mL</td>
   </tr>
       <tr>
   <th>10 uM NileRed solution</th>
   <td>0.13 mL</td>
   </tr>
   <tr>
   <th>Chloroform</th>
   <td>2.67 mL</td>
   </tr>        
   </table>

<!--Procedure-->

  <div class="zairyou-heading">[Procedure]</div>
   <ul class="procedure-list">
     <li>A lipid film was formed by evaporating 3 ml of lipid mix in a 50 ml

eggplant flask, using a rotational evaporator (EYELA, model#N1110) for 10 mins. </li>

     <li>The flask was kept under vacuum overnight to evaporate remaining chloroform.</li>
     <li>The lipid film was resuspended in 1 mL of 150 mM KCl solution.</li>
     <li>Lipid suspended solution was filtered through 100nm polar filter using extruder (Avanti) to prepare uniformly-sized liposome.</li>
     <li>The size of liposome was measured with DLS (Viscotek 802 DLS).</li>
     <li>The solution was kept at 3℃ until usage.</li>
   </ul>
   </article>
   

<!--◆◆Hybridization of cholesterol modified oligo◆◆-->

  <article>
  <div class="mini-title">
      <a name="Protocol_Hybridization_of_cholesterol_oligo_with_OCK">4) Hybridization of cholesterol oligo with OCK</a>
  </div>

<!--Reagent-->

     <div class="zairyou-heading">[Reagent]</div>
     <br>
   <table>
   <tr>
   <th>OCK</th>
   <td>48 µL</td>
   </tr>
   <tr>
   <th>Cholesterol oligo (0.32, 0.64, 3.2, 6.4 µM)</th>
   <td>100 µL</td>
   </tr>
   </table>

<center> Marker; GeneRuler DNA Ladder Mix (Fermentas, GeneRuler DNA Ladder Mix #SM0331) </center>

<!--Procedure-->

  <div class="zairyou-heading">[Procedure]</div>
   <ul class="procedure-list">
     <li>Centrifuge for 16 minutes at 100 krpm at 4 ℃ using TLA 100.2 rotor (BECKMAN COULTER) with Ultracentrifuge (BECKMAN COULTER, Optima MAX-XP).</li>
     <li>Each sample was mixed and incubated as shown below:<sup>*</sup></li>
   </table>
   <br>

&nbsp;*...Table1. <table> <center>

   <tr>
   <th>Sample No.</th>
   <td>1</td>
   <td>2</td>
   <td>3</td>
   <td>4</td>
   <td>5</td>
   <td>6</td>
   <td>7</td>
   <td>8</td>
   </tr>
   <tr>
   <th>Purified OCK (40 µM)</th>
   <td>6 µL</td>
   <td>6 µL</td>
   <td>6 µL</td>
   <td>6 µL</td>
   <td>6 µL</td>
   <td>6 µL</td>
   <td>6 µL</td>
   <td>6 µL</td>
   </tr>
       <tr>
   <th>0.32 µM Cholesterol oligo</th>
   <td>1.5 µL</td>
   <td>1.5 µL</td>
   <td>-</td>
   <td>-</td>
   <td>-</td>
   <td>-</td>
   <td>-</td>
   <td>-</td>
   </tr>
   <tr>
   <th>0.64 µM Cholesterol oligo</th>
   <td>-</td>
   <td>-</td>
   <td>1.5 µL</td>
   <td>1.5 µL</td>
   <td>-</td>
   <td>-</td>
   <td>-</td>
   <td>-</td>
   </tr>
   <tr>
   <th>3.2 µM Cholesterol oligo</th>
   <td>-</td>
   <td>-</td>
   <td>-</td>
   <td>-</td>
   <td>1.5 µL</td>
   <td>1.5 µL</td>
   <td>-</td>
   <td>-</td>
   </tr>
       <tr>
   <th>6.4 µM Cholesterol oligo</th>
   <td>-</td>
   <td>-</td>
   <td>-</td>
   <td>-</td>
   <td>-</td>
   <td>-</td>
   <td>1.5 µL</td>
   <td>1.5 µL</td>
   </tr>
       <tr>
   <th>[Cholesterol oligo]/[OCK] (see #Note)</th>
   <td>1/2</td>
   <td>1/2</td>
   <td>1</td>
   <td>1</td>
   <td>5</td>
   <td>5</td>
   <td>10</td>
   <td>10</td>
   </tr>

<tr>

   <th>Incubation time [min]</th>
   <td>60</td>
   <td>30</td>
   <td>60</td>
   <td>30</td>
   <td>60</td>
   <td>30</td>
   <td>60</td>
   <td>30</td>
   </tr>

</center>

   </table>
  1. Note; OCK has 4 cholesterol oligo binding sites. Therefore, we devided the molar ratio of cholesterol oligo to OCK with 4.
               <figure>
       <center>
        <img src="http://openwetware.org/images/6/66/OCK_Cholesterol-Todai.png" width=300px height=300px>
       
       </center>
      </figure>
     <li>Each sample was analyzed by 1% agarose gel electrophoresis (100V, 1 hour).</li>
   </ul>


   </article>
   <br>



<!--◆◆STEP3◆◆--> <h2 class="PS_title"><a name="STEP3">&nbsp;STEP 3: Subunits recognize cancer-specific proteins.</a></h2> <article> <!--◆◆Reaction_of_a_biotinized_oligo_to_streptavidin◆◆-->

  <article>
  <div class="mini-title">
      <a name="Reaction_of_a_biotinized_oligo_to_streptavidin">1) Reaction of a biotinized oligo to streptavidin</a>
  </div>

<!--Reagent-->

     <div class="zairyou-heading">[Reagent]</div>
     <br>

<li>materials for hybridization</li>

   	    <li>5ap_M-3t4e_T0 (1uM) (oligo): 5’ to 3’</li>

TACTCAGGGCACTGCAAGCAATTGTGGTCCCAATGGGCTGAGTACGCGACCTCATCTTTGACCCCCAGGCAGGGAG

   	    <li>5ap_5B_16 (10uM) (oligo, which has biotin in 5’ end):</li>

Biotin-TACTCAGCCCATTGGG

   	    <li>10x tile buffer<sup>*</sup></li>
   	    <li>MilliQ</li>
   </table>
   <br>

&nbsp;*...10x tile buffer(f.100 µl) <table>

   <tr>
   <th>Mg(OAc)<sub>2</sub></th>
   <td>f.100 mM</td>
   </tr>
   <tr>
   <th>Tris-HCl (pH7.5)</th>
   <td>f.200 mM</td>
   </tr>
       <tr>
   <th>EDTA</th>
   <td>f.10 mM</td>
   </tr>
   </table>

<li>5ap_tile</li>

   	    <li>M13mp18 (scaffold)</li>
   	    <li>Cy5_Rmix (staples)</li>
   	    <li>10x tile buffer</li>
   	    <li>Cy3 streptavidin (800nM)</li>

<!--Procedure-->

  <div class="zairyou-heading">[Procedure]</div>
   <ul class="procedure-list">
     <li>Hybridization</li>

<li>Mix materials (mentioned above in [hybridization]) in 0.2 ml PCR-tubes.</li> <li>Incubate the mixture at the room temperature (25 ℃) for 1 hour.</li>

     <li>Making 5ap_tile</li>

<li>Mix materials (mentioned above in [5ap_tile]) in 0.2 ml PCR-tubes.</li> <li>Anneal the mixture using PCR machine (from 85 ℃ to 25 ℃, -2 ℃/min).</li>

     <li>Insertion of hybridized double-stranded DNA into 5ap_tile</li>

<li>Mix hybridized double-stranded DNA and 5ap_tile in 0.2 ml PCR-tubes.</li> <li>Incubate the mixture at 48 ℃ for 1 hour.</li> <li>Mix the mixture and Cy3 streptavidin.</li>

     <li>1wt% Agarose-gel Electrophoresis</li>

<li>Electrophoresis the inserted 5ap_tile for 50 minutes at 100 V at 4 ℃.</li> <li>Take photographs of the electrophoresed gel by LAS-4000.</li>


   </ul>
   </article>
   <br>

<!--◆◆Reaction_between_aptamer_embedded_in_rect_tile_and_PDGF10◆◆-->

  <article>
  <div class="mini-title">
      <a name="Reaction_between_aptamer_embedded_in_rect_tile_and_PDGF10">2) Reaction between aptamer embedded in rect tile and PDGF</a>
  </div>

<!--Reagent-->

     <div class="zairyou-heading">[Reagent]</div>
     <br>

<li>materials for hybridization</li>

   	    <li>5ap_M-3t4e_T0 (1uM) (oligo): 5’ to 3’</li>

TACTCAGGGCACTGCAAGCAATTGTGGTCCCAATGGGCTGAGTACGCGACCTCATCTTTGACCCCCAGGCAGGGAG

 <li>5ap_M-3t4e_T-3(1µM) (oligo):</li>  

TACTCAGGGCACTGCAAGCAATTGTGGTCCCAATGGGCTGAGTAGACCTCATCTTTGACCCCCAGGCAGGGAG

<li>5ap_M-3t4e_T-1(1µM) (oligo):</li> TACTCAGGGCACTGCAAGCAATTGTGGTCCCAATGGGCTGAGTAGCGACCTCATCTTTGACCCCCAGGCAGGGAG

 <li>5ap_M-3t4e_T7(1µM) (oligo):</li>

TACTCAGGGCACTGCAAGCAATTGTGGTCCCAATGGGCTGAGTAtttttttCGCGACCTCATCTTTGACCCCCAGGCAGGGAG

<li>5ap_5B_16 (10uM) (oligo, which has biotin in 5’ end):</li>

Biotin-TACTCAGCCCATTGGG

   	    <li>10x tile buffer<sup>*</sup></li>
   	    <li>MilliQ</li>
   </table>
   <br>

&nbsp;*...10x tile buffer(f.100 µl) <table>

   <tr>
   <th>Mg(OAc)<sub>2</sub></th>
   <td>f.100 mM</td>
   </tr>
   <tr>
   <th>Tris-HCl (pH7.5)</th>
   <td>f.200 mM</td>
   </tr>
       <tr>
   <th>EDTA</th>
   <td>f.10 mM</td>
   </tr>
   </table>

<li>5ap_tile</li>

   	    <li>M13mp18 (scaffold)</li>
   	    <li>5ap_Rmix (staples)</li>
   	    <li>10x tile buffer</li>
   	    <li>PDGF(dye 45nM)</li>

<!--Procedure-->

  <div class="zairyou-heading">[Procedure]</div>
   <ul class="procedure-list">
     <li>Hybridization</li>

<li>Mix materials (mentioned above in [hybridization]) in 0.2 ml PCR-tubes.</li> <li>Incubate the mixture at the room temperature (25 ℃) for 1 hour.</li>

     <li>Making 5ap_tile</li>

<li>Mix materials (mentioned above in [5ap_tile]) in 0.2 ml PCR-tubes.</li> <li>Anneal the mixture using PCR machine (from 85 ℃ to 25 ℃, -2 ℃/min).</li>

     <li>Insertion of hybridized double-stranded DNA into 5ap_tile</li>

<li>Mix hybridized double-stranded DNA and 5ap_tile in 0.2 ml PCR-tubes.</li> <li>Incubate the mixture at 48 ℃ for 1 hour.</li> <li>Mix the mixture and PDGF.</li>

        <li>Incubate at 37 ℃ for 30 min and then at 4 ℃ for 25 hours.</li>
     <li>1wt% Agarose-gel Electrophoresis</li>

<li>Electrophoresis the inserted 5ap_tile for 50 minutes at 100 V at 4 ℃.</li>

        <li>Stain the gel by SYBR Gold in TBE.</li>

<li>Take photographs of the electrophoresed gel by LAS-4000.</li>


   </ul>
   </article>
   <br>


<!--◆◆efficient_hybridization_(changing_mixture_ratio)_Added◆◆-->

  <article>
  <div class="mini-title">
      <a name="efficient_hybridization_(changing_mixture_ratio)">3) Efficient hybridization (changing mixture ratio)</a>
  </div>

<!--Reagent-->

     <div class="zairyou-heading">[Reagent]</div>
     <br>
   	<li>5ap_M-3t4e_T0 (1 µM): 5’ to 3’</li>

TACTCAGGGCACTGCAAGCAATTGTGGTCCCAATGGGCTGAGTACGCGACCTCATCTTTGACCCCCAGGCAGGGAG

   	<li>5ap_5B_16 (1 or 10 µM): Biotin in 5’ end</li>

Biotin - TACTCAGCCCATTGGG

   	<li>10x tile buffer<sup>*</sup></li>
   	<li>MilliQ</li>
   </table>
   <br>

&nbsp;*...10x tile buffer <table>

   <tr>
   <th>Mg(OAc)<sub>2</sub></th>
   <td>f.100 mM</td>
   </tr>
   <tr>
   <th>Tris-HCl (pH7.5)</th>
   <td>f.200 mM</td>
   </tr>
       <tr>
   <th>EDTA</th>
   <td>f.10 mM</td>
   </tr>
   </table>


<!--Procedure-->

  <div class="zairyou-heading">[Procedure]</div>
   <ul class="procedure-list">

<li>Mix materials to make samples, following the ratio written in Table.3.<sup>*</sup></li>

   </table>
   <br>

&nbsp;*...Table.3 <table> <center>

   <tr>
   <th>Sample No.</th>
   <td>1</td>
   <td>2</td>
   <td>3</td>
   <td>4</td>
   <td>5</td>
   <td>6</td>
   <td>7</td>
   <td>8</td>
   </tr>
   <tr>
   <th>5ap_M-3t4e_T0 (1 µM)</th>
   <td>3 µL</td>
   <td>-</td>
   <td>-</td>
   <td>3 µL</td>
   <td>3 µL</td>
   <td>3 µL</td>
   <td>3 µL</td>
   <td>3 µL</td>
   </tr>
       <tr>
   <th>5ap_5B_16 (10 µM)</th>
   <td>-</td>
   <td>-</td>
   <td>-</td>
   <td>-</td>
   <td>-</td>
   <td>1.5 µL</td>
   <td>3 µL</td>
   <td>6 µL</td>
   </tr>
   <tr>
   <th>5ap_5B_16 (1 µM)</th>
   <td>-</td>
   <td>3 µL</td>
   <td>-</td>
   <td>3 µL</td>
   <td>6 µL</td>
   <td>-</td>
   <td>-</td>
   <td>-</td>
   </tr>
   <tr>
   <th>10x tile buffer</th>
   <td>1 µL</td>
   <td>1 µL</td>
   <td>1 µL</td>
   <td>1 µL</td>
   <td>1 µL</td>
   <td>1 µL</td>
   <td>1 µL</td>
   <td>1 µL</td>
   </tr>
       <tr>
   <th>MilliQ</th>
   <td>6 µL</td>
   <td>6 µL</td>
   <td>9 µL</td>
   <td>3 µL</td>
   <td>-</td>
   <td>4.5 µL</td>
   <td>3 µL</td>
   <td>-</td>
   </tr>
       <tr>
   <th>Ratio of concentration of 5ap_5B_16 to 5ap_M-3t4e_T0</th>
   <td>-</td>
   <td>-</td>
   <td>-</td>
   <td>1 %</td>
   <td>2 %</td>
   <td>5 %</td>
   <td>10 %</td>
   <td>20 %</td>    
   </tr>

</center>

   </table>

<li>Apply the samples to 10 % Native-PAGE for 85 minutes at 100 V at 4 ℃.</li> <li>Stain the gel by SYBR Gold in TBE.</li> <li>Take a photograph of the gel by LAS-4000.</li>

   </ul>
   </article>
   <br>
   <!--◆◆efficient hybridization (incubation time)◆◆-->
  <article>
  <div class="mini-title">
      <a name="efficient_hybridization_(incubation_time)">4) efficient hybridization (incubation time)</a>
  </div>

<!--Reagent-->

     <div class="zairyou-heading">[Reagent]</div>
     <br>
   	<li>5ap_M-3t4e_T0 (1 µM): 5’ to 3’</li>

TACTCAGGGCACTGCAAGCAATTGTGGTCCCAATGGGCTGAGTACGCGACCTCATCTTTGACCCCCAGGCAGGGAG

   	<li>5ap_5B_16 (10 µM): (biotin in 5’ end)</li>

Biotin - TACTCAGCCCATTGGG

   	<li>10x tile buffer<sup>*</sup></li>
   	<li>MilliQ</li>
   </table>
   <br>

&nbsp;*...10x tile buffer(f.100 µl) <table>

   <tr>
   <th>Mg(OAc)<sub>2</sub></th>
   <td>f.100 mM</td>
   </tr>
   <tr>
   <th>Tris-HCl (pH7.5)</th>
   <td>f.200 mM</td>
   </tr>
       <tr>
   <th>EDTA</th>
   <td>f.10 mM</td>
   </tr>
   </table>


<!--Procedure-->

  <div class="zairyou-heading">[Procedure]</div>
   <ul class="procedure-list">

<li>Mix the materials in 0.2 ml PCR-tubes.</li> <li>Denature the oligos at 95 ℃ for 30 seconds.</li> <li>Incubate the mixture at room temperature (25 ℃).</li> <li>Freeze the samples into nitrogen liquid at planned incubation time.</li> <li>Pick up the samples out from nitrogen liquid immediately before applying into gel.</li> <li>Apply the samples to 10 % Native PAGE for 85 minutes at 100 V at 4 ℃.</li> <li>Take a photograph of the electrophoresed gel by LAS-4000.</li>


   </ul>
   </article>
   <br>

<!-- ◆◆Insertion of hybridized double-stranded DNA into tile◆◆-->

  <article>
  <div class="mini-title">
      <a name="insertion_of__hybridized_double-stranded_DNA_into_tile">5) Insertion of hybridized double-stranded DNA into tile</a>
  </div>

<!--Reagent-->

     <div class="zairyou-heading">[Reagent]</div>
     <br>

hybridization

   	<li>5ap_M-3t4e_T0 (1 µM) (oligo): 5’ to 3’</li>

TACTCAGGGCACTGCAAGCAATTGTGGTCCCAATGGGCTGAGTACGCGACCTCATCTTTGACCCCCAGGCAGGGAG

   	<li>5ap_5B3G_16 (1 µM) (oligo, which has biotin in 5’ end and Cy3 in 3’ end):</li>

Biotin–TACTCAGCCCATTGGG–Cy3

   	<li>10x tile buffer<sup>*</sup></li>
   	<li>MilliQ</li>
   </table>
   <br>

&nbsp;*...10x tile buffer(f.100 µl) <table>

   <tr>
   <th>Mg(OAc)<sub>2</sub></th>
   <td>f.100 mM</td>
   </tr>
   <tr>
   <th>Tris-HCl (pH7.5)</th>
   <td>f.200 mM</td>
   </tr>
       <tr>
   <th>EDTA</th>
   <td>f.10 mM</td>
   </tr>
   </table>

5ap_tile

   	<li>M13mp18 (scaffold)</li>
   	<li>5ap_Rmix (staples)</li>
   	<li>10x tile buffer</li>

<!--Procedure-->

  <div class="zairyou-heading">[Procedure]</div>
   <ul class="procedure-list">

Hybridization <li>Mix materials (mentioned above in [hybridization]) in 0.2 ml PCR-tubes.</li> <li>Incubating the mixture at the room temperature (25 ℃) for 1 hour.</li>

Making 5ap_tile <li>Mix materials (mentioned above in [5ap_tile]) in 0.2 ml PCR-tubes.</li> <li>Anneal the mixture using PCR machine (from 85 ℃ to 25 ℃, -2 ℃/min).</li>

Insertion of hybridized double-stranded DNA into 5ap_tile <li>Mix hybridized double-stranded DNA and 5ap_tile in 0.2 ml PCR-tubes.</li> <li>Incubate the mixture at 48 ℃ for 1 hour.</li>

1 wt% Agarose-gel Electrophoresis <li>Electrophoresis the inserted 5ap_tile for 50 minutes at 100 V at 4 ℃.</li>

       <li>Take photographs of the electrophoresed gel by LAS-4000.</li>
   </ul>
   </article>
   <br>

<!--Reaction_between_aptamer_(3ap-M5t10f-T0)_and_PDGF_Added-->

  <article>
  <div class="mini-title">
      <a name="Reaction_between_aptamer_(3ap-M5t10f-T0)_and_PDGF">6) Reaction between aptamer (3ap-M5t10f-T0) and PDGF</a>
  </div>

<!--Reagent-->

     <div class="zairyou-heading">[Reagent]</div>
     <br>

<li>materials for the reaction</li>

   <table>
   <tr>
   <th>10x tile buffer </th>
   <td>f. 1x tile buffer</td>
   </tr>
   <tr>
   <th>1 µM aptamer</th>
   <td>f. 0.3µM</td>
   </tr>
   <tr>
   <th>1 µM Ladder151515_1</th>
   <td>f. 0.3µM</td>
   </tr>
   <tr>
   <th>PDGF (dye 45nM)</th>
   <td>f. 20mM</td>
   </tr>
   </table>

<li>materials for the electrophoresis</li>

   <gel for 10% Native-PAGE>
   <table>
   <tr>
   <th>MilliQ</th>
   <td>7.9 mL</td>
   </tr>
   <tr>
   <th>30% Acrylamide mix</th>
   <td>6.7 mL</td>
   </tr>
   <tr>
   <th>1.5 M Tris-HCl (pH 8.8)</th>
   <td>5 mL</td>
   </tr>
   <tr>
   <th>1 M MgCl2</th>
   <td>200 µL</td>
   </tr>
   <tr>
   <th>10 % APS</th>
   <td>80 µL</td>
   </tr>
   <tr>
   <th>TEMED</th>
   <td>80 µL</td>
   </tr>
   </table>
   <Electrophoresis buffer for 10 % Native-PAGE>
     1x TBE
   <materials for stain>
   <table>
   <tr>
   <th>Electrophoresis buffer for 10 % Native-PAGE</th>
   <td>50 mL</td>
   </tr>
   <tr>
   <th>SYBR Gold</th>
   <td>5 µL</td>
   </tr>
   </table>
   <others>
   <table>
   <tr>
   <th>Loading buffer</th>
   <th>20 % glycerol (as used 6x)</th>
   </tr>
   <tr>
   <th>Marker</th>
   <th>Cy5 38 mer</th>
   </tr>
   </table>


<!--Procedure-->

  <div class="zairyou-heading">[Procedure]</div>
   <ul class="procedure-list">
     <li>Mix the solutions as shown below:<sup>*</sup></li>
   </table>
   <br>

&nbsp;*...Table.2 <table> <center>

   <tr>
   <th>Sample No.</th>
   <td>1</td>
   <td>2</td>
   <td>3</td>
   <td>4</td>
   <td>5</td>
   <td>6</td>
   </tr>
   <tr>
   <th>10x tile buffer</th>
   <td>1 µL</td>
   <td>1 µL</td>
   <td>1 µL</td>
   <td>1 µL</td>
   <td>1 µL</td>
   <td>1 µL</td>
   </tr>
       <tr>
   <th>1 µM aptamer</th>
   <td>3 µL</td>
   <td>-</td>
   <td>-</td>
   <td>3 µL</td>
   <td>-</td>
   <td>-</td>
   </tr>
   <tr>
   <th>1 µM Ladder151515_1</th>
   <td>-</td>
   <td>-</td>
   <td>-</td>
   <td>-</td>
   <td>3 µL</td>
   <td>3 µL</td>
   </tr>
   <tr>
   <th>PDGF (dye 45 nM)</th>
   <td>-</td>
   <td>6 µL</td>
   <td>-</td>
   <td>6 µL</td>
   <td>-</td>
   <td>6 µL</td>
   </tr>
       <tr>
   <th>MilliQ</th>
   <td>6 µL</td>
   <td>3 µL</td>
   <td>9 µL</td>
   <td>-</td>
   <td>6 µL</td>
   <td>-</td>
   </tr>

</center>

   </table>
     <li>Incubate at 37 ℃ for 30 min and then at 4 ℃ for 30 min.</li>
     <li>Make 10 % Native PAGE gel as mentioned above.</li>
     <li>Add loading buffer into each samples.</li>
     <li>Apply the samples to 10 % Native-PAGE for 85 minutes at 100V at 4℃.</li>
     <li>Take a photograph of the electrophoresed gel by LAS-4000 for Cy5.</li>
     <li>Stain the gel by SYBR Gold for 20 min.</li>
     <li>Take a photograph by LAS-4000.</li>
   </ul>
   </article>
   <br>


</article>

<!-- ◆◆Double insertion of hybridized double-stranded DNA into tile◆◆-->

  <article>
  <div class="mini-title">
      <a name="Double insertion_of__hybridized_double-stranded_DNA_into_tile">7) Double insertion of hybridized double-stranded DNA into tile</a>
  </div>

<!--Reagent-->

     <div class="zairyou-heading">[Reagent]</div>
     <br>

hybridization

   	<li>5ap_R125_T-3_Cy5 (1 µM) (oligo):</li>

TACTCAGGGCACTGCAAGCAATTGTGGTCCCAATGGGCTGAGTAGACCTCATCTTTGACCCCCAGGCAGGGAG

   	<li>5ap_5B3T30_16_Cy3 (1 µM) (oligo, which has Cy3 in 5’ end):</li>

TACTCAGCCCATTGGGttttttttttttttttttttttttttttttAAAACACTGCTCCATGTTACTTAACAAAGCT

   	<li>10x tile buffer<sup>*</sup></li>
   	<li>MilliQ</li>
   </table>
   <br>

&nbsp;*...10x tile buffer(f.100 µl) <table>

   <tr>
   <th>Mg(OAc)<sub>2</sub></th>
   <td>f.100 mM</td>
   </tr>
   <tr>
   <th>Tris-HCl (pH7.5)</th>
   <td>f.200 mM</td>
   </tr>
       <tr>
   <th>EDTA</th>
   <td>f.10 mM</td>
   </tr>
   </table>

5ap_tile

   	<li>M13mp18 (scaffold)</li>
   	<li>5ap_Rmix (staples)</li>
   	<li>10x tile buffer</li>

<!--Procedure-->

  <div class="zairyou-heading">[Procedure]</div>
   <ul class="procedure-list">

Hybridization <li>Mix materials (mentioned above in [hybridization]) in 0.2 ml PCR-tubes.</li> <li>Incubating the mixture at the room temperature (25 ℃) for 1 hour.</li>

Making 5ap_tile <li>Mix materials (mentioned above in [5ap_tile]) in 0.2 ml PCR-tubes.</li> <li>Anneal the mixture using PCR machine (from 85 ℃ to 25 ℃, -2 ℃/min).</li>

Insertion of hybridized double-stranded DNA into 5ap_tile <li>Mix hybridized double-stranded DNA and 5ap_tile in 0.2 ml PCR-tubes.</li> <li>Incubate the mixture at 48 ℃, 46℃, 44℃, 42℃,or 40℃ for 1 hour.</li>

1 wt% Agarose-gel Electrophoresis <li>Electrophoresis the inserted 5ap_tile for 50 minutes at 100 V at 4 ℃.</li>

       <li>Stain the gel by SYBR Gold in TBE.</li>
       <li>Take photographs of the electrophoresed gel by LAS-4000.</li>
   </ul>
   </article>
   <br>


<!--◆◆STEP4◆◆--> <h2 class="PS_title"><a name="STEP4">&nbsp;STEP 4: The formed subunits oligomerize in solution.</a></h2>

<!--◆◆SA dimer◆◆-->

  <article>
  <div class="mini-title">
      <a name="Dimerization_of_OCK--using_biotin,_streptavidin_and_click_ reaction">1) Dimerization of OCK--using biotin, 	streptavidin and click reaction</a>
  </div>

<!--Reagent-->

     <div class="zairyou-heading">[Reagent]</div>
     <br>
   <table>
   <tr>
   <th>OCK (90 nM)</th>
   <td>8 µL</td>
   </tr>
   <tr>
   <th>Streptavidin (190 nM)</th>
   <td>2 µL</td>
   </tr>
   <tr>
   <th>CuSO4 aq (8 mM)</th>
   <td>1 µL</td>
   </tr>
   <tr>
   <th>THTA (32.5 mM)</th>
   <td>1 µL</td>
   </tr>
   <tr>
   <th>Sodium ascorbate (3.25 mM)</th>
   <td>1 µL</td>
   </tr>


   </table>
   <br>


<!--Procedure-->

  <div class="zairyou-heading">[Procedure]</div>
   <ul class="procedure-list">
     <li>7.4 µL of OCK and 1 µL Streptavidin (190 nM) were mixed and kept at room temperature (27 ℃) for an hour. 		(Mix1)</li>
     <li>10 µL of Mix1 and 1 µL of Sodium ascorbate (3.25 mM) were mixed and then 1 µL of CuSO4 aq (8 mM) was added 		into that solution.</li>
     <li>The solution was mixed and 1µL of THTA (20 mM) was added in it and mixed.</li>
     <li>That solution was kept at room temperature (27 ℃) for a day.</li>
   </ul>
   </article>
   <br>


<!--◆◆Click_reaction◆◆-->

     <div class="mini-title">
      <a name="Click_reaction_via_(3+2)_cycloaddition">
      2) Click reaction via (3+2) cycloaddition
      </a>
  </div>
  <article>

<!--Reagent-->

     <div class="zairyou-heading">[Reagent]</div>
     <br>
   <table>
   <tr>
   <th>azide solution (10μM)</th>
   <td>3μL</td>
   </tr>
   <tr>
   <th>alkyne solution (10μM)</th>
   <td>3μL</td>
   </tr>
   <tr>
   <th>CuSO<sub>4</sub> solution (50mM)</th>
   <td>1μL</td>
   </tr>
   <tr>
   <th>THTA solution (100mM)</th>
   <td>1μL</td>
   </tr>
   <tr>
   <th>sodium ascorbate solution (100mM)</th>
   <td>1μL</td>
   </tr>
   </table>
   <br>

<!--Procedure-->

  <div class="zairyou-heading">[Procedure]<sup>[4]</sup></div>
   <ul class="procedure-list">
     <li>The above all solutions were mixed, using a vortex.</li>
     <li>The solution was kept at room temperature.</li>
   </ul>
   </article>
   <br>

<!--◆◆Accelerated_Click_reaction◆◆-->

  <article>
  <div class="mini-title">
      <a name="Accelerated_Click_reaction">3) Accelerated Click reaction (using streptavidin to make the aklyne and azide 	reactive groups close) </a>
  </div>

<!--Reagent-->

     <div class="zairyou-heading">[Reagent]</div>
     <br>
   <table>
   <tr>
   <th>2x barrel buffer</th>
   <td>6 µL</td>
   </tr>
   <tr>
   <th>alkyne oligo (carrying biotin) (15 µM)</th>
   <td>1 µL</td>
   </tr>
   <tr>
   <th>azide oligo (carrying biotin) (15 µM)</th>
   <td>1 µL</td>
   </tr>


   <tr>
   <th>streptavidin (500 µM)</th>
   <td>1 µL</td>
   </tr>
   </table>
   <br>


<!--Procedure-->

  <div class="zairyou-heading">[Procedure]</div>
   <ul class="procedure-list">
     <li>mix reagents</li>
     <li>incubate the tube at 37 ℃ for indicated reaction time.</li>
     <li>boil at 95 ℃ for 30 minutes to break down streptavidin</li>
   </ul>
   </article>
   <br>

   

<!--◆◆Click_reaction_hybridization◆◆-->

  <article>
  <div class="mini-title">
      <a name="Click_reaction_(using_hybridization_to_make_the_aklyne_and_azide_reactive_groups close)">4) Click reaction 	(using hybridization to make the aklyne and azide reactive 	groups close) </a>
  </div>

<!--Reagent-->

     <div class="zairyou-heading">[Reagent]</div>
     <br>
   <table>
   <tr>
   <th>2x barrel buffer</th>
   <td>7 µL</td>
   </tr>
   <tr>
   <th>alkyne oligo (15 µM)</th>
   <td>1 µL</td>
   </tr>
   <tr>
   <th>azide oligo (15 µM)</th>
   <td>1 µL</td>
   </tr>


   <tr>
   <th>scaffold (15 µM)</th>
   <td>1 µL</td>
   </tr>
   </table>
   <br>


<!--Procedure-->

  <div class="zairyou-heading">[Procedure]</div>
   <ul class="procedure-list">
     <li>mix reagents</li>
     <li>incubate the tube at 37 ℃.</li>
     <li>add loading buffer into the reaction mixture and boil at 95 ℃ for 5 minutes to denature the double strand to 	single strand.</li>
   </ul>

<!--◆◆Click reaction cupper free◆◆-->

  <div class="mini-title">
      <a name="Click_reaction_(copper_catalyst-free)">5) Click reaction (copper catalyst-free)</a>
  </div>

<!--Reagent-->

     <div class="zairyou-heading">[Reagent]</div>
     <br>
   <table>
   <tr>
   <th>2x barrel buffer</th>
   <td>7 µL</td>
   </tr>
   <tr>
   <th>alkyne oligo (15 µM)</th>
   <td>1 µL</td>
   </tr>
   <tr>
   <th>azide oligo (15 µM)</th>
   <td>1 µL</td>
   </tr>


   <tr>
   <th>scaffold (15 µM)</th>
   <td>1 µL</td>
   </tr>
   </table>
   <br>

<!--※10x OCK buffer (f. 100 µl)-->

  <div class="zairyou-heading">[※※	2x barrel buffer]</div>
      <br>
   <table>
   <tr>
   <th>1M Tris (pH 7.5)</th>
   <td>5 µL</td>
   </tr>


   <tr>
   <th>0.5M EDTA</th>
   <td>2 µL</td>
   </tr>


   <tr>
   <th>5M NaCl</th>
   <td>1 µL</td>
   </tr>


   <tr>
   <th>MQ</th>
   <td>32 µL</td>
   </tr>
   </table>
   <br>

<!--Procedure-->

  	<div class="zairyou-heading">[Procedure]</div>
   <ul class="procedure-list">
     <li>mix reagents</li>
     <li>incubate the tube at 37 ℃.</li>
     <li>add loading buffer into the reaction mixture and boil at 95 ℃ for 5 minutes to denature the double strand to 	single strand.</li>
   </ul>
   </article>


<article>
  <div class="mini-title">
      <a name="Synthesis_of_streptavidin_mutants">
      6) Synthesis of streptavidin mutants
      </a>
  </div>


<!--Procedure-->

  <div class="zairyou-heading">[Procedure]<sup>[7],[8]</sup></div>
   <p class="paragraph">Mono-, di-, tri-, tetra-valent streptavidin were prepared as described [7,8] with some modifications. Shortly, BL21 Star (DE3) pLysSRARE and C43 (DE3) was transformed with pET21a(+) SA-Alive-his or pET21a(+) SA-Dead plasmids and cultured in LB at 37℃. Collected cells were resuspended in B-PER (Pierce) and inclusion bodies were purified, and dissolved in 6M guanidinium hydrochloride (GuHCl; pH 1.5). After mixing the unfolded subunits in desired ratio, the unfolded subunits were refolded by rapid dilution into PBS, then concentrated by ammonium sulfate precipitation. After dialyzed 3 x against PBS, refolded streptavidin were purified by Ni-NTA column (GE 17-5248-02) using AKTA system (GE AKTAexplorer 10S). Fractionized samples were concentrated by Amicon Ultra (Millipore).
   </p>
   </article>

<!--Reference-->

    <h1 class="title"><a name="Reference">&nbsp;Reference</a></h1>
    <div>     
       <div class="reference-title">
       <a name="proref-1">
       [1] Folding DNA to create nanoscale shapes and patterns
       </a>
       </div>
          <div class="reference-author">
          Rothemund, P. W.
          </div>
             <div class="reference-journal">
             Nature 440, 297–302 (2006)
             </div>
    </div>
    <div>
       <div class="reference-title">
       <a name="proref-1">
       [2] Rapid Folding of DNA into Nanoscale Shapes at Constant Temperature
       </a>
       </div>
          <div class="reference-author">
           Jean-Philippe J. Sobczak, Thomas G. Martin, Thomas Gerling, Hendrik Dietz
          </div>
             <div class="reference-journal">
             Science, 2012, 338, 1458
             </div>
    </div>
    <div>     
       <div class="reference-title">
       <a name="proref-1">
       [3] Transcription Regulation System Mediated by Mechanical Operation of a DNA &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbs	p;Nanostructure
       </a>
       </div>
          <div class="reference-author">
          Masayuki Endo, Ryoji Miyazaki, Tomoko Emura, Kumi Hidaka, and Hiroshi Sugiyama
          </div>
             <div class="reference-journal">
             Journal of the American Chemical Society, 2012, 134, 2852-2855
             </div>
    </div>
    <div>     
       <div class="reference-title">
       <a name="proref-1">
       [4] the protocol of Jena Bioscience GmbH
       </a>
       </div>
         <div class="reference-journal">
          <a target="_blank" href="http://www.jenabioscience.com" style="color:#e00000">
          http://www.jenabioscience.com</a>
         </div>
    </div>
    <div>
       <div class="reference-title">
       <a name="proref-1">
       [5] Substrate-Assisted Assembly of Interconnected Single-Duplex DNA Nanostructures
       </a>
       </div>
          <div class="reference-author">
          Shogo Hamada, Satoshi Murata Prof.
          </div>
             <div class="reference-journal">
             Angewandte Chemie International Edition,2009,48(37),6820–6823
             </div>
    </div>
    <div>     
       <div class="reference-title">
       <a name="proref-1">
       [6] A primer to scaffolded DNA origami.</a>
       </div>
          <div class="reference-author">
          Castro CE, Kilchherr F, Kim DN, Shiao EL, Wauer T, Wortmann P, Bathe M

and Dietz H.

          </div>
             <div class="reference-journal">
             Nat Methods 221-229 (2011, Mar;8(3))
             </div>
    </div>
         <div>     
       <div class="reference-title">
       <a name="proref-1">
       [7] A monovalent streptavidin with a single femtomolar biotin binding site.</a>
       </div>
          <div class="reference-author">
          Howarth M, Chinnapen DJ, Gerrow K, Dorrestein PC, Grandy MR, Kelleher NL, El-Husseini A and Ting AY.
          </div>
             <div class="reference-journal">
             Nat Methods 267-273 (2006, Apr;3(4))
             </div>
    </div>
         <div>     
       <div class="reference-title">
       <a name="proref-1">
       [8] Imaging proteins in live mammalian cells with biotin ligase and monovalent streptavidin.</a>
       </div>
          <div class="reference-author">
         Howarth M and Ting AY.
          </div>
             <div class="reference-journal">
             Nat Protoc 534-545 (2008, Mar;3(3)); doi: 10.1038/nprot.2008.20.
             </div>
    </div>
             
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