Biomod/2013/Todai/Experiment

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<title>Result-Todai nanORFEVRE-</title>
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  <h1 class="big-title"><a name="Result">&nbsp;Result</a></h1>
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        <img src="
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http://openwetware.org/images/1/15/Todai_Projectsteps_map.png" width=720px height=480px >
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        </center>
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  <h1 class="heading"><a name="Contents">&nbsp;Contents</a></h1>
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<ul>
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<li><h3><a href="#STEP1">STEP 1: DNA strands assemble to form designed structures</a></h3>
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<body>
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<!--Experiment-->
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    <h1 class="big-title"><a name="Experiment">&nbsp;Experiment</a></h1>
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    <div id="Explist">
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    <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>
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-
      <li><div class="mokuji"><a href="#Protocols">Protocols</a></div></li>
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    </ul>
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    </div>
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    <br>
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<!--Contents-->
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  <article>
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    <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;">
<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="#Optimize_the_condition_to_assemble_OCK">1)Optimize the condition to assemble OCK</a></li>
-
<li><a href="#Preparation_of_liposome">2) Preparation of liposome</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="#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>
-
<li><div class="mini-title"><a href="#STEP4">STEP 4: The formed subunits oligomerize in solution.</a></div>
+
<li><h3><a href="#STEP2">STEP 2: Penetration into the membrane</a></h3>
<ul style="list-style: none;">
<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="#Flotation_assay">1)Flotation assay</a></li>
-
 
+
<li><a href="#Preparation_of_GUVs">2)Preparation of GUVs</a></li>
-
<li><a href="#OptimumConc_SA">2) OptimumConc SA</a></li>
+
</ul>
</ul>
</li>
</li>
-
</ul>
+
<li><h3><a href="#STEP3">STEP 3: Recognition of cancer-specific proteins</a></h3>
-
  </article>
+
<ul style="list-style:none;">
-
</article>
+
<li><a href="#Optimization_of_aptamer_lock_system">1) Optimization of aptamer-lock system</a>
-
+
</li>
-
+
<li><a href="#Embedding_of_recognition_system_to_OCK">2) Embedding of recognition system to OCK</a></li>
-
  <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>
</ul>
</li>
</li>
-
<br>
+
<li><h3><a href="#STEP4">STEP 4: Oligomerization in solution</a></h3>
-
<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_[OCK]">1) Flotation assay [OCK]</a></li>
+
<li><a href="#Oligomerization_by_streptavidin-biotin_complex">1)Oligomerization by streptavidin-biotin complex</a></li>
-
<li><a href="#Preparation_of_GUVs">2) Preparation of GUVs</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="Preparation_of_SUVs">3) Preparation of SUVs</a>
+
<li><a href="#Oligomerization_by_Click_reaction">3)Oligomerization by Click reaction</a></li>
-
<li><a href="#hybridization_of_Cholesterol_Oligo_with_OCK">4) hybridization of cholesterol oligo with OCK</a></li>
+
</ul>
</ul>
</li>
</li>
-
<br>
+
-
<li><div class="mini-title"><a href="#STEP3">STEP 3: Recognition of target cells</a></div>
+
  <h1 class="heading"><a name="Oligomeric_Cell_Killer_(OCK)">&nbsp;Oligomeric Cell Killer (OCK)</a></h1>
-
<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◆◆-->
+
<!--◆◆STEP 1: DNA strands assemble to form designed structures◆◆-->
-
    <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>
+
    <article>
-
      </figure>
+
      <h2 class="small-title"><a name="STEP1">&nbsp;STEP 1: DNA strands assemble to form designed structures</a></h2>
 +
     
 +
<!--◆◆Optimize the condition to assemble OCK◆◆-->
 +
<h3><a name ="Optimize_the_condition_to_assemble_OCK"></a>1)Optimize the condition to assemble OCK</h3>
 +
<!--Method-->
 +
    <div class="zairyou-heading">[Method]</div>
 +
    <figure>
 +
        <iframe style="float:left; margin:0;margin-right:-10px;margin-bottom:10px; position:relative;left:-20px;" width="420" height="315" src="//www.youtube.com/embed/1ci93_QI6QA" frameborder="0" allowfullscreen></iframe>
 +
       
 +
</figure>
 +
    <div id ="step0_1)">
 +
      <p class="paragraph">
 +
      The DNA nanostructure,
 +
      <a target="_bramk" href="http://openwetware.org/wiki/Biomod/2013/Todai/Design#Oligomeric_Cell_Killer " style="color:#e00000">
 +
      "Oligomeric Cell Killer"
 +
      </a>
 +
      , was designed to achieve our goal(--><a href="http://openwetware.org/wiki/Biomod/2013/Todai/Project">Project</a>).
 +
      </p>
 +
      <p class ="paragraph">
 +
      The result of simulation by "CanDo<sup>[1]</sup>" showed the shape and flexibility of OCK.
 +
To know the optimum condition of the structure assembly, we did experiments in three conditions as follows.
 +
<ul style="position:relative;left:30px;">
 +
  <li>At different concentration of MgCl<sub>2</sub></li>
 +
  <li>At different incubate temperature</li>
 +
  <li>At different length of incubate time</li>
 +
        </ul>
 +
        (<a target="_blank" href="http://openwetware.org/wiki/Biomod/2013/Todai/Experiment#Protocols" style="color:#e00000;">
 +
        protocols
 +
        </a>
 +
        )
 +
        </p>
 +
    </div>
 +
    <br>
 +
    <br>
 +
<br>
 +
<br>
<br>
<br>
 +
<br>
 +
<!--Result&Discussion1-->
-
  <div class="zairyou-heading">[Discussion]</div>
+
      <div class="zairyou-heading">[Result & 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>
+
    <div class="res-conclusion">
-
        <center>
+
    Optimum concentration of MgCl<sub>2</sub>
-
        <img src="http://openwetware.org/images/7/71/640px_suv_dls_popg50r60-Todai.png" width=640px height=360px >
+
    </div>
-
<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>
       <figure>
         <center>
         <center>
-
         <img src="http://openwetware.org/images/1/1d/640pxflotationassay-Todai.jpg" width=300px height=300px >
+
         <img src="http://openwetware.org/images/a/af/OOCK_Optimize_MgConc.png" width=640px height=360px>
-
<figcaption> <b>Result of agarose gel electrophoresis of the sample of flotation assay</b> <br>
+
            <figcaption> <b>Agarose-gel electrophoresis to research the optimum concentration of MgCl<sub>2</sub>
-
The result of 1% agarose gel electrophoresis(100V,30min). In this measurement, the fluorescence of Cy5, which is
+
</b>
-
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>
-
</figcaption>
+
         </center>
         </center>
       </figure>
       </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">
     <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
+
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.
-
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>
     </p>
-
  <br>
+
    <div class="res-conclusion">
-
</article>
+
    --->> Optimum concentration of MgCl<sub>2</sub>: 10mM 
 +
    </div>
 +
    <br>
 +
    <br>
-
<!--◆◆STEP3◆◆-->
+
<!--Result&Discussion2-->
-
<h2 class="PS_title"><a name="STEP3">&nbsp;STEP 3: Subunits recognize cancer-specific proteins</a></h2>
+
-
<article>
+
-
</article>
+
-
<!--◆◆STEP4◆◆-->
+
    <div class="res-conclusion">
-
<h2 class="PS_title"><a name="STEP4">&nbsp;STEP 4: The formed subunits oligomerize in solution</a></h2>
+
    Optimum incubate temparature
-
 
+
    </div>
-
<!--◆◆Click reaction ◆◆-->
+
 
-
  <div class="mini-title">
+
    <figure>
-
      <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>
         <center>
-
        <img src="http://openwetware.org/images/a/ab/450pxclick0828-Todai.jpg" width=480px height=360px >
+
          <img src="http://openwetware.org/images/9/95/OOCK_Optimize_Temp-Todai.png" width=640px height=360px >
-
<figcaption> <b>Result of urea gel electrophoresis of the sample of click reaction</b><br>
+
          <figcaption>
-
 
+
          <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>
+
-
<!--◆◆Optimum_Conc_SA◆◆-->
+
    <p class="paragraph">
-
  <article>
+
Fast-migrating species upon agarose-gel electrophoresis was yielded at 52.0 °C.  
-
  <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>
+
    </p>
-
        </center>
+
    <div class="res-conclusion">
-
      </figure>
+
    --->> Optimum temparature : 52.0 °C 
-
  <br>
+
    </div>
 +
    <br>
 +
    <br>
-
  <br>
+
<!--Result&Discussion2-->
-
</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 >
+
 +
    <div class="res-conclusion">
 +
    To decide optimum length of incubate time
 +
    </div>
 +
 +
      <figure>
 +
        <center>
 +
          <img src="http://openwetware.org/images/9/94/OOCK_Optimize_Time-Todai.png" width=640px height=360px >
 +
          <figcaption>
 +
          <b>Agarose-gel electrophoresis to research the optimum time</b>
 +
          </figcaption>
         </center>
         </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 procedure of TEM was refered to previous researches<sup>[6]</sup>.  
+
The band for 3h is fast migrated and sharp.
-
    </p>
+
 
-
    </article>
+
    </p>
-
<!--◆◆STEP2◆◆-->
+
-
<h2 class="PS_title"><a name="STEP2">&nbsp;STEP 2: Subunits penetrate into the membrane.</a></h2>
+
-
<!--◆◆flotation asssay of OCK◆◆-->
+
    <div class="res-conclusion">
 +
    --->> Optimum incubate time : 3h
 +
    </div>
-
  <div class="mini-title">
+
    <br>
-
      <a name="Flotation_assay_[OCK]">1) Flotation assay [OCK]</a>
+
-
  </div>
+
-
  <article>
+
-
<!--Reagent-->
+
-
      <div class="zairyou-heading">[Reagent]</div>
+
-
      <br>
+
-
    <table>
+
<!--◆◆1.2 Conformation of the 3D structure of OCK by TEM◆◆-->  
-
    <tr>
+
    <h3><a name="Conformation_of_the_3D_structure_of_OCK_by_TEM"></a>2) TEM imaging of the 3D structure of OCK</h3>
-
    <th>OCK</th>
+
    <!--Method-->
-
    <td>100 µL</td>
+
   
-
    </tr>
+
    <div class="zairyou-heading">[Method]</div>
-
    <tr>
+
    <div id ="step0_2)">
-
    <th>Cholesterol hybridized OCK</th>
+
      <p class="paragraph">
-
    <td>100 µL</td>
+
Gel electrophoresis cannot visualize the 3D structure of OCK, so it was confirmed by Transmission electron microscopy (TEM).
-
    </tr>
+
        (<a target="_blank" href="http://openwetware.org/wiki/Biomod/2013/Todai/Experiment#Protocols" style="color:#e00000;">
-
 
+
        protocols
-
    <tr>
+
        </a>
-
    <th>Liposome (1 mg/mL SUVs)</th>
+
        )
-
    <td>100 µL</td>
+
        </p>
-
    </tr>
+
     </div>
-
 
+
-
    <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>
     <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>
 +
    <br>
 +
<!--Result&Discussion1-->
-
    </table>
+
      <div class="zairyou-heading">[Result & Discussion]</div>
-
    <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>
+
 +
    <div class="res-conclusion">
 +
    TEM imaging of OCK
 +
    </div>
-
<!--Procedure-->
+
      <figure>
-
  <div class="zairyou-heading">[Procedure]</div>
+
        <center>
-
    <ul class="procedure-list">
+
        <img src="http://openwetware.org/images/e/ea/Monomer-Todai.png" width=480px height=360px>
-
      <li>Each sample was mixed as shown below:<sup>****</sup></li>
+
            <figcaption> <b>TEM image of OCK</b>
-
    </table>
+
            Three monomers of OCK were observed in this figure.
-
    <br>
+
       
-
&nbsp;****...Table1. Breakdown of Samples
+
            </figcaption>
-
    <table>
+
         </center>
-
    <tr>
+
      </figure>
-
    <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
+
    <p class="paragraph">
-
mixture in centrifuge tubes (Beckman, cat#343778, 11 x 34 mm).
+
    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.
-
</li>
+
    </p>
-
      <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="res-conclusion">
-
      <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>
+
    </div>
-
      <li>Fraction 1-4 of each sample were analyzed by 1 % agaraose gel
+
    </article>
-
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>
+
 +
<!--◆◆2. Penetration◆◆-->
 +
<!--editting-->
-
    </article>
+
      <h2 class="small-title"><a name="STEP2">&nbsp;STEP 2: Penetration into the membrane</a></h2>
-
    <br>
+
      <article>
-
 
+
-
    <br>
+
 +
<!--◆◆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>
 
-
<!--Procedure-->
+
    <br>
-
  <div class="zairyou-heading">[Procedure]</div>
+
<!--Result&Discussion1-->
-
    <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>
+
      <div class="zairyou-heading">[Result & Discussion]</div>
-
    <br>
+
 +
    <div class="res-conclusion">
 +
   
 +
    </div>
-
<!--◆◆Preparation of SUVs◆◆-->
+
      <figure>
 +
        <center>
 +
        <img src="http://openwetware.org/images/c/ca/Todai_result_step2_fluolescence.JPG" width=450px height=350px>
 +
            <figcaption> <b>The fluorescence intensity of NIL (in liposome) in each fraction </b><br>
 +
            The result of fluorescence spectrophotometer (JASCO, FP-6500) showed that liposome distributed mostly in fraction 3(lower layer).
 +
            </figcaption>
 +
        </center>
 +
      </figure>
 +
          <figure>
 +
        <center>
 +
        <img src="http://openwetware.org/images/f/fe/FlotationOCK12gel-Todai.png" width=480px height=360px>
 +
            <figcaption> <b>1% Agarose gel electrophoresis of each fraction in sample 1, 2</b>
 +
            </figcaption>
 +
        </center>
 +
      </figure>
 +
          <figure>
 +
        <center>
 +
        <img src="http://openwetware.org/images/0/0d/FlotationOCK34gel-Todai.png" width=480px height=360px>
 +
            <figcaption> <b>1% Agarose gel electrophoresis of each fraction in sample 3, 4</b>
 +
            </figcaption>
 +
        </center>
 +
      </figure>
 +
          <figure>
 +
        <center>
 +
        <img src="http://openwetware.org/images/f/ff/450px_FloatingOCKprofile-Todai.jpg" width=450px height=300px>
 +
<figcaption>The ratios of OCK in each fraction were analyzed by the density of band.</figcaption>
 +
        </center>
 +
      </figure>
-
  <div class="mini-title">
+
    <p class="paragraph">
-
      <a name="Preparation_of_SUVs">3) Preparation of SUVs</a>
+
    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.
-
  </div>
+
-
  <article>
+
-
<b>Type 1: POPC 100%</b>
+
-
<!--Reagent-->
+
-
      <div class="zairyou-heading">[Reagent]</div>
+
-
      <br>
+
-
    <table>
+
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>150mM KCl solution</th>
+
-
    <td>3mL</td>
+
-
    </tr>
+
-
    <tr>
+
    </p>
-
    <th>POPC</th>
+
    <div class="res-conclusion">
-
    <td>3mg</td>
+
    --> OCK stack in liposome.
-
    </tr>
+
    </div>
 +
    </article>
 +
<!--◆◆2.2 Preparation of GUVs◆◆-->
-
    <tr>
+
      <article>
-
    <th>Chloroform (99.0%)</th>
+
    <h3><a name="Preparation_of_GUVs"></a>2) Preparation of GUVs</h3>
-
    <td>3mL</td>
+
    <!--Method-->
-
    </tr>
+
    <div class="zairyou-heading">[Method]</div>
-
    <tr>
+
    <div id ="step2_2)">
-
    <th>40μM Nile Red solution</th>
+
      <p class="paragraph">
-
    <td>0.1mL</td>
+
      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.
-
    </tr>
+
        (<a target="_blank" href="http://openwetware.org/wiki/Biomod/2013/Todai/Experiment#Protocols" style="color:#e00000;">
-
 
+
        protocols
-
     </table>
+
        </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-->
+
    <br>
-
      <div class="zairyou-heading">[Reagent]</div>
+
<!--Result&Discussion1-->
-
      <br>
+
-
    <table>
+
      <div class="zairyou-heading">[Result & Discussion]</div>
-
    <tr>
+
-
    <th>Lipid mix<sup>*</sup></th>
+
-
    <td>3 ml</td>
+
-
    </tr>
+
-
    <tr>
+
    <div class="res-conclusion">
-
    <th>150 mM KCl solution</th>
+
   
-
    <td>1 µL</td>
+
    </div>
-
    </tr>
+
 +
          <figure>
 +
        <center>
 +
        <img src="http://openwetware.org/images/b/be/GUVconfocal_scale-Todai.png" width=450px height=350px>
-
    </table>
+
         </center>
-
    <br>
+
       </figure>
-
&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-->
+
    <p class="paragraph">
-
  <div class="zairyou-heading">[Procedure]</div>
+
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.
-
    <ul class="procedure-list">
+
About 10 um of GUVs were observed.
-
      <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>
+
    </p>
-
   
+
-
<!--◆◆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>
+
    </article>
-
    <tr>
+
-
    <th>OCK</th>
+
-
    <td>48 µL</td>
+
-
    </tr>
+
-
    <tr>
+
<!--◆◆STEP 3: Recognition of cancer-specific proteins◆◆-->
-
    <th>Cholesterol oligo (0.32, 0.64, 3.2, 6.4 µM)</th>
+
-
    <td>100 µL</td>
+
-
    </tr>
+
-
    </table>
+
    <article>
-
<center>
+
      <h2 class="small-title"><a name="STEP3">&nbsp;STEP3: Subunits recognize cancer-specific proteins. </a></h2>
-
Marker; GeneRuler DNA Ladder Mix (Fermentas, GeneRuler DNA Ladder Mix #SM0331)
+
    <h3><a name="Optimization_of_aptamer_lock_system"></a>1) Optimization of aptamer-lock system</h3>
-
</center>
+
<!--Method-->
-
 
+
    <div class="zairyou-heading">[Method]</div>
-
<!--Procedure-->
+
            <figure>
-
  <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>
+
-
#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/6/66/OCK_Cholesterol-Todai.png" width=300px height=300px>
+
         <img src="http://openwetware.org/images/5/50/Todai_Recognition_ideal_mod.png" width=480px height=270px>
-
       
+
         </center>
         </center>
       </figure>
       </figure>
-
      <li>Each sample was analyzed by 1% agarose gel electrophoresis (100V, 1 hour).</li>
 
-
    </ul>
+
    <div id ="step0_1)">
 +
      <p class="paragraph">
 +
     
 +
      We did pilot study of oligomerization process triggered by membrane
 +
      protein recognition. We used cholesterol modified PDGF as model membrane
 +
      protein, as DNA origami embedded aptamer system recognizing PDGF was
 +
      reported (Douglass et al. (2012)).
 +
     
 +
      </p>
 +
      <p class ="paragraph">
 +
      Our simplified model lock system is consisted with two steps: 1)
 +
Blocking of streptavidin binding to biotin by steric hindrance. Our lock
 +
system consists of two strands: biotin attached strands (biotin strands)
 +
and aptamer attached strands (aptamer strands). These two strands
 +
hybridize each other in inactive form and hide biotin moiety from the
 +
streptavidin by steric hindrance effect. 2) Upon binding of ligands
 +
(PDGF in this study) to aptamer strands, the complementary strand(biotin
 +
strands) is released from the DNA aptamer, because ligands take over the
 +
DNA strands of DNA aptamer from the complementally strands, and biotin
 +
can now bind to streptavidin. Therefore, the cancer cell recognition and
 +
OCK oligomerization are achieved simultaneously in the future study.
 +
      </p>
-
     </article>
+
     </div>
     <br>
     <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>
     <br>
-
&nbsp;*...10x tile buffer(f.100 µl)
+
<br>
-
    <table>
+
    </article>
-
    <tr>
+
    <br>
-
    <th>Mg(OAc)<sub>2</sub></th>
+
<!--Result&Discussion1-->
-
    <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>
+
      <div class="zairyou-heading">[Result & Discussion]</div>
-
<li>5ap_tile</li>
+
    <div class="res-conclusion">
-
        <li>M13mp18 (scaffold)</li>
+
    Integration of aptamer strands into DNA origami tile
-
        <li>Cy5_Rmix (staples)</li>
+
    </div>
-
        <li>10x tile buffer</li>
+
                          <figure>
-
        <li>Cy3 streptavidin (800nM)</li>
+
        <center>
 +
        <img src="http://openwetware.org/images/d/d3/Todai_intofapt_1.png" width=600px height=450px>
 +
        </center>
 +
        </figure>
 +
                                  <figure>
 +
        <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>
-
<!--Procedure-->
+
            </figcaption>
-
  <div class="zairyou-heading">[Procedure]</div>
+
        </center>
-
    <ul class="procedure-list">
+
      </figure>
-
      <li>Hybridization</li>
+
      <p class="paragraph">                               We confirmed the integration of aptamer attached strands (aptamer
-
<li>Mix materials (mentioned above in [hybridization]) in 0.2 ml PCR-tubes.</li>
+
strands) into rectangle DNA origami tile (rect-tile).
-
<li>Incubate the mixture at the room temperature (25 ℃) for 1 hour.</li>
+
</p>
 +
<br>
 +
    <div class="res-conclusion">
 +
    Responsibility of aptamer
 +
    </div>
 +
                <figure>
 +
        <center>
 +
        <img src="http://openwetware.org/images/4/42/Todai_Recognition_tile_B_PDGF_mod.png" width=600px height=450px>
-
      <li>Making 5ap_tile</li>
+
        </center>
-
<li>Mix materials (mentioned above in [5ap_tile]) in 0.2 ml PCR-tubes.</li>
+
      </figure>
-
<li>Anneal the mixture using PCR machine (from 85 ℃ to 25 ℃, -2 ℃/min).</li>
+
      <figure>
 +
        <center>
 +
        <img src="http://openwetware.org/images/f/f5/Figure9_10-Todai.png" width=480px height=270px>
 +
            <figcaption> <b></b>
-
      <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>
+
            </figcaption>
-
<li>Electrophoresis the inserted 5ap_tile for 50 minutes at 100 V at 4 ℃.</li>
+
        </center>
-
<li>Take photographs of the electrophoresed gel by LAS-4000.</li>
+
      </figure>
 +
      <p class ="paragraph">
 +
            We confirmed the responsibility of aptamer sequence embedded in
 +
rect-tile (shown above). The position of Cy5-PDGF band coincided
 +
with that of DNA tile, showing that the aptamers work also on rect-tile.
 +
Furthermore, the linker length between aptamer sequence and staple
 +
sequence, the latter staple sequence is embedded into rect-tile, does
 +
not affect the binding ability of aptamer to PDGF.
 +
</p>
 +
<br>
 +
    <div class="res-conclusion">
 +
    Blocking capability of lock system  by aptamer
 +
    </div>
 +
                    <figure>
 +
        <center>
 +
        <img src="http://openwetware.org/images/9/96/Todai_Recognition_tile_B_SA_mod.png" width=600px height=450px>
 +
        </center>
 +
      </figure>
 +
      <table cellpadding="0" style="position:relative;left:-50px;">
 +
        <tbody><tr>
 +
        <td>
 +
        <figure>
 +
          <img src="http://openwetware.org/images/3/30/Figure11_12-Todai.png" width="360px" height="240px">
-
    </ul>
+
        </figure>
 +
        </td>
-
    </article>
+
        <td>
-
    <br>
+
        <figure  style="position:relative;left:-50px;">
 +
          <img src="http://openwetware.org/images/2/24/Koyama_131027_1-Todai.JPG" width="240px" height="240px">
-
<!--◆◆Reaction_between_aptamer_embedded_in_rect_tile_and_PDGF10◆◆-->
+
        </figure>
-
  <article>
+
        </td>
-
  <div class="mini-title">
+
        </tr>
-
      <a name="Reaction_between_aptamer_embedded_in_rect_tile_and_PDGF10">2) Reaction between aptamer embedded in rect tile and PDGF</a>
+
      </tbody></table>
-
  </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>
+
    <p class="paragraph">
-
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>
+
We confirmed the blocking capability of our lock system for streptavidin
-
    <br>
+
binding (left figure, the image of gel electrophoresis). Our lock system consists of two strands:
-
&nbsp;*...10x tile buffer(f.100 µl)
+
biotin attached strands (biotin strands) and aptamer attached strands
-
    <table>
+
(aptamer strands). These two strands hybridize each other in inactive
-
    <tr>
+
form and hide biotin moiety from the streptavidin by steric hindrance
-
    <th>Mg(OAc)<sub>2</sub></th>
+
effect. We confirmed this blocking capability by mixing Cy3 labeled
-
    <td>f.100 mM</td>
+
streptavidin with lock system embedded rect-tile. Data indicates that
-
    </tr>
+
the slight blocking capability upon shorten the polyT linker between
-
    <tr>
+
aptamer sequence and staple sequence. Recently, we tried other sequence
-
    <th>Tris-HCl (pH7.5)</th>
+
and have better results, which may be presented in the Jamboree in Boston.
-
    <td>f.200 mM</td>
+
<br>
-
    </tr>
+
    <div class="res-conclusion">
-
         <tr>
+
Optimum embedding condition of our lock system into rect-tile
-
    <th>EDTA</th>
+
    </div>
-
    <td>f.10 mM</td>
+
                                      <figure>
-
    </tr>
+
        <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>
-
    </table>
 
-
<li>5ap_tile</li>
+
            </figcaption>
-
        <li>M13mp18 (scaffold)</li>
+
        </center>
-
        <li>5ap_Rmix (staples)</li>
+
      </figure>
-
        <li>10x tile buffer</li>
+
Next, we optimize the embedding condition of our lock system into
-
        <li>PDGF(dye 45nM)</li>
+
rect-tile. This time full length of biotin strands were used instead of
-
<!--Procedure-->
+
truncated ones used in above figure. Data indicate that the integrate
-
  <div class="zairyou-heading">[Procedure]</div>
+
efficiency of both biotin strands and aptamer strands into rect-tile is
-
    <ul class="procedure-list">
+
independent on the incubate temperature.
-
      <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>
+
We improve our lock system everyday. Don't miss our presentation in
-
<li>Mix materials (mentioned above in [5ap_tile]) in 0.2 ml PCR-tubes.</li>
+
Jaboree in Boston !
-
<li>Anneal the mixture using PCR machine (from 85 ℃ to 25 ℃, -2 ℃/min).</li>
+
    </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>
-
      <li>Insertion of hybridized double-stranded DNA into 5ap_tile</li>
+
    <div id ="step3_1)">
-
<li>Mix hybridized double-stranded DNA and 5ap_tile in 0.2 ml PCR-tubes.</li>
+
      <p class="paragraph">
-
<li>Incubate the mixture at 48 ℃ for 1 hour.</li>
+
To embed recognition system to OCK, we equiped PDGF aptamer used in rect-tile to OCK and confirmed the association of aptamer and PDGF .
-
<li>Mix the mixture and PDGF.</li>
+
        (<a target="_blank" href="http://openwetware.org/wiki/Biomod/2013/Todai/Experiment#Protocols" style="color:#e00000;">
-
        <li>Incubate at 37 ℃ for 30 min and then at 4 ℃ for 25 hours.</li>
+
        protocols
 +
        </a>
 +
        )
 +
        </p>
 +
    </div>
 +
<!--Result&Discussion1-->
-
       <li>1wt% Agarose-gel Electrophoresis</li>
+
       <div class="zairyou-heading">[Result & Discussion]</div>
-
<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>
+
 +
    <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>
-
    </ul>
+
        </center>
 +
      </figure>
 +
     
 +
          <div class="res-conclusion">
 +
    -->PDGF was recognized by the aptamer of OCK
 +
    </div>
 +
<!--◆◆STEP 4: Oligomerization in solution◆◆-->
-
    </article>
+
    <article>
-
    <br>
+
      <h2 class="small-title"><a name="STEP4">&nbsp;STEP 4: Oligomerization in solution</a></h2>
 +
     
 +
<!--◆◆4.1 Oligomerization by streptavidin-biotin complex◆◆-->
 +
    <h3><a name="Oligomerization_by_streptavidin-biotin_complex"></a>1) Oligomerization by streptavidin-biotin complex</h3>
 +
    <!--Method-->
 +
    <div class="zairyou-heading">[Method]</div>
-
 
+
    <div id ="step3_1)">
-
<!--◆◆efficient_hybridization_(changing_mixture_ratio)_Added◆◆-->
+
      <p class="paragraph">
-
  <article>
+
       Biotins are equipped to OCK for oligomerization. The experiment which confirmed that streptavidins induced oligomeriation.
-
  <div class="mini-title">
+
        (<a target="_blank" href="http://openwetware.org/wiki/Biomod/2013/Todai/Experiment#Protocols" style="color:#e00000;">
-
       <a name="efficient_hybridization_(changing_mixture_ratio)">3) Efficient hybridization (changing mixture ratio)</a>
+
        protocols
-
  </div>
+
        </a>
-
 
+
        )
-
<!--Reagent-->
+
        </p>
-
      <div class="zairyou-heading">[Reagent]</div>
+
     </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>
     <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>
     <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>
+
    </article>
-
    <th>5ap_M-3t4e_T0 (1 µM)</th>
+
    <br>
-
    <td>3 µL</td>
+
<!--Result&Discussion1-->
-
    <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>
+
      <div class="zairyou-heading">[Result & Discussion]</div>
-
    <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>
+
    <div class="res-conclusion">
-
    <th>5ap_5B_16 (1 µM)</th>
+
    Streptavidins induced oligomerization
-
    <td>-</td>
+
    </div>
-
    <td>3 µL</td>
+
-
    <td>-</td>
+
-
    <td>3 µL</td>
+
-
    <td>6 µL</td>
+
-
    <td>-</td>
+
-
    <td>-</td>
+
-
    <td>-</td>
+
-
    </tr>
+
-
    <tr>
+
      <figure>
-
    <th>10x tile buffer</th>
+
        <center>
-
    <td>1 µL</td>
+
        <img src="http://openwetware.org/images/5/5b/Streptavidin_dimer-Todai.png" width=600px height=450px>
-
    <td>1 µL</td>
+
<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>
-
    <td>1 µL</td>
+
         </center>
-
    <td>1 µL</td>
+
      </figure>
-
    <td>1 µL</td>
+
    </article>
-
    <td>1 µL</td>
+
   
-
    <td>1 µL</td>
+
<!--◆◆4.2 EMm imaging of dimers by streptavidin-biotin complex◆◆-->
-
    <td>1 µL</td>
+
    <article>
-
    </tr>
+
          <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>
-
         <tr>
+
    <!--Method-->
-
    <th>MilliQ</th>
+
    <div class="zairyou-heading">[Method]</div>
-
    <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>
+
    <div id ="step4_2)">
-
<li>Stain the gel by SYBR Gold in TBE.</li>
+
      <p class="paragraph">
-
<li>Take a photograph of the gel by LAS-4000.</li>
+
      Dimers of OCKs were also imaged by TEM to confirm the bands observed in the experiment 3.1) originated from the dimers.
-
 
+
        (<a target="_blank" href="http://openwetware.org/wiki/Biomod/2013/Todai/Experiment#Protocols" style="color:#e00000;">
-
    </ul>
+
        protocols
-
 
+
        </a>
-
     </article>
+
        )
 +
        </p>
 +
     </div>
     <br>
     <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>
     <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>
+
    </article>
 +
    <br>
 +
<!--Result&Discussion1-->
 +
      <div class="zairyou-heading">[Result & Discussion]</div>
-
<!--Procedure-->
+
    <div class="res-conclusion">
-
  <div class="zairyou-heading">[Procedure]</div>
+
    </div>
-
    <ul class="procedure-list">
+
    <center>
-
<li>Mix the materials in 0.2 ml PCR-tubes.</li>
+
      <table cellpadding="0" style ="position:relative;left:-30px;">
-
<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>
         <tr>
-
    <th>EDTA</th>
+
        <td>
-
    <td>f.10 mM</td>
+
        <figure>
-
    </tr>
+
          <img src="http://openwetware.org/images/c/c1/Dimerv2-Todai.png" width="300px" height="300px" >
 +
        </figure>
 +
        </td>
-
    </table>
 
-
5ap_tile
+
        <td>
-
    <li>M13mp18 (scaffold)</li>
+
        <figure>
-
    <li>5ap_Rmix (staples)</li>
+
          <img src="http://openwetware.org/images/5/53/Dimer_2v2-Todai.png" width="300px" height="300px" >
-
    <li>10x tile buffer</li>
+
        </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◆◆-->
-
<!--Procedure-->
+
    <article>
-
  <div class="zairyou-heading">[Procedure]</div>
+
    <h3><a name="Oligomerization_by_Click_reaction"></a>3) Oligomerization by Click reaction</h3>
-
    <ul class="procedure-list">
+
    <!--Method-->
-
Hybridization
+
    <div class="zairyou-heading">[Method]</div>
-
<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
+
    <div id ="step4_3)">
-
<li>Mix materials (mentioned above in [5ap_tile]) in 0.2 ml PCR-tubes.</li>
+
      <p class="paragraph">
-
<li>Anneal the mixture using PCR machine (from 85 ℃ to 25 ℃, -2 ℃/min).</li>
+
      Azide and alkyne, which function as a reactive group of click reaction, are also equiped to OCK. It demands Cu<sup>+</sup> as catalyst, but too high concentration of Cu<sup>+</sup> (cation) might denaturate OCK like Mg<sup>2+</sup>. Therefore, we optimized the concentration of Cu<sup>+</sup> to OCK first, and then the optimum Cu<sup>+</sup> concentration to click reaction was investigated.
 +
        (<a target="_blank" href="http://openwetware.org/wiki/Biomod/2013/Todai/Experiment#Protocols" style="color:#e00000;">
 +
        protocols
 +
        </a>
 +
        )
 +
        </p>
 +
    </div>
 +
   
 +
      <div class="zairyou-heading">[Result & Discussion]</div>
 +
      <div class="res-conclusion">
 +
      a) Optimum concentration of CuSO<sub>4</sub> to OCK
 +
    </div>
 +
          <figure>
 +
        <center>
 +
        <img src="http://openwetware.org/images/5/5e/CuAlive-Todai.png" width=600px height=450px>
-
Insertion of hybridized double-stranded DNA into 5ap_tile
+
        </center>
-
<li>Mix hybridized double-stranded DNA and 5ap_tile in 0.2 ml PCR-tubes.</li>
+
      </figure>
-
<li>Incubate the mixture at 48 ℃ for 1 hour.</li>
+
              <div class="res-conclusion">
-
 
+
      -->Optimum concentration of CuSO<sub>4</sub>: 625 uM or less
-
1 wt% Agarose-gel Electrophoresis
+
    </div>
-
<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>
 
-
 
-
    <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>
     <br>
-
&nbsp;*...Table.2
+
     </div>
-
    <table>
+
      <div class="res-conclusion">
-
<center>
+
      b) Optimum concentration of CuSO<sub>4</sub> to click reaction
-
     <tr>
+
    </div>
-
    <th>Sample No.</th>
+
          <figure>
-
    <td>1</td>
+
        <center>
-
    <td>2</td>
+
        <img src="http://openwetware.org/images/9/94/ClickResult-Todai.png" width=600px height=450px>
-
    <td>3</td>
+
-
    <td>4</td>
+
-
    <td>5</td>
+
-
    <td>6</td>
+
-
    </tr>
+
-
    <tr>
+
        </center>
-
    <th>10x tile buffer</th>
+
      </figure>
-
    <td>1 µL</td>
+
      <p class ="paragraph">
-
    <td>1 µL</td>
+
      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.
-
    <td>1 µL</td>
+
      </p>
-
    <td>1 µL</td>
+
              <div class="res-conclusion">
-
    <td>1 µL</td>
+
              --> Optimum concentration of CuSO<sub>4</sub>: 625 uM
-
    <td>1 µL</td>
+
    </div>
-
    </tr>
+
    </article>
 +
    <!--◆◆4.4 Cupper-free click chemistry◆◆-->
-
        <tr>
+
    <article>
-
    <th>1 µM aptamer</th>
+
    <h3><a name="Cupper-free_click_reaction"></a>4) Cupper-free click reaction</h3>
-
    <td>3 µL</td>
+
    <!--Method-->
-
    <td>-</td>
+
    <div class="zairyou-heading">[Method]</div>
-
    <td>-</td>
+
-
    <td>3 µL</td>
+
-
    <td>-</td>
+
-
    <td>-</td>
+
-
    </tr>
+
-
    <tr>
+
    <div id ="step4_3)">
-
    <th>1 µM Ladder151515_1</th>
+
       <p class="paragraph">
-
    <td>-</td>
+
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.
-
    <td>-</td>
+
        (<a target="_blank" href="http://openwetware.org/wiki/Biomod/2013/Todai/Experiment#Protocols" style="color:#e00000;">
-
    <td>-</td>
+
        protocols
-
    <td>-</td>
+
        </a>
-
    <td>3 µL</td>
+
        )
-
    <td>3 µL</td>
+
        </p>
-
    </tr>
+
     </div>
-
 
+
-
    <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◆◆-->
+
      <div class="zairyou-heading">[Result & Discussion]</div>
-
  <article>
+
      <div class="res-conclusion">
-
  <div class="mini-title">
+
       a) Optimum concentration of CuSO<sub>4</sub> to OCK
-
       <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>
-
  </div>
+
-
<!--Reagent-->
+
<center>
-
       <div class="zairyou-heading">[Reagent]</div>
+
      <table cellpadding="0" style="position:relative;left:-25px;">
-
       <br>
+
        <tbody><tr>
 +
        <td>
 +
        <figure>
 +
          <img src="http://openwetware.org/images/2/25/Gelphoto1-Todai.png" width="400px" height="225px">
 +
        </figure>
 +
        </td>
 +
        <td>
 +
        <figure  style="position:relative;left:-50px;">
 +
          <img src="http://openwetware.org/images/2/2f/Yatagai_131027_1.JPG" width="225px" height="225px">
 +
<figcaption>
 +
<b>The reaction rate of cupper-free click reaction with no accelerator</b>
 +
</figcaption>
 +
        </figure>
 +
        </td>
 +
       
 +
        </tr>
 +
       </tbody></table>
 +
</center>
 +
      <p class="paragraph">
 +
We first measured the Cu-free click reaction in solution (without no
 +
catalyst nor accelerator). The association time at 2 uM oligonucleotide
 +
condition was 17.1 h, and appearent association time was estimated as
 +
8.1 [1/M/s].
 +
</p>
 +
<center>
 +
        <table cellpadding="0" style="position:relative;left:-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>
-
    <table>
 
-
    <tr>
 
-
    <th>2x barrel buffer</th>
 
-
    <td>7 µL</td>
 
-
    </tr>
 
-
    <tr>
+
        <td>
-
    <th>alkyne oligo (15 µM)</th>
+
        <figure style="position:relative;left:-35px;">
-
    <td>1 µL</td>
+
          <img src="http://openwetware.org/images/c/c6/Yatagaiclickhybri-Todai.png" width="270px" height="270px">
-
    </tr>
+
-
    <tr>
+
        </figure>
-
    <th>azide oligo (15 µM)</th>
+
        </td>
-
    <td>1 µL</td>
+
        </tr>
-
    </tr>
+
      </tbody></table>
 +
</center>
 +
                <figure  style="position:relative;left:-45px;">
 +
          <img src="http://openwetware.org/images/a/a2/Yatagai_131027_2.JPG" width="270px" height="270px">
-
    <tr>
+
        </figure>
-
    <th>scaffold (15 µM)</th>
+
       
-
    <td>1 µL</td>
+
        <p class="paragraph">
-
    </tr>
+
We add accelerator, which can work as a scaffold and make alkyne and
-
 
+
azide reactive group close. Acceleration of the click reaction was
-
    </table>
+
observed. In other words, azide and alkyne reactive groups do not react
-
    <br>
+
each other in solution, but easy to react each other after
-
 
+
proximally-positioned. This character is very suitable to prevent
-
 
+
non-specific oligomerization, while accelerating the specific
-
<!--Procedure-->
+
oligomerization in OCK.
-
  <div class="zairyou-heading">[Procedure]</div>
+
</p>
-
    <ul class="procedure-list">
+
      <p class ="paragraph">
-
      <li>mix reagents</li>
+
Note: Streptavidin has 4 identical subunits. So we can not control the
-
      <li>incubate the tube at 37 ℃.</li>
+
binding order of alkyne and azide oligo with streptavidin method.
-
      <li>add loading buffer into the reaction mixture and boil at 95 ℃ for 5 minutes to denature the double strand to single strand.</li>
+
Therefore, vicinity subunit may have identical reactive groups (e.g.
-
    </ul>
+
alkyne-alkyne or azide-azide, instead of alkyne-azide or azide-alkyne),
-
 
+
and may reduce the yield.
-
<!--◆◆Click reaction cupper free◆◆-->
+
</p>
-
  <div class="mini-title">
+
               <div class="res-conclusion">
-
      <a name="Click_reaction_(copper_catalyst-free)">5) Click reaction (copper catalyst-free)</a>
+
               --> Cu-free click reaction has suitable character for specific oligomerization.
-
  </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>
 +
   
 +
    </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">
-
         [3] Transcription Regulation System Mediated by Mechanical Operation of a DNA &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbs p;Nanostructure
+
         [1] CanDo(<a href="http://cando-dna-origami.org/usersguide">http://cando-dna-origami.org/usersguide</a>)
         </a>
         </a>
         </div>
         </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>
-
 
+
     <br>
-
     <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">
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          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">
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-
              Nat Protoc 534-545 (2008, Mar;3(3)); doi: 10.1038/nprot.2008.20.
+
-
              </div>
+
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    </div>
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Revision as of 01:46, 27 October 2013


Result-Todai nanORFEVRE-

 Result

 Contents

  • STEP 1: DNA strands assemble to form designed structures

  • STEP 2: Penetration into the membrane

  • STEP 3: Recognition of cancer-specific proteins

  • STEP 4: Oligomerization in solution

  •  Oligomeric Cell Killer (OCK)

     STEP 1: DNA strands assemble to form designed structures

    1)Optimize the condition to assemble OCK

    [Method]

    The DNA nanostructure, "Oligomeric Cell Killer" , was designed to achieve our goal(-->Project).

    The result of simulation by "CanDo[1]" showed the shape and flexibility of OCK. To know the optimum condition of the structure assembly, we did experiments in three conditions as follows.

    • At different concentration of MgCl2
    • At different incubate temperature
    • At different length of incubate time
    ( protocols )







    [Result & Discussion]
    Optimum concentration of MgCl2
    Agarose-gel electrophoresis to research the optimum concentration of MgCl2

    Fast-migrating species upon agarose-gel electrophoresis was yielded at 10~20mM MgCl2 condition. At higher MgCl2 concentration, a sub-band, which might be a dimer, appeared.

    --->> Optimum concentration of MgCl2: 10mM


    Optimum incubate temparature
    Agarose-gel electrophoresis to research the optimum temperature

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

    --->> Optimum temparature : 52.0 °C


    To decide optimum length of incubate time
    Agarose-gel electrophoresis to research the optimum time

    The band for 3h is fast migrated and sharp.

    --->> Optimum incubate time : 3h

    2) TEM imaging of the 3D structure of OCK

    [Method]

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




    [Result & Discussion]
    TEM imaging of OCK
    TEM image of OCK Three monomers of OCK were observed in this figure.

    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.

     STEP 2: Penetration into the membrane

    1) Flotation assay

    [Method]

    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. ( protocols )




    [Result & Discussion]
    The fluorescence intensity of NIL (in liposome) in each fraction
    The result of fluorescence spectrophotometer (JASCO, FP-6500) showed that liposome distributed mostly in fraction 3(lower layer).
    1% Agarose gel electrophoresis of each fraction in sample 1, 2
    1% Agarose gel electrophoresis of each fraction in sample 3, 4
    The ratios of OCK in each fraction were analyzed by the density of band.

    With the condition of cholesterol +/ liposome+, the peak fraction was No.3, which coinced with peak fraction of liposome. In contrast, lacking of cholesterol or liposome, OCK exist mainly in fraction No.2. As the peak fraction of OCK shifted from fraction No.2 to No.3, with the attachment of cholesterol and existence of liposome, we concluded that OCK stack in liposome.

    --> OCK stack in liposome.

    2) Preparation of GUVs

    [Method]

    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. ( protocols )




    [Result & Discussion]

    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.

     STEP3: Subunits recognize cancer-specific proteins.

    1) Optimization of aptamer-lock system

    [Method]

    We did pilot study of oligomerization process triggered by membrane protein recognition. We used cholesterol modified PDGF as model membrane protein, as DNA origami embedded aptamer system recognizing PDGF was reported (Douglass et al. (2012)).

    Our simplified model lock system is consisted with two steps: 1) Blocking of streptavidin binding to biotin by steric hindrance. Our lock system consists of two strands: biotin attached strands (biotin strands) and aptamer attached strands (aptamer strands). These two strands hybridize each other in inactive form and hide biotin moiety from the streptavidin by steric hindrance effect. 2) Upon binding of ligands (PDGF in this study) to aptamer strands, the complementary strand(biotin strands) is released from the DNA aptamer, because ligands take over the DNA strands of DNA aptamer from the complementally strands, and biotin can now bind to streptavidin. Therefore, the cancer cell recognition and OCK oligomerization are achieved simultaneously in the future study.





    [Result & Discussion]
    Integration of aptamer strands into DNA origami tile

    We confirmed the integration of aptamer attached strands (aptamer strands) into rectangle DNA origami tile (rect-tile).


    Responsibility of aptamer

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


    Blocking capability of lock system by aptamer

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

    Optimum embedding condition of our lock system into rect-tile
    Next, we optimize the embedding condition of our lock system into rect-tile. This time full length of biotin strands were used instead of truncated ones used in above figure. Data indicate that the integrate efficiency of both biotin strands and aptamer strands into rect-tile is independent on the incubate temperature. We improve our lock system everyday. Don't miss our presentation in Jaboree in Boston !

    2) Embedding of recognition system to OCK

    [Method]

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

    [Result & Discussion]
    Recognition of PDGF by DNA aptamer on OCK
    -->PDGF was recognized by the aptamer of OCK

    [Result & Discussion]
    Streptavidins induced oligomerization
    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).

    2) TEM imaging of OCK dimers connected by streptavidin-biotin interaction

    [Method]

    Dimers of OCKs were also imaged by TEM to confirm the bands observed in the experiment 3.1) originated from the dimers. ( protocols )




    [Result & Discussion]

    Dimers of OCKs were observed in this experiment and two of them were shown above.

    -->The dimerization by streptavidin-biotin complex was confirmed.

    3) Oligomerization by Click reaction

    [Method]

    Azide and alkyne, which function as a reactive group of click reaction, are also equiped to OCK. It demands Cu+ as catalyst, but too high concentration of Cu+ (cation) might denaturate OCK like Mg2+. Therefore, we optimized the concentration of Cu+ to OCK first, and then the optimum Cu+ concentration to click reaction was investigated. ( protocols )

    [Result & Discussion]
    a) Optimum concentration of CuSO4 to OCK
    -->Optimum concentration of CuSO4: 625 uM or less


b) Optimum concentration of CuSO4 to click reaction

Product of click reaction appeared over 375 uM CuSO4 concentration. Combining with the stability data, we decided to use 625 uM CuSO4 condition.

--> Optimum concentration of CuSO4: 625 uM

4) Cupper-free click reaction

[Method]

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. ( protocols )

[Result & Discussion]
a) Optimum concentration of CuSO4 to OCK
The reaction rate of cupper-free click reaction with no accelerator

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].

We add accelerator, which can work as a scaffold and make alkyne and azide reactive group close. Acceleration of the click reaction was observed. In other words, azide and alkyne reactive groups do not react each other in solution, but easy to react each other after proximally-positioned. This character is very suitable to prevent non-specific oligomerization, while accelerating the specific oligomerization in OCK.

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.

--> Cu-free click reaction has suitable character for specific oligomerization.

 Reference










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