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<title>Experiment-Todai nanORFEVRE-</title>
<title>Experiment-Todai nanORFEVRE-</title>
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</style>
<!--Experiment-->
</head>
    <h1 class="big-title"><a name="Experiment">&nbsp;Experiment</a></h1>
    <div id="Explist">
    <ul>
      <li><div class="mokuji"><a href="#Contents">Contents of pilot study</a></div></li>
      <li><div class="mokuji"><a href="#Contents">Contents of Protocols</a></div></li>
      <li><div class="mokuji"><a href="#PilotStudy">Pilot Study</a></div></li>
      <li><div class="mokuji"><a href="#Protocols">Protocols</a></div></li>
    </ul>
    </div>
    <br>


<body>
<!--Contents-->


<!--◆◆Experiment◆◆-->
  <article>
    <h1 class="big-title"><a name="Experiment">&nbsp;Experiment</a></h1>
    <h1 class="title"><a name="Contents">&nbsp;Contents of pilot study</a></h1>
    <div id="Explist">
    <ul >


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


<!--◆◆Contents◆◆-->
<li><a href="#OptimumConc_SA">2) OptimumConc SA</a></li>
 
</ul>
  <article>
</li>
    <h1 class="title"><a name="Contents">&nbsp;Contents</a></h1>
</ul>
 
  </article>
  <article>
</article>
    <ul class="Contents-list">
 
 
  <article>
  <li><a href ="#Assembling_of_DNA_structure"><b>Assembly of DNA nanostructure</b> -->(See <a href="http://openwetware.org/wiki/Biomod/2013/Todai/Result">Result</a>)
    <h1 class="title"><a name="Contents">&nbsp;Contents of protocols</a></h1>
<div class="preparation">Optimize the assembly condition of the DNA nanostructure,"Cylinder in barrel"</div><br>
      <article>
</li>
        <ul>
<li><a href ="#flotation_assay"><b>Flotation assay</b></a>
<li><div class="mini-title"><a href="#STEP1">STEP 1: DNA strands assemble to form designed structures.</a></div> <ul style="list-style: none;">
    <ul class="Contents-list">
<li><a href="#Assembling_of_OCK">1) Assembly of OCK</a></li>
      <li><a href ="#Preparation_of_liposome">Preparation of liposome</a><br>
<li><a href="#Transmission_electron_microscopy">2) Transmission electron microscopy(TEM)</a></li>
       Making of liposome for floatation assay
</ul>
       <br>
</li>
      </li>
<br>
      <li><a href ="#Flotation_assay_of_liposome_and_DNA_origami">Floatation assay of liposome and DNA origami</a>
<li><div class="mini-title"><a href="#STEP2">STEP 2: Subunits stick in the membrane.</a></div>
<br> Assay to check the penetration of DNA origami
<ul style="list-style: none;">
<br>
<li><a href="#Flotation_assay_[OCK]">1) Flotation assay [OCK]</a></li>
</li>
<li><a href="#Preparation_of_GUVs">2) Preparation of GUVs</a></li>
</ul><br></li>
<li><a href="Preparation_of_SUVs">3) Preparation of SUVs</a>
<li>
<li><a href="#hybridization_of_Cholesterol_Oligo_with_OCK">4) Hybridization of cholesterol oligo with OCK</a></li>
<a href ="#Comparision_of_dimerization"><b>Comparision of dimerization method</b> </a>
</ul>
</li>
<br>
<li><div class="mini-title"><a href="#STEP3">STEP 3: Recognition of target cells</a></div>
<ul style="list-style: none;">
<li><a href="#Reaction_of_a_biotinized_oligo_to_streptavidin">1) Reaction of a biotinized oligo to streptavidin</a></li>
<li>      <a href="Reaction_between_aptamer_embedded_in_rect_tile_and_PDGF10">2) Reaction between aptamer embedded in rect tile and PDGF</a>
<li><a href="#efficient_hybridization_(changing_mixture_ratio)">3) Efficient hybridization (changing mixture ratio)</a></li>
<li><a href="#efficient_hybridization_(incubation_time)">4) Efficient hybridization (incubation time)</a></li>
<li><a href="#insertion_of__hybridized_double-stranded_DNA_into_tile">5) Insertion of hybridized double-stranded DNA into tile</a></li>
<li> <a href="#Reaction_between_aptamer_(3ap-M5t10f-T0)_and_PDGF">6) Reaction between aptamer (3ap-M5t10f-T0) and PDGF</a></li>
<li><a href="Double insertion_of__hybridized_double-stranded_DNA_into_tile">7) Double insertion of hybridized double-stranded DNA into tile</a></li>
</ul>
</li>
<br>
<li><div class="mini-title"><a href="#STEP4">STEP 4: The formed subunits oligomerize in solution.</a></div>
<ul style="list-style: none;">
<li><a href="#Oligomerization_by_streptavidin-biotin_complex">1) Oligomerization by streptavidin-biotin complex</a></li>
<li>       <a href="#Click_reaction_via_(3+2)_cycloaddition"> 2) Click reaction via (3+2) cycloaddition
       </a>
      </li>
<li>
      <a href="#Accelerated_Click_reaction">3) Accelerated Click reaction (using streptavidin to make the alkyne 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 alkyne 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>


<ul class="Contents-list">
      <li><a href ="#Click_reaction_via_(3+2)_cycloaddition">Click reaction via (3+2) cycloaddition</a>
<br>Optimize the reaction condition for click chemistry
<br>
</li>
<li><a href ="#Azobenzene">Azobenzene</a>
<ul class="Contents-list">
      <li><a href ="#Synthesis_of_Tube(Research_for_azobenzene)">Synthesis of tube</a>
<br> Optimize the assembly condition pf the DNA origami tube to be equipped with azobenzene
<br>
</li>
<li><a href ="#Synthesis_of_Motif(Research_for_azobenzene)">Synthesis of tube</a>
<br>Optimize the assembly condition of the DNA motif to be equipped with azobenzene<br>
</li>
</ul>
</li>
</ul>
</li>
     
    </ul>
</article>
</article>
<!--◆◆Pilot Study◆◆-->
<!--◆◆Pilot Study◆◆-->
  <article>
 
     <h1 class="title"><a name="PilotStudy">&nbsp;Pilot Study</a></h1>
     <h1 class="title"><a name="PilotStudy">&nbsp;Pilot Study</a></h1>
      
      
<!--★Liposome making★-->
<!--◆◆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>
   <article>
 
  <h2><a name="Assembling_of_DNA_structure">&nbsp;1.Assembly of DNA nanostructure</a></h2>
  <p class="paragraph">
  The assembly of DNA structure is explained in <a href="http://openwetware.org/wiki/Biomod/2013/Todai/Result">result</a>.
  <h2><a name="flotation_assay">&nbsp;2.Flotation assay</a></h2>
   <div class="mini-title">
   <div class="mini-title">
    <div style="width:512px">
       <a name="hybridization_of_Cholesterol_Oligo_with_OCK">1) Hybridization of cholesterol Oligo with OCK</a>
       <a name="Preparation_of_liposome">2.1.Preparation of liposome</a>
</div>
    </div>
  </div>
 
       <figure>
       <figure>
         <center>
         <center>
         <img src="http://openwetware.org/images/b/b5/640px_liposomeDLS-Todai.png" width=640px height=360px >
         <img src="http://openwetware.org/images/d/d7/480px_OCKchol-Todai.png" width=480px height=300px >
<figcaption> <b>The result of DLS </b>
 
      </figcaption>
         </center>
         </center>
       </figure>
       </figure>
<div class="zairyou-heading">[Discussion]</div>
    <p class="paragraph">In flotation assay, uniformly-sized liposomes are required because liposomes should have


same buoyancy. Moreover, liposomes must have enough radiuses (about 100nm in radius) to float in sucrose buffer.
<br>


Using Extruder device(Avanti), we prepared liposome of 120nm in radius.
  <div class="zairyou-heading">[Discussion]</div>
    <p class="paragraph">
The result of 1 % agarose gel electrophoresis showed that the band of
sample 5 and 7 were smeared, showing the successful of hybridization of
cholesterol oligo with OCK (Langecker et al. (2012)). We concluded that
the optimized condition for hybridization is: 1 hour incubation at room
temperature with 5 times excess cholesterol oligo to OCK.
    </p>
  <br>
</article>
<!--◆◆Preparation_of_liposome◆◆-->
<article
  <div class="mini-title">
      <a name="Preparation_of_liposome">2) Preparation of liposome</a>
  </div>


      <figure>
        <center>
        <img src="http://openwetware.org/images/7/71/640px_suv_dls_popg50r60-Todai.png" width=640px height=360px >
<figcaption> <b>The result of DLS (Viscotek, 802 DLS)</b>
      </figcaption>
        </center>
      </figure>
<div class="zairyou-heading">[Discussion]</div>
    <p class="paragraph">For floating assay, uniformly-sized liposome were prepared. DLS data shows sharp peak with the mean radius of 60 nm, indicating the homogenity of liposomes.
     </p>
     </p>
   </article>
   </article>
   <br>
   <br>
<!--◆◆Flotation assay(Rect-tile)◆◆-->


<!--★Flotation assay★-->
   <article>
   <article>
   <div class="mini-title">
   <div class="mini-title">
    <div style="width:512px">
       <a name="Flotation_assay_of_liposome_and_DNA_origami">3) Floatation assay of liposome and Rectangular tile(DNA origami)</a>
       <a name="Flotation_assay_of_liposome_and_DNA_origami">2.2.Floatation assay of liposome and DNA origami</a>
      </div>
  </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/1/1d/640pxflotationassay-Todai.jpg" width=300px height=300px >
<figcaption> <b>Result of agarose gel electrophoresis of the sample of flotation assay</b> <br>
<figcaption> <b>Result of agarose gel electrophoresis of the sample of flotation assay</b> <br>
The result of 1% agarose gel electrophoresis(100V,30min). In this measurement, the fluorescence of Cy5, which is  
The result of 1% agarose gel electrophoresis(100V,30min). In this measurement, the fluorescence of Cy5, which is  
integrated into DNA origami(Rect tile<sup>[1]</sup>) ,is observed. Fraction1 is the liquid in the top layer, and fraction 5 is in the bottom layer. Fraction 6 is the sample retrieved from precipitation. DNA origami solely was also loaded on the extreme right lane.
integrated into DNA origami(Rect tile<sup>[1]</sup>) ,is observed. Fraction1 is the liquid in the top layer, and fraction 5 is in the bottom layer. Fraction 6 is the sample retrieved from precipitation. DNA origami solely was also loaded on the extreme right lane.
</figcaption>
</figcaption>
         </center>
         </center>
       </figure>
       </figure>
Line 207: Line 257:
         <center>
         <center>
         <img src="http://openwetware.org/images/0/0d/300pxNILGraph-Todai.PNG" width=350px height=350px >
         <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>
<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 fluorescence of NIL(Nile Red, ex 500nm, em 550~700nm )  
Although the size of liposome might change during the flotation assay(data not shown), the intensity of the fluorescence 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
suggests the amount of lipid membrane,liposome. The fluorescence spectrum of water was subtracted as background
       </figcaption>
       </figcaption>
         </center>
         </center>
       </figure>
       </figure>
<br>
<br>


   <div class="zairyou-heading">[Discussion]</div>
   <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  
To confirm the flotation assay, mixed tiles(DNA origami) and liposomes were assayed. Five samples (fraction 1,2,..., 5, from the top) were  
retrieved from supermetant liquid and a sample(fraction 6) from precipitation by the addition of buffer used in  
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 distribution of liposomes is observed by the fluorescence of NIL(Nile Red).  
assay. When the sample, tile mixed with liposomes, were assayed, tiles were observed in the top layer. The distribution of liposomes is observed by the fluorescence of NIL(Nile Red).  
     </p>
     </p>
  <br>
  <br>
</article>
</article>
<!--★Click Reaction★-->
 
<h2><a name="Comparision_of_dimerization">3.Comparision of dimerization method</a></h2>
 
  <article>
<!--◆◆STEP3◆◆-->
  <div class="mini-title">
<h2 class="PS_title"><a name="STEP3">&nbsp;STEP 3: Subunits recognize cancer-specific proteins</a></h2>
    <div style="width:500px">
<article>
</article>
 
<!--◆◆STEP4◆◆-->
<h2 class="PS_title"><a name="STEP4">&nbsp;STEP 4: The formed subunits oligomerize in solution</a></h2>
 
<!--◆◆Click reaction ◆◆-->
  <div class="mini-title">
       <a name="Click_reaction_via_(3+2)_cycloaddition">
       <a name="Click_reaction_via_(3+2)_cycloaddition">
      3-1.Click reaction via (3+2) cycloaddition<sup>[4]</sup>
        1)Optimum time of Click reaction via (3+2) cycloaddition<sup>[4]</sup>
       </a>
       </a>
    </div>
  </div>
  </div>
 
  <article>
       <figure>
       <figure>
         <center>
         <center>
         <img src="http://openwetware.org/images/a/ab/450pxclick0828-Todai.jpg" width=480px height=360px >
         <img src="http://openwetware.org/images/a/ab/450pxclick0828-Todai.jpg" width=480px height=360px >
<figcaption> <b>Result of urea gel electrophoresis of the sample of click reaction</b><br>
<figcaption> <b>Result of urea gel electrophoresis of the sample of click reaction</b><br>


       </figcaption>
       </figcaption>
         </center>
         </center>
       </figure>
       </figure>
<div class="zairyou-heading">[Discussion]</div>
<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 class="paragraph">Copper(Ⅰ) catalyzed click reaction was used to dimerize of oligo DNA(length of 20bp and 14bp) . The time cause of the reaction indicate that the click reaction is so quick(<5min).</p>


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


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


    </p>
   <br>
   <br>
 
</article>
<!--★Synthesis of tube (Research for azobenzene)★-->
<!--◆◆Evaluation of streptavidin mutants◆◆-->
   <article>
   <article>
      <div class="mini-title">
    <div style="width:150px">
      <a name ="Azobenzene">3-2.Azobenzene</a>
    </div>
  </div>
   <div class="mini-title">
   <div class="mini-title">
    <div style="width:512px">
      <a name="Evaluation_of_streptavidin_mutants">3) Evaluation of streptavidin mutants</a>
      <a name="Synthesis_of_Tube(Research_for_azobenzene)">3-2-1.Synthesis of tube<sup>[2],[3]</sup>(Research for
azobenzene)</a>
    </div>
   </div>
   </div>
 
          <figure>
      <figure>
         <center>
         <center>
         <img src="http://openwetware.org/images/b/b2/640x360px_tube_result-Todai.png" width=640px height=360px >
         <img src="http://openwetware.org/images/1/1e/SA_result-Todai.png" width=400px height=400px >
<figcaption> <b>results of the electrophoresis of DNA-tube</b><br>


      </figcaption>
         </center>
         </center>
       </figure>
       </figure>
<div class="zairyou-heading">[Discussion]</div>
  <br>
    <p class="paragraph">  
<p class="paragraph">
We examined how DNA-tube was synthesized efficiently by using the method which is introduced in “Rapid Folding of
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◆◆-->


DNA into Nanoscale Shapes at Constant Temperature” (Jean-Philippe J. Sobczak et al, Science, 2012, 338, 1458)
    <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>


<sup>[2]</sup>. In
<!--◆◆Assembly of OCK◆◆-->


the figure above, two bands derived from scaffold or DNA-tube were showed with cursors. At 56.4℃, the scaffold
  <div class="mini-title">
      <a name="Assembling_of_OCK">1) Assembly of OCK<sup>[2]</sup></a>
      </div>
    <br>
  <article>


band was diminished. In contrast, the DNA-tube band was concentrated. In fact, the ratio of DNA- tube band


to scaffold band was the greatest at this temperature, which means we succeeded in synthesizing DNA-
<!--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>


tube efficiently and improving the yield of DNA-tube.


    </p>
    </table>
  <br>
    <br>
<!--★Synthesis of Motif (Research for azobenzene)★-->
  <article>
  <div class="mini-title">
    <div style="width:256px">
      <a name="Synthesis_of_Motif(Research_for_azobenzene)">3.2.2.Synthesis of Motif<sup>[5]</sup></a>
    </div>
  </div>


      <figure>
&nbsp;*...10x OCKbuffer(f.100 ul)
         <center>
    <table>
        <img src="http://openwetware.org/images/a/a8/480px_electrophoresis_of_T-motif_improved-Todai.png"
    <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>


width=480px height=360px >
<!--Procedure-->
<figcaption> <b>Result of agarose gel electrophoresis of T-motif (wheel)</b><br>
  <div class="zairyou-heading">[Procedure]</div>
The result of 1.5% agarose gel electrophoresis(100V) for 33min.
    <ul class="procedure-list">
      </figcaption>
      <li>mix the solutions.</li>
        </center>
      <li>It was annealed at 85 °C for 25 min and then at 52 °C for 3 or 4 hours.</li>
      </figure>
    </ul>
<div class="zairyou-heading">[Discussion]</div>
    <p class="paragraph">  
In this measurement, it is difficult to distinguish between final structure band and monomer band.  
Next time, Native PAGE will be used instead of agarose gel.  
    </p>
  <br>


    </article>
    <br>


<!--◆◆Protocols◆◆-->


  <article>
<!--◆◆TEM◆◆-->
    <h1 class="title"><a name="Protocols">&nbsp;Protocols</a></h1>


<!--★定温法　構造体(staple+コレステ)組立プロトコル★-->
   <article>
   <article>
   <div class="mini-title">
   <div class="mini-title">
    <div style="width:515px">
      <a name="Transmission_electron_microscopy">
      <a name="Assembling_of_DNA structure (scaffold+staple+cholesterol)">1.Assembly of DNA structure (scaffold
      2) Transmission electron microscopy(TEM)
      </a>
  </div>
<!--Procedure-->
  <div class="zairyou-heading">[Procedure]<sup>[6]</sup></div>
    <p class="paragraph">
    The procedure of TEM was refered to previous researches<sup>[6]</sup>.
    </p>
    </article>
<!--◆◆STEP2◆◆-->
<h2 class="PS_title"><a name="STEP2">&nbsp;STEP 2: Subunits penetrate into the membrane.</a></h2>


+staple)<sup>[2]</sup></a>
<!--◆◆flotation asssay of OCK◆◆-->
    </div>
 
  <div class="mini-title">
      <a name="Flotation_assay_[OCK]">1) Flotation assay [OCK]</a>
   </div>
   </div>
 
  <article>
<!--Reagent-->
<!--Reagent-->
       <div class="zairyou-heading">[Reagent]</div>
       <div class="zairyou-heading">[Reagent]</div>
       <br>
       <br>
Line 336: Line 454:
     <table>
     <table>
     <tr>
     <tr>
     <th>M13mp18ss (0.1μM)</th>
     <th>OCK</th>
     <td>5μL</td>
    <td>100 µL</td>
    </tr>
 
    <tr>
    <th>Cholesterol hybridized OCK</th>
    <td>100 µL</td>
    </tr>
 
    <tr>
    <th>Liposome (1 mg/mL SUVs)</th>
    <td>100 µL</td>
    </tr>
 
    <tr>
    <th>2.25 M Sucrose buffer<sup>*</sup></th>
    <td>500 µL</td>
    </tr>
 
    <tr>
    <th>1.6 M Sucrose buffer<sup>**</sup></th>
    <td>900 µL</td>
    </tr>
 
    <tr>
    <th>150 mM KCl solution</th>
     <td>100 µL</td>
     </tr>
     </tr>


     <tr>
     <tr>
     <th>staple mix (0.68μM)</th>
     <th>1×Flotation buffer<sup>***</sup></th>
     <td>4μL</td>
     <td>600 µL</td>
     </tr>
     </tr>


    </table>
    <br>
&nbsp;*...2.25 M Sucrose buffer
    <table>
    <tr>
    <th>HEPES-KOH (pH 7.6)</th>
    <td>50 mM</td>
    </tr>
    <tr>
    <th>KCl</th>
    <td>100 mM</td>
    </tr>
        <tr>
    <th>MgCl<sub>2</sub></th>
    <td>20 mM</td>
    </tr>
    <tr>
    <th>Sucrose</th>
    <td>2.25 M</td>
    </tr>       
    </table>
    </table>
    <br>
&nbsp;**...1.6 M Sucrose buffer
    <table>
    <tr>
    <th>HEPES-KOH (pH 7.6)</th>
    <td>50 mM</td>
    </tr>
     <tr>
     <tr>
     <th>10x buffer</th>
     <th>KCl</th>
     <td>1μL</td>
     <td>100 mM</td>
    </tr>
        <tr>
    <th>MgCl<sub>2</sub></th>
    <td>20 mM</td>
     </tr>
     </tr>
    <tr>
    <th>Sucrose</th>
    <td>1.6 M</td>
    </tr>       
    </table>


     </table>
     </table>
     <br>
     <br>
&nbsp;***...1×Flotation buffer
    <table>
    <tr>
    <th>HEPES-KOH (pH 7.6)</th>
    <td>50 mM</td>
    </tr>
    <tr>
    <th>KCl</th>
    <td>100 mM</td>
    </tr>
        <tr>
    <th>MgCl<sub>2</sub></th>
    <td>20 mM</td>
    </tr>   
    </table>


<!--Procedure-->
 
   <div class="zairyou-heading">[Procedure]<sup>[2]</sup></div>
<!--Procedure-->
   <div class="zairyou-heading">[Procedure]</div>
     <ul class="procedure-list">
     <ul class="procedure-list">
       <li>5μL of M13mp18ss, 4μL of staple mix and 1μL of 10×buffer were mixed, pipetting.</li>
       <li>Each sample was mixed as shown below:<sup>****</sup></li>
       <li>The solution was kept at 45 degree C for 4h.</li>
    </table>
    <br>
&nbsp;****...Table1. Breakdown of Samples
    <table>
    <tr>
    <th>Sample No.</th>
    <td>1</td>
    <td>2</td>
    <td>3</td>
    <td>4</td>
    </tr>
    <tr>
    <th>Cholesterol hybridized OCK</th>
    <td>50 µL</td>
    <td>50 µL</td>
    <td>-</td>
    <td>-</td>
    </tr>
        <tr>
    <th>OCK</th>
    <td>-</td>
    <td>-</td>
    <td>50 µL</td>
    <td>50 µL</td>
    </tr>
    <tr>
    <th>Liposome</th>
    <td>50 µL</td>
    <td>-</td>
    <td>50 µL</td>
    <td>-</td>
    </tr>
    <tr>
    <th>150 mM aqueous KCl solution</th>
    <td>-</td>
    <td>50 µL</td>
    <td>-</td>
    <td>50 µL</td>
    </tr>
        <tr>
    <th>2.25 M Sucrose buffer</th>
    <td>125 µL</td>
    <td>125 µL</td>
    <td>125 µL</td>
    <td>125 µL</td>
    </tr>
    </table>
 
      <li>225 µL of 1.6 M sucrose buffer was overlaid with 225 µL of sample
mixture in centrifuge tubes (Beckman, cat#343778, 11 x 34 mm).
</li>
      <li>Centrifuge for 16 minutes at 100 krpm at 4 ℃ using TLA 100.2 rotor (BECKMAN COULTER) with Ultracentrifuge (BECKMAN COULTER, Optima MAX-XP).</li>
      <li>150 µL of supernatant was extracted from top to bottom for 3 times (Fraction 1 to 3) and the pellet was retrieved with 150 µL of 1×Flotation buffer (Fraction 4).</li>
      <li>Fraction 1-4 of each sample were analyzed by 1 % agaraose gel
electrophoresis (100V, 1 hour).
</li>
       <li>The Intensity of fluorescence of NileRed (Liposome) was measured with fluorescence spectrophotometer (JASCO, FP-6500) to investigate the existence of liposome in each Fraction.</li>
      <li>The radiuses of liposome of each fraction were measured with DLS (Viscotek, 802 DLS).</li>
     </ul>
     </ul>


     </article>
     </article>
     <br>
     <br>


<!--★リポソーム作製★-->
    <br>
 
 
    <br>
 
<!--◆◆Preparation of GUVs◆◆-->
 
  <div class="mini-title">
      <a name="Preparation_of_GUVs">2) Preparation of GUVs</a>
  </div>
   <article>
   <article>
   <h2>2.Flotation assay</h2>
<!--Reagent-->
      <div class="zairyou-heading">[Reagent]</div>
      <br>
 
    <table>
    <tr>
    <th>Lipid mix<sup>*</sup></th>
    <td>3 ml</td>
    </tr>
 
    <tr>
    <th>150 mM KCl solution</th>
    <td>1 µL</td>
    </tr>
 
 
    </table>
    <br>
&nbsp;*...Lipid mix
    <table>
    <tr>
    <th>5 mg/mL POPC</th>
    <td>0.1 mL</td>
    </tr>
    <tr>
    <th>5 mg/mL POPG</th>
    <td>0.1 mL</td>
    </tr>
        <tr>
    <th>10 uM NileRed solution</th>
    <td>0.13 mL</td>
    </tr>
    <tr>
    <th>Chloroform</th>
    <td>2.67 mL</td>
    </tr>       
    </table>
 
<!--Procedure-->
   <div class="zairyou-heading">[Procedure]</div>
    <ul class="procedure-list">
      <li>A lipid film was formed by evaporating 3 ml of lipid mix in a 50 ml
eggplant flask, using a rotational evaporator (EYELA, model#N1110) for
10 mins.
</li>
      <li>The flask was kept under vacuum overnight to evaporate remaining chloroform.</li>
      <li>The lipid film was resuspended in 1 mL of 150 mM KCl solution.</li>
    </ul>
 
    </article>
    <br>
 
 
<!--◆◆Preparation of SUVs◆◆-->
 
   <div class="mini-title">
   <div class="mini-title">
    <div style="width:215px">
       <a name="Preparation_of_SUVs">3) Preparation of SUVs</a>
       <a name="Preparation_of_liposome">2.1.Preparation of liposome</a>
    </div>
   </div>
   </div>
 
  <article>
<!--Reagent-->
<b>Type 1: POPC 100%</b>
<!--Reagent-->
       <div class="zairyou-heading">[Reagent]</div>
       <div class="zairyou-heading">[Reagent]</div>
       <br>
       <br>
Line 378: Line 699:
     <table>
     <table>
     <tr>
     <tr>
     <th>150mM aqueous KCl solution</th>
     <th>150mM KCl solution</th>
     <td>3mL</td>
     <td>3mL</td>
     </tr>
     </tr>
Line 400: Line 721:
     <br>
     <br>


<!--Procedure-->
<!--Procedure-->
   <div class="zairyou-heading">[Procedure]</div>
   <div class="zairyou-heading">[Procedure]</div>
     <ul class="procedure-list">
     <ul class="procedure-list">
Line 406: Line 727:
       <li>A lipid film was formed by evaporating 3mL of POPC solution in a 50mL eggplant flask, using a rotational  
       <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>
evaporator for 5 minutes.</li>
       <li>The flask was kept under vacuum overnight to evaporate remaining chloroform.</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 aqueous KCl solution.</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 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 size of liposome was measured with DLS (Viscotek 802 DLS).</li>
Line 416: Line 737:
     </article>
     </article>
     <br>
     <br>
<!--Preparation of SUVs_Added-->
  <article>
<b>Type 2: POPC 50%, POPG 50%</b>
<!--Reagent-->
      <div class="zairyou-heading">[Reagent]</div>
      <br>
    <table>
    <tr>
    <th>Lipid mix<sup>*</sup></th>
    <td>3 ml</td>
    </tr>
    <tr>
    <th>150 mM KCl solution</th>
    <td>1 µL</td>
    </tr>
    </table>
    <br>
&nbsp;*...Lipid mix
    <table>
    <tr>
    <th>5 mg/mL POPC</th>
    <td>0.1 mL</td>
    </tr>
    <tr>
    <th>5 mg/mL POPG</th>
    <td>0.1 mL</td>
    </tr>
        <tr>
    <th>10 uM NileRed solution</th>
    <td>0.13 mL</td>
    </tr>
    <tr>
    <th>Chloroform</th>
    <td>2.67 mL</td>
    </tr>       
    </table>
<!--Procedure-->
  <div class="zairyou-heading">[Procedure]</div>
    <ul class="procedure-list">
      <li>A lipid film was formed by evaporating 3 ml of lipid mix in a 50 ml
eggplant flask, using a rotational evaporator (EYELA, model#N1110) for
10 mins.
</li>
      <li>The flask was kept under vacuum overnight to evaporate remaining chloroform.</li>
      <li>The lipid film was resuspended in 1 mL of 150 mM KCl solution.</li>
      <li>Lipid suspended solution was filtered through 100nm polar filter using extruder (Avanti) to prepare uniformly-sized liposome.</li>
      <li>The size of liposome was measured with DLS (Viscotek 802 DLS).</li>
      <li>The solution was kept at 3℃ until usage.</li>
    </ul>
    </article>
   
<!--◆◆Hybridization of cholesterol modified oligo◆◆-->
  <article>
  <div class="mini-title">
      <a name="Protocol_Hybridization_of_cholesterol_oligo_with_OCK">4) Hybridization of cholesterol oligo with OCK</a>
  </div>
<!--Reagent-->
      <div class="zairyou-heading">[Reagent]</div>
      <br>
    <table>
    <tr>
    <th>OCK</th>
    <td>48 µL</td>
    </tr>
    <tr>
    <th>Cholesterol oligo (0.32, 0.64, 3.2, 6.4 µM)</th>
    <td>100 µL</td>
    </tr>
    </table>
<center>
Marker; GeneRuler DNA Ladder Mix (Fermentas, GeneRuler DNA Ladder Mix #SM0331)
</center>
<!--Procedure-->
  <div class="zairyou-heading">[Procedure]</div>
    <ul class="procedure-list">
      <li>Centrifuge for 16 minutes at 100 krpm at 4 ℃ using TLA 100.2 rotor (BECKMAN COULTER) with Ultracentrifuge (BECKMAN COULTER, Optima MAX-XP).</li>
      <li>Each sample was mixed and incubated as shown below:<sup>*</sup></li>
    </table>
    <br>
&nbsp;*...Table1.
    <table>
<center>
    <tr>
    <th>Sample No.</th>
    <td>1</td>
    <td>2</td>
    <td>3</td>
    <td>4</td>
    <td>5</td>
    <td>6</td>
    <td>7</td>
    <td>8</td>
    </tr>
    <tr>
    <th>Purified OCK (40 µM)</th>
    <td>6 µL</td>
    <td>6 µL</td>
    <td>6 µL</td>
    <td>6 µL</td>
    <td>6 µL</td>
    <td>6 µL</td>
    <td>6 µL</td>
    <td>6 µL</td>
    </tr>
        <tr>
    <th>0.32 µM Cholesterol oligo</th>
    <td>1.5 µL</td>
    <td>1.5 µL</td>
    <td>-</td>
    <td>-</td>
    <td>-</td>
    <td>-</td>
    <td>-</td>
    <td>-</td>
    </tr>
    <tr>
    <th>0.64 µM Cholesterol oligo</th>
    <td>-</td>
    <td>-</td>
    <td>1.5 µL</td>
    <td>1.5 µL</td>
    <td>-</td>
    <td>-</td>
    <td>-</td>
    <td>-</td>
    </tr>
    <tr>
    <th>3.2 µM Cholesterol oligo</th>
    <td>-</td>
    <td>-</td>
    <td>-</td>
    <td>-</td>
    <td>1.5 µL</td>
    <td>1.5 µL</td>
    <td>-</td>
    <td>-</td>
    </tr>
        <tr>
    <th>6.4 µM Cholesterol oligo</th>
    <td>-</td>
    <td>-</td>
    <td>-</td>
    <td>-</td>
    <td>-</td>
    <td>-</td>
    <td>1.5 µL</td>
    <td>1.5 µL</td>
    </tr>
        <tr>
    <th>[Cholesterol oligo]/[OCK] (see #Note)</th>
    <td>1/2</td>
    <td>1/2</td>
    <td>1</td>
    <td>1</td>
    <td>5</td>
    <td>5</td>
    <td>10</td>
    <td>10</td>
    </tr>
<tr>
    <th>Incubation time [min]</th>
    <td>60</td>
    <td>30</td>
    <td>60</td>
    <td>30</td>
    <td>60</td>
    <td>30</td>
    <td>60</td>
    <td>30</td>
    </tr>
</center>
    </table>
#Note; OCK has 4 cholesterol oligo binding sites. Therefore, we devided the molar ratio of cholesterol oligo to OCK with 4.
                <figure>
        <center>
        <img src="http://openwetware.org/images/6/66/OCK_Cholesterol-Todai.png" width=300px height=300px>
       
        </center>
      </figure>
      <li>Each sample was analyzed by 1% agarose gel electrophoresis (100V, 1 hour).</li>
    </ul>
    </article>
    <br>
 
 
<!--◆◆STEP3◆◆-->
<h2 class="PS_title"><a name="STEP3">&nbsp;STEP 3: Subunits recognize cancer-specific proteins.</a></h2>
<article>
<!--◆◆Reaction_of_a_biotinized_oligo_to_streptavidin◆◆-->
  <article>
  <div class="mini-title">
      <a name="Reaction_of_a_biotinized_oligo_to_streptavidin">1) Reaction of a biotinized oligo to streptavidin</a>
  </div>
<!--Reagent-->
      <div class="zairyou-heading">[Reagent]</div>
      <br>
<li>materials for hybridization</li>
        <li>5ap_M-3t4e_T0 (1uM) (oligo): 5’ to 3’</li>
TACTCAGGGCACTGCAAGCAATTGTGGTCCCAATGGGCTGAGTACGCGACCTCATCTTTGACCCCCAGGCAGGGAG
        <li>5ap_5B_16 (10uM) (oligo, which has biotin in 5’ end):</li>
Biotin-TACTCAGCCCATTGGG
        <li>10x tile buffer<sup>*</sup></li>
        <li>MilliQ</li>
    </table>
    <br>
&nbsp;*...10x tile buffer(f.100 µl)
    <table>
    <tr>
    <th>Mg(OAc)<sub>2</sub></th>
    <td>f.100 mM</td>
    </tr>
    <tr>
    <th>Tris-HCl (pH7.5)</th>
    <td>f.200 mM</td>
    </tr>
        <tr>
    <th>EDTA</th>
    <td>f.10 mM</td>
    </tr>
    </table>
<li>5ap_tile</li>
        <li>M13mp18 (scaffold)</li>
        <li>Cy5_Rmix (staples)</li>
        <li>10x tile buffer</li>
        <li>Cy3 streptavidin (800nM)</li>
<!--Procedure-->
  <div class="zairyou-heading">[Procedure]</div>
    <ul class="procedure-list">
      <li>Hybridization</li>
<li>Mix materials (mentioned above in [hybridization]) in 0.2 ml PCR-tubes.</li>
<li>Incubate the mixture at the room temperature (25 ℃) for 1 hour.</li>
      <li>Making 5ap_tile</li>
<li>Mix materials (mentioned above in [5ap_tile]) in 0.2 ml PCR-tubes.</li>
<li>Anneal the mixture using PCR machine (from 85 ℃ to 25 ℃, -2 ℃/min).</li>
      <li>Insertion of hybridized double-stranded DNA into 5ap_tile</li>
<li>Mix hybridized double-stranded DNA and 5ap_tile in 0.2 ml PCR-tubes.</li>
<li>Incubate the mixture at 48 ℃ for 1 hour.</li>
<li>Mix the mixture and Cy3 streptavidin.</li>
      <li>1wt% Agarose-gel Electrophoresis</li>
<li>Electrophoresis the inserted 5ap_tile for 50 minutes at 100 V at 4 ℃.</li>
<li>Take photographs of the electrophoresed gel by LAS-4000.</li>
    </ul>
    </article>
    <br>
<!--◆◆Reaction_between_aptamer_embedded_in_rect_tile_and_PDGF10◆◆-->
  <article>
  <div class="mini-title">
      <a name="Reaction_between_aptamer_embedded_in_rect_tile_and_PDGF10">2) Reaction between aptamer embedded in rect tile and PDGF</a>
  </div>
<!--Reagent-->
      <div class="zairyou-heading">[Reagent]</div>
      <br>
<li>materials for hybridization</li>
        <li>5ap_M-3t4e_T0 (1uM) (oligo): 5’ to 3’</li>
TACTCAGGGCACTGCAAGCAATTGTGGTCCCAATGGGCTGAGTACGCGACCTCATCTTTGACCCCCAGGCAGGGAG
  <li>5ap_M-3t4e_T-3(1µM) (oligo):</li> 
TACTCAGGGCACTGCAAGCAATTGTGGTCCCAATGGGCTGAGTAGACCTCATCTTTGACCCCCAGGCAGGGAG
<li>5ap_M-3t4e_T-1(1µM) (oligo):</li>
TACTCAGGGCACTGCAAGCAATTGTGGTCCCAATGGGCTGAGTAGCGACCTCATCTTTGACCCCCAGGCAGGGAG
  <li>5ap_M-3t4e_T7(1µM) (oligo):</li>
TACTCAGGGCACTGCAAGCAATTGTGGTCCCAATGGGCTGAGTAtttttttCGCGACCTCATCTTTGACCCCCAGGCAGGGAG
<li>5ap_5B_16 (10uM) (oligo, which has biotin in 5’ end):</li>
Biotin-TACTCAGCCCATTGGG
        <li>10x tile buffer<sup>*</sup></li>
        <li>MilliQ</li>
    </table>
    <br>
&nbsp;*...10x tile buffer(f.100 µl)
    <table>
    <tr>
    <th>Mg(OAc)<sub>2</sub></th>
    <td>f.100 mM</td>
    </tr>
    <tr>
    <th>Tris-HCl (pH7.5)</th>
    <td>f.200 mM</td>
    </tr>
        <tr>
    <th>EDTA</th>
    <td>f.10 mM</td>
    </tr>
    </table>
<li>5ap_tile</li>
        <li>M13mp18 (scaffold)</li>
        <li>5ap_Rmix (staples)</li>
        <li>10x tile buffer</li>
        <li>PDGF(dye 45nM)</li>
<!--Procedure-->
  <div class="zairyou-heading">[Procedure]</div>
    <ul class="procedure-list">
      <li>Hybridization</li>
<li>Mix materials (mentioned above in [hybridization]) in 0.2 ml PCR-tubes.</li>
<li>Incubate the mixture at the room temperature (25 ℃) for 1 hour.</li>
      <li>Making 5ap_tile</li>
<li>Mix materials (mentioned above in [5ap_tile]) in 0.2 ml PCR-tubes.</li>
<li>Anneal the mixture using PCR machine (from 85 ℃ to 25 ℃, -2 ℃/min).</li>
      <li>Insertion of hybridized double-stranded DNA into 5ap_tile</li>
<li>Mix hybridized double-stranded DNA and 5ap_tile in 0.2 ml PCR-tubes.</li>
<li>Incubate the mixture at 48 ℃ for 1 hour.</li>
<li>Mix the mixture and PDGF.</li>
        <li>Incubate at 37 ℃ for 30 min and then at 4 ℃ for 25 hours.</li>
      <li>1wt% Agarose-gel Electrophoresis</li>
<li>Electrophoresis the inserted 5ap_tile for 50 minutes at 100 V at 4 ℃.</li>
        <li>Stain the gel by SYBR Gold in TBE.</li>
<li>Take photographs of the electrophoresed gel by LAS-4000.</li>
    </ul>
    </article>
    <br>
<!--◆◆efficient_hybridization_(changing_mixture_ratio)_Added◆◆-->
  <article>
  <div class="mini-title">
      <a name="efficient_hybridization_(changing_mixture_ratio)">3) Efficient hybridization (changing mixture ratio)</a>
  </div>
<!--Reagent-->
      <div class="zairyou-heading">[Reagent]</div>
      <br>
    <li>5ap_M-3t4e_T0 (1 µM): 5’ to 3’</li>
TACTCAGGGCACTGCAAGCAATTGTGGTCCCAATGGGCTGAGTACGCGACCTCATCTTTGACCCCCAGGCAGGGAG
    <li>5ap_5B_16 (1 or 10 µM): Biotin in 5’ end</li>
Biotin - TACTCAGCCCATTGGG
    <li>10x tile buffer<sup>*</sup></li>
    <li>MilliQ</li>
    </table>
    <br>
&nbsp;*...10x tile buffer
    <table>
    <tr>
    <th>Mg(OAc)<sub>2</sub></th>
    <td>f.100 mM</td>
    </tr>
    <tr>
    <th>Tris-HCl (pH7.5)</th>
    <td>f.200 mM</td>
    </tr>
        <tr>
    <th>EDTA</th>
    <td>f.10 mM</td>
    </tr>
    </table>
<!--Procedure-->
  <div class="zairyou-heading">[Procedure]</div>
    <ul class="procedure-list">
<li>Mix materials to make samples, following the ratio written in Table.3.<sup>*</sup></li>
    </table>
    <br>
&nbsp;*...Table.3
    <table>
<center>
    <tr>
    <th>Sample No.</th>
    <td>1</td>
    <td>2</td>
    <td>3</td>
    <td>4</td>
    <td>5</td>
    <td>6</td>
    <td>7</td>
    <td>8</td>
    </tr>
    <tr>
    <th>5ap_M-3t4e_T0 (1 µM)</th>
    <td>3 µL</td>
    <td>-</td>
    <td>-</td>
    <td>3 µL</td>
    <td>3 µL</td>
    <td>3 µL</td>
    <td>3 µL</td>
    <td>3 µL</td>
    </tr>
        <tr>
    <th>5ap_5B_16 (10 µM)</th>
    <td>-</td>
    <td>-</td>
    <td>-</td>
    <td>-</td>
    <td>-</td>
    <td>1.5 µL</td>
    <td>3 µL</td>
    <td>6 µL</td>
    </tr>
    <tr>
    <th>5ap_5B_16 (1 µM)</th>
    <td>-</td>
    <td>3 µL</td>
    <td>-</td>
    <td>3 µL</td>
    <td>6 µL</td>
    <td>-</td>
    <td>-</td>
    <td>-</td>
    </tr>
    <tr>
    <th>10x tile buffer</th>
    <td>1 µL</td>
    <td>1 µL</td>
    <td>1 µL</td>
    <td>1 µL</td>
    <td>1 µL</td>
    <td>1 µL</td>
    <td>1 µL</td>
    <td>1 µL</td>
    </tr>
        <tr>
    <th>MilliQ</th>
    <td>6 µL</td>
    <td>6 µL</td>
    <td>9 µL</td>
    <td>3 µL</td>
    <td>-</td>
    <td>4.5 µL</td>
    <td>3 µL</td>
    <td>-</td>
    </tr>
        <tr>
    <th>Ratio of concentration of 5ap_5B_16 to 5ap_M-3t4e_T0</th>
    <td>-</td>
    <td>-</td>
    <td>-</td>
    <td>1 %</td>
    <td>2 %</td>
    <td>5 %</td>
    <td>10 %</td>
    <td>20 %</td>   
    </tr>
</center>
    </table>


<!--★フローテーションアッセイ★-->
<li>Apply the samples to 10 % Native-PAGE for 85 minutes at 100 V at 4 ℃.</li>
<li>Stain the gel by SYBR Gold in TBE.</li>
<li>Take a photograph of the gel by LAS-4000.</li>


    </ul>


    </article>
    <br>
    <!--◆◆efficient hybridization (incubation time)◆◆-->
   <article>
   <article>
   <div class="mini-title">
   <div class="mini-title">
    <div style="width:512px">
       <a name="efficient_hybridization_(incubation_time)">4) efficient hybridization (incubation time)</a>
       <a name="Flotation_assay_of_liposome">2.2.Flotation assay of liposome and DNA origami</a>
    </div>
   </div>
   </div>


<!--Reagent-->
      <div class="zairyou-heading">[Reagent]</div>
      <br>
    <li>5ap_M-3t4e_T0 (1 µM): 5’ to 3’</li>
TACTCAGGGCACTGCAAGCAATTGTGGTCCCAATGGGCTGAGTACGCGACCTCATCTTTGACCCCCAGGCAGGGAG
    <li>5ap_5B_16 (10 µM): (biotin in 5’ end)</li>
Biotin - TACTCAGCCCATTGGG
    <li>10x tile buffer<sup>*</sup></li>
    <li>MilliQ</li>
    </table>
    <br>
&nbsp;*...10x tile buffer(f.100 µl)
    <table>
    <tr>
    <th>Mg(OAc)<sub>2</sub></th>
    <td>f.100 mM</td>
    </tr>
    <tr>
    <th>Tris-HCl (pH7.5)</th>
    <td>f.200 mM</td>
    </tr>
        <tr>
    <th>EDTA</th>
    <td>f.10 mM</td>
    </tr>
    </table>
<!--Procedure-->
  <div class="zairyou-heading">[Procedure]</div>
    <ul class="procedure-list">
<li>Mix the materials in 0.2 ml PCR-tubes.</li>
<li>Denature the oligos at 95 ℃ for 30 seconds.</li>
<li>Incubate the mixture at room temperature (25 ℃).</li>
<li>Freeze the samples into nitrogen liquid at planned incubation time.</li>
<li>Pick up the samples out from nitrogen liquid immediately before applying into gel.</li>
<li>Apply the samples to 10 % Native PAGE for 85 minutes at 100 V at 4 ℃.</li>
<li>Take a photograph of the electrophoresed gel by LAS-4000.</li>
    </ul>


<!--Reagent-->
    </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>
       <div class="zairyou-heading">[Reagent]</div>
      <br>
hybridization
    <li>5ap_M-3t4e_T0 (1 µM) (oligo): 5’ to 3’</li>
TACTCAGGGCACTGCAAGCAATTGTGGTCCCAATGGGCTGAGTACGCGACCTCATCTTTGACCCCCAGGCAGGGAG
    <li>5ap_5B3G_16 (1 µM) (oligo, which has biotin in 5’ end and Cy3 in 3’ end):</li>
Biotin–TACTCAGCCCATTGGG–Cy3
    <li>10x tile buffer<sup>*</sup></li>
    <li>MilliQ</li>
    </table>
    <br>
&nbsp;*...10x tile buffer(f.100 µl)
    <table>
    <tr>
    <th>Mg(OAc)<sub>2</sub></th>
    <td>f.100 mM</td>
    </tr>
    <tr>
    <th>Tris-HCl (pH7.5)</th>
    <td>f.200 mM</td>
    </tr>
        <tr>
    <th>EDTA</th>
    <td>f.10 mM</td>
    </tr>
    </table>
5ap_tile
    <li>M13mp18 (scaffold)</li>
    <li>5ap_Rmix (staples)</li>
    <li>10x tile buffer</li>
<!--Procedure-->
  <div class="zairyou-heading">[Procedure]</div>
    <ul class="procedure-list">
Hybridization
<li>Mix materials (mentioned above in [hybridization]) in 0.2 ml PCR-tubes.</li>
<li>Incubating the mixture at the room temperature (25 ℃) for 1 hour.</li>
Making 5ap_tile
<li>Mix materials (mentioned above in [5ap_tile]) in 0.2 ml PCR-tubes.</li>
<li>Anneal the mixture using PCR machine (from 85 ℃ to 25 ℃, -2 ℃/min).</li>
Insertion of hybridized double-stranded DNA into 5ap_tile
<li>Mix hybridized double-stranded DNA and 5ap_tile in 0.2 ml PCR-tubes.</li>
<li>Incubate the mixture at 48 ℃ for 1 hour.</li>
1 wt% Agarose-gel Electrophoresis
<li>Electrophoresis the inserted 5ap_tile for 50 minutes at 100 V at 4 ℃.</li>
        <li>Take photographs of the electrophoresed gel by LAS-4000.</li>
    </ul>


  <div class="zairyou-heading">・Sucrosebuffer</div>
    </article>
    <div class="zairyou-heading-sub">a) 0.375M Sucrose buffer</div>
    <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>
     <table>
     <tr>
     <tr>
     <th>HEPES-KOH(pH7.6)</th>
     <th>10x tile buffer </th>
     <td>50mM</td>
     <td>f. 1x tile buffer</td>
     </tr>
     </tr>


     <tr>
     <tr>
     <th>KCl</th>
     <th>1 µM aptamer</th>
     <td>100mM</td>
     <td>f. 0.3µM</td>
     </tr>
     </tr>


     <tr>
     <tr>
     <th>MgCl<sub>2</sub></th>
     <th>1 µM Ladder151515_1</th>
     <td>10mM</td>
     <td>f. 0.3µM</td>
     </tr>
     </tr>


     <tr>
     <tr>
     <th>Sucrose</th>
     <th>PDGF (dye 45nM)</th>
     <td>0.375M</td>
     <td>f. 20mM</td>
     </tr>
     </tr>
     </table>
     </table>


    <div class="zairyou-heading-sub">b) 1.25M Sucrose buffer</div>
<li>materials for the electrophoresis</li>
    <gel for 10% Native-PAGE>
     <table>
     <table>
     <tr>
     <tr>
     <th>HEPES-KOH(pH7.6)</th>
     <th>MilliQ</th>
     <td>50mM</td>
    <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>


     <tr>
     <tr>
     <th>KCl</th>
     <th>10 % APS</th>
     <td>100mM</td>
     <td>80 µL</td>
     </tr>
     </tr>


     <tr>
     <tr>
     <th>MgCl<sub>2</sub></th>
     <th>TEMED</th>
     <td>10mM</td>
    <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>


     <tr>
     <tr>
     <th>Sucrose</th>
     <th>SYBR Gold</th>
     <td>1.25M</td>
     <td>5 µL</td>
     </tr>
     </tr>
     </table>
     </table>


     <div class="zairyou-heading-sub">c) 1.6M Sucrose buffer</div>
     <others>
     <table>
     <table>
     <tr>
     <tr>
     <th>HEPES-KOH(pH7.6)</th>
     <th>Loading buffer</th>
     <td>50mM</td>
    <th>20 % glycerol (as used 6x)</th>
    </tr>
 
    <tr>
    <th>Marker</th>
    <th>Cy5 38 mer</th>
    </tr>
 
    </table>
 
 
<!--Procedure-->
  <div class="zairyou-heading">[Procedure]</div>
    <ul class="procedure-list">
      <li>Mix the solutions as shown below:<sup>*</sup></li>
    </table>
    <br>
&nbsp;*...Table.2
    <table>
<center>
    <tr>
    <th>Sample No.</th>
     <td>1</td>
    <td>2</td>
    <td>3</td>
    <td>4</td>
    <td>5</td>
    <td>6</td>
     </tr>
     </tr>


     <tr>
     <tr>
     <th>KCl</th>
     <th>10x tile buffer</th>
     <td>100mM</td>
     <td>1 µL</td>
    <td>1 µL</td>
    <td>1 µL</td>
    <td>1 µL</td>
    <td>1 µL</td>
    <td>1 µL</td>
    </tr>
 
        <tr>
    <th>1 µM aptamer</th>
    <td>3 µL</td>
    <td>-</td>
    <td>-</td>
    <td>3 µL</td>
    <td>-</td>
    <td>-</td>
     </tr>
     </tr>


     <tr>
     <tr>
     <th>MgCl<sub>2</sub></th>
     <th>1 µM Ladder151515_1</th>
     <td>10mM</td>
    <td>-</td>
    <td>-</td>
    <td>-</td>
    <td>-</td>
    <td>3 µL</td>
     <td>3 µL</td>
     </tr>
     </tr>


     <tr>
     <tr>
     <th>Sucrose</th>
     <th>PDGF (dye 45 nM)</th>
     <td>1.6M</td>
     <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>
     </tr>
</center>
     </table>
     </table>
      <li>Incubate at 37 ℃ for 30 min and then at 4 ℃ for 30 min.</li>
      <li>Make 10 % Native PAGE gel as mentioned above.</li>
      <li>Add loading buffer into each samples.</li>
      <li>Apply the samples to 10 % Native-PAGE for 85 minutes at 100V at 4℃.</li>
      <li>Take a photograph of the electrophoresed gel by LAS-4000 for Cy5.</li>
      <li>Stain the gel by SYBR Gold for 20 min.</li>
      <li>Take a photograph by LAS-4000.</li>
    </ul>
    </article>
     <br>
     <br>
    <div class="zairyou-heading-sub">・liposome </div>    <div style="font-


size:110%;position:relative;left:60px;">
1mg/ml( -->
        <a href="#Preparation_of_liposome" style="color:#e00000">Preparation of liposome</a>)
    </div>


    <div class="zairyou-heading-sub">・DNA origami(Rect tile<sup>[1]</sup>) </div>
</article>
    <div style="font-size:110%;position:relative;left:60px;">~30nM
 
    </div>
<!-- ◆◆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>
     <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
<!--Procedure-->
    <li>M13mp18 (scaffold)</li>
    <li>5ap_Rmix (staples)</li>
    <li>10x tile buffer</li>


<!--Procedure-->
   <div class="zairyou-heading">[Procedure]</div>
   <div class="zairyou-heading">[Procedure]</div>
     <ul class="procedure-list">
     <ul class="procedure-list">
      <li>sample + sucrose buffer were layered
Hybridization
in this order on centrifuge tubes.</li>
<li>Mix materials (mentioned above in [hybridization]) in 0.2 ml PCR-tubes.</li>
      <li>The tubes were centrifugalized.</li>
<li>Incubating the mixture at the room temperature (25 ℃) for 1 hour.</li>
      <li>Samples were fructionized.</li>
 
      <li>Precipitation was resuspended with sucrose buffer.
Making 5ap_tile
      <li>Samples were measured by the fluorescence of NIL(ex 500nm, em 550nm~700nm) and agarose gel  
<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>


electrophoresis.</li>
     </ul>
     </ul>
     </article>
     </article>
     <br>
     <br>




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


<!--★Click Reaction★-->
<!--◆◆SA dimer◆◆-->
   <article>
   <article>
  <h2>3.Comparision of dimerization method</h2>
   <div class="mini-title">
   <div class="mini-title">
    <div style="width:350px">
      <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">
       <a name="Click_reaction_via_(3+2)_cycloaddition">
       3.1.Click reaction via (3+2) cycloaddition
       2) Click reaction via (3+2) cycloaddition
       </a>
       </a>
    </div>
   </div>
   </div>
  <article>


<!--Reagent-->
<!--Reagent-->
       <div class="zairyou-heading">[Reagent]</div>
       <div class="zairyou-heading">[Reagent]</div>
       <br>
       <br>
Line 563: Line 1,679:


     <tr>
     <tr>
     <th>CuSO4 solution (50mM)</th>
     <th>CuSO<sub>4</sub> solution (50mM)</th>
     <td>1μL</td>
     <td>1μL</td>
     </tr>
     </tr>
Line 580: Line 1,696:
     <br>
     <br>


<!--Procedure-->
<!--Procedure-->
   <div class="zairyou-heading">[Procedure]<sup>[4]</sup></div>
   <div class="zairyou-heading">[Procedure]<sup>[4]</sup></div>
     <ul class="procedure-list">
     <ul class="procedure-list">
Line 588: Line 1,704:
     </article>
     </article>
     <br>
     <br>
<!--★Research for azobenzene★-->
<!--◆◆Accelerated_Click_reaction◆◆-->
   <article>
   <article>
   <div class="mini-title">
   <div class="mini-title">
    <div style="width:350px">
       <a name="Accelerated_Click_reaction">3) Accelerated Click reaction (using streptavidin to make the alkyne and azide reactive groups close) </a>
       <a name="Research_for_azobenzene)">
      3.2.Research for azobenzene
      </a>
    </div>
   </div>
   </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>


<!--Procedure-->
    </table>
  <div class="zairyou-heading">[Procedure]<sup>[2],[3],[5]</sup></div>
    <br>
  <p class="paragraph">
The procedure of synthesis of tubes and motifs were refered to previous reseachs([2],[3],[5])




<!--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>
     </article>
     <br>
     <br>
   
<!--◆◆Click_reaction_hybridization◆◆-->
  <article>
  <div class="mini-title">
      <a name="Click_reaction_(using_hybridization_to_make_the_aklyne_and_azide_reactive_groups close)">4) Click reaction (using hybridization to make the alkyne and azide reactive groups close) </a>
  </div>
<!--Reagent-->
      <div class="zairyou-heading">[Reagent]</div>
      <br>
    <table>
    <tr>
    <th>2x barrel buffer</th>
    <td>7 µL</td>
    </tr>
    <tr>
    <th>alkyne oligo (15 µM)</th>
    <td>1 µL</td>
    </tr>
    <tr>
    <th>azide oligo (15 µM)</th>
    <td>1 µL</td>
    </tr>
    <tr>
    <th>scaffold (15 µM)</th>
    <td>1 µL</td>
    </tr>
    </table>
    <br>
<!--Procedure-->
  <div class="zairyou-heading">[Procedure]</div>
    <ul class="procedure-list">
      <li>mix reagents</li>
      <li>incubate the tube at 37 ℃.</li>
      <li>add loading buffer into the reaction mixture and boil at 95 ℃ for 5 minutes to denature the double strand to single strand.</li>
    </ul>
<!--◆◆Click reaction cupper free◆◆-->
  <div class="mini-title">
      <a name="Click_reaction_(copper_catalyst-free)">5) Click reaction (copper catalyst-free)</a>
  </div>
<!--Reagent-->
      <div class="zairyou-heading">[Reagent]</div>
      <br>
    <table>
    <tr>
    <th>2x barrel buffer</th>
    <td>7 µL</td>
    </tr>
    <tr>
    <th>alkyne oligo (15 µM)</th>
    <td>1 µL</td>
    </tr>
    <tr>
    <th>azide oligo (15 µM)</th>
    <td>1 µL</td>
    </tr>
    <tr>
    <th>scaffold (15 µM)</th>
    <td>1 µL</td>
    </tr>
    </table>
    <br>
<!--※10x OCK buffer (f. 100 µl)-->
  <div class="zairyou-heading">[※※ 2x barrel buffer]</div>
      <br>
    <table>
    <tr>
    <th>1M Tris (pH 7.5)</th>
    <td>5 µL</td>
    </tr>
    <tr>
    <th>0.5M EDTA</th>
    <td>2 µL</td>
    </tr>
    <tr>
    <th>5M NaCl</th>
    <td>1 µL</td>
    </tr>
    <tr>
    <th>MQ</th>
    <td>32 µL</td>
    </tr>
    </table>
    <br>
<!--Procedure-->
  <div class="zairyou-heading">[Procedure]</div>
    <ul class="procedure-list">
      <li>mix reagents</li>
      <li>incubate the tube at 37 ℃.</li>
      <li>add loading buffer into the reaction mixture and boil at 95 ℃ for 5 minutes to denature the double strand to single strand.</li>
    </ul>
    </article>
<article>
   <div class="mini-title">
   <div class="mini-title">
    <div style="width:200px">
       <a name="Synthesis_of_streptavidin_mutants">
       <a name="Reference">
       6) Synthesis of streptavidin mutants
       Reference
       </a>
       </a>
    </div>
   </div>
   </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>     
Line 630: Line 1,902:
               Nature 440, 297–302 (2006)
               Nature 440, 297–302 (2006)
               </div>
               </div>
     <div>    
    </div>
 
     <div>
         <div class="reference-title">
         <div class="reference-title">
         <a name="proref-1">
         <a name="proref-1">
Line 642: Line 1,916:
               Science, 2012, 338, 1458
               Science, 2012, 338, 1458
               </div>
               </div>
    </div>
     <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 Nanostructure
         [3] Transcription Regulation System Mediated by Mechanical Operation of a DNA &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbs p;Nanostructure
         </a>
         </a>
         </div>
         </div>
           <div class="reference-author">
           <div class="reference-author">
           >Masayuki Endo, Ryoji Miyazaki, Tomoko Emura, Kumi Hidaka, and Hiroshi Sugiyama
           Masayuki Endo, Ryoji Miyazaki, Tomoko Emura, Kumi Hidaka, and Hiroshi Sugiyama
           </div>
           </div>
               <div class="reference-journal">
               <div class="reference-journal">
               Journal of the American Chemical Society, 2012, 134, 2852-2855
               Journal of the American Chemical Society, 2012, 134, 2852-2855
               </div>
               </div>
    </div>
     <div>     
     <div>     
         <div class="reference-title">
         <div class="reference-title">
Line 660: Line 1,938:
         </a>
         </a>
         </div>
         </div>
              <div class="reference-journal">
          <div class="reference-journal">
             
          <a target="_blank" href="http://www.jenabioscience.com" style="color:#e00000">
      <a target="_blank" href="http://www.jenabioscience.com" style="color:#e00000">
          http://www.jenabioscience.com</a>
      http://www.jenabioscience.com</a>
          </div>
              </div>
    </div>
     <div>    
 
     <div>
         <div class="reference-title">
         <div class="reference-title">
         <a name="proref-1">
         <a name="proref-1">
Line 677: Line 1,956:
               Angewandte Chemie International Edition,2009,48(37),6820–6823
               Angewandte Chemie International Edition,2009,48(37),6820–6823
               </div>
               </div>
    </div>
    <div>   
        <div class="reference-title">
        <a name="proref-1">
        [6] A primer to scaffolded DNA origami.</a>
        </div>
          <div class="reference-author">
          Castro CE, Kilchherr F, Kim DN, Shiao EL, Wauer T, Wortmann P, Bathe M
and Dietz H.
          </div>
              <div class="reference-journal">
              Nat Methods 221-229 (2011, Mar;8(3))
              </div>
    </div>
          <div>   
        <div class="reference-title">
        <a name="proref-1">
        [7] A monovalent streptavidin with a single femtomolar biotin binding site.</a>
        </div>
          <div class="reference-author">
          Howarth M, Chinnapen DJ, Gerrow K, Dorrestein PC, Grandy MR, Kelleher NL, El-Husseini A and Ting AY.
          </div>
              <div class="reference-journal">
              Nat Methods 267-273 (2006, Apr;3(4))
              </div>
    </div>
          <div>   
        <div class="reference-title">
        <a name="proref-1">
        [8] Imaging proteins in live mammalian cells with biotin ligase and monovalent streptavidin.</a>
        </div>
          <div class="reference-author">
          Howarth M and Ting AY.
          </div>
              <div class="reference-journal">
              Nat Protoc 534-545 (2008, Mar;3(3)); doi: 10.1038/nprot.2008.20.
              </div>
    </div>
                
                
  </article>
   <br>
   <br>
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   <br>
    
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  <footer style="position:relative;left:400px">
    <small>
    Copyright &copy; Todai nanORFEVRE, all rights reserved.
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Latest revision as of 23:54, 26 October 2013

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<!--Experiment--> <h1 class="big-title"><a name="Experiment">&nbsp;Experiment</a></h1> 

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

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

      	<article>
       		<ul>

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

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

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

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

      </a>
      </li>

<li> 

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

      </li>

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

 		</article>
		</article>

<!--◆◆Pilot Study◆◆--> 

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

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

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

</div> 

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

       </center>
      </figure>

<br> 

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

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

    </p>
  	<br>

</article>

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

  	<div class="mini-title">
      <a name="Preparation_of_liposome">2) Preparation of liposome</a>
  	</div>

      <figure>
       <center>
        <img src="http://openwetware.org/images/7/71/640px_suv_dls_popg50r60-Todai.png" width=640px height=360px >

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

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

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

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

<!--◆◆Flotation assay(Rect-tile)◆◆--> 

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

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

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

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

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

<br> 

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

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

    </p>
  	<br>

</article>


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

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

<!--◆◆Click reaction ◆◆--> 

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

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

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

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

<!--◆◆Optimum_Conc_SA◆◆--> 

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

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

  <br>

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

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

       </center>
      </figure>
  <br>

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

  <br>

<!--◆◆Protocols◆◆--> 

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

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

<!--◆◆Assembly of OCK◆◆--> 

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


<!--Reagent--> 

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

   <table>
   <tr>
   <th>M13mp18ss</th>
   <td>4.5 ul</td>
   </tr>

   <tr>
   <th>Staple mix</th>
   <td>4.5 µL</td>
   </tr>

   <tr>
   <th>10x OCK buffer<sup>*</sup></th>
   <td>1 µL</td>
   </tr>



   </table>
   <br>

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

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

<!--Procedure--> 

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

   </article>
   <br>


<!--◆◆TEM◆◆--> 

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

<!--Procedure--> 

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

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

<!--◆◆flotation asssay of OCK◆◆--> 

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

<!--Reagent--> 

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

   <table>
   <tr>
   <th>OCK</th>
   <td>100 µL</td>
   </tr>

   <tr>
   <th>Cholesterol hybridized OCK</th>
   <td>100 µL</td>
   </tr>

   <tr>
   <th>Liposome (1 mg/mL SUVs)</th>
   <td>100 µL</td>
   </tr>

   <tr>
   <th>2.25 M Sucrose buffer<sup>*</sup></th>
   <td>500 µL</td>
   </tr>

   <tr>
   <th>1.6 M Sucrose buffer<sup>**</sup></th>
   <td>900 µL</td>
   </tr>

   <tr>
   <th>150 mM KCl solution</th>
   <td>100 µL</td>
   </tr>

   <tr>
   <th>1×Flotation buffer<sup>***</sup></th>
   <td>600 µL</td>
   </tr>



   </table>
   <br>

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

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



   </table>
   <br>

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

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



   </table>
   <br>

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

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


<!--Procedure--> 

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

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

   <tr>
   <th>Sample No.</th>
   <td>1</td>
   <td>2</td>
   <td>3</td>
   <td>4</td>
   </tr>
   <tr>
   <th>Cholesterol hybridized OCK</th>
   <td>50 µL</td>
   <td>50 µL</td>
   <td>-</td>
   <td>-</td>
   </tr>
       <tr>
   <th>OCK</th>
   <td>-</td>
   <td>-</td>
   <td>50 µL</td>
   <td>50 µL</td>
   </tr>
   <tr>
   <th>Liposome</th>
   <td>50 µL</td>
   <td>-</td>
   <td>50 µL</td>
   <td>-</td>
   </tr>
   <tr>
   <th>150 mM aqueous KCl solution</th>
   <td>-</td>
   <td>50 µL</td>
   <td>-</td>
   <td>50 µL</td>
   </tr>
       <tr>
   <th>2.25 M Sucrose buffer</th>
   <td>125 µL</td>
   <td>125 µL</td>
   <td>125 µL</td>
   <td>125 µL</td>
   </tr>
   </table>

     <li>225 µL of 1.6 M sucrose buffer was overlaid with 225 µL of sample

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

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

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

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



   </article>
   <br>

   <br>



   <br>

<!--◆◆Preparation of GUVs◆◆--> 

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

<!--Reagent--> 

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

   <table>
   <tr>
   <th>Lipid mix<sup>*</sup></th>
   <td>3 ml</td>
   </tr>

   <tr>
   <th>150 mM KCl solution</th>
   <td>1 µL</td>
   </tr>



   </table>
   <br>

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

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

<!--Procedure--> 

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

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

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

   </article>
   <br>


<!--◆◆Preparation of SUVs◆◆--> 

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

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

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

   <table>
   <tr>
   <th>150mM KCl solution</th>
   <td>3mL</td>
   </tr>

   <tr>
   <th>POPC</th>
   <td>3mg</td>
   </tr>

   <tr>
   <th>Chloroform (99.0%)</th>
   <td>3mL</td>
   </tr>

   <tr>
   <th>40μM Nile Red solution</th>
   <td>0.1mL</td>
   </tr>

   </table>
   <br>

<!--Procedure--> 

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

evaporator for 5 minutes.</li> 

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

   </article>
   <br>

<!--Preparation of SUVs_Added--> 

  <article>

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

<!--Reagent--> 

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

   <table>
   <tr>
   <th>Lipid mix<sup>*</sup></th>
   <td>3 ml</td>
   </tr>

   <tr>
   <th>150 mM KCl solution</th>
   <td>1 µL</td>
   </tr>



   </table>
   <br>

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

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

<!--Procedure--> 

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

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

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

   </article>

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

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

<!--Reagent--> 

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

   <table>
   <tr>
   <th>OCK</th>
   <td>48 µL</td>
   </tr>

   <tr>
   <th>Cholesterol oligo (0.32, 0.64, 3.2, 6.4 µM)</th>
   <td>100 µL</td>
   </tr>

   </table>

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

<!--Procedure--> 

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

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

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

<tr> 

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

</center> 

   </table>
  1. Note; OCK has 4 cholesterol oligo binding sites. Therefore, we devided the molar ratio of cholesterol oligo to OCK with 4.
               <figure>
       <center>
        <img src="http://openwetware.org/images/6/66/OCK_Cholesterol-Todai.png" width=300px height=300px>
       
       </center>
      </figure>

     <li>Each sample was analyzed by 1% agarose gel electrophoresis (100V, 1 hour).</li>

   </ul>



   </article>
   <br>



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

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

<!--Reagent--> 

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

<li>materials for hybridization</li> 

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

TACTCAGGGCACTGCAAGCAATTGTGGTCCCAATGGGCTGAGTACGCGACCTCATCTTTGACCCCCAGGCAGGGAG 

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

Biotin-TACTCAGCCCATTGGG 

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

   </table>
   <br>

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

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

   </table>

<li>5ap_tile</li> 

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

<!--Procedure--> 

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

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

     <li>Making 5ap_tile</li>

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

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

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

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

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


   </ul>

   </article>
   <br>

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

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

<!--Reagent--> 

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

<li>materials for hybridization</li> 

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

TACTCAGGGCACTGCAAGCAATTGTGGTCCCAATGGGCTGAGTACGCGACCTCATCTTTGACCCCCAGGCAGGGAG 

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

TACTCAGGGCACTGCAAGCAATTGTGGTCCCAATGGGCTGAGTAGACCTCATCTTTGACCCCCAGGCAGGGAG

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

 <li>5ap_M-3t4e_T7(1µM) (oligo):</li>

TACTCAGGGCACTGCAAGCAATTGTGGTCCCAATGGGCTGAGTAtttttttCGCGACCTCATCTTTGACCCCCAGGCAGGGAG 

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

Biotin-TACTCAGCCCATTGGG 

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

   </table>
   <br>

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

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

   </table>

<li>5ap_tile</li> 

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

<!--Procedure--> 

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

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

     <li>Making 5ap_tile</li>

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

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

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

        <li>Incubate at 37 ℃ for 30 min and then at 4 ℃ for 25 hours.</li>

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

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

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

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


   </ul>

   </article>
   <br>


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

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

<!--Reagent--> 

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

TACTCAGGGCACTGCAAGCAATTGTGGTCCCAATGGGCTGAGTACGCGACCTCATCTTTGACCCCCAGGCAGGGAG 

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

Biotin - TACTCAGCCCATTGGG 

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

   </table>
   <br>

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

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

   </table>


<!--Procedure--> 

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

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

   </table>
   <br>

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

   <tr>
   <th>Sample No.</th>
   <td>1</td>
   <td>2</td>
   <td>3</td>
   <td>4</td>
   <td>5</td>
   <td>6</td>
   <td>7</td>
   <td>8</td>
   </tr>

   <tr>
   <th>5ap_M-3t4e_T0 (1 µM)</th>
   <td>3 µL</td>
   <td>-</td>
   <td>-</td>
   <td>3 µL</td>
   <td>3 µL</td>
   <td>3 µL</td>
   <td>3 µL</td>
   <td>3 µL</td>
   </tr>

       <tr>
   <th>5ap_5B_16 (10 µM)</th>
   <td>-</td>
   <td>-</td>
   <td>-</td>
   <td>-</td>
   <td>-</td>
   <td>1.5 µL</td>
   <td>3 µL</td>
   <td>6 µL</td>
   </tr>

   <tr>
   <th>5ap_5B_16 (1 µM)</th>
   <td>-</td>
   <td>3 µL</td>
   <td>-</td>
   <td>3 µL</td>
   <td>6 µL</td>
   <td>-</td>
   <td>-</td>
   <td>-</td>
   </tr>

   <tr>
   <th>10x tile buffer</th>
   <td>1 µL</td>
   <td>1 µL</td>
   <td>1 µL</td>
   <td>1 µL</td>
   <td>1 µL</td>
   <td>1 µL</td>
   <td>1 µL</td>
   <td>1 µL</td>
   </tr>
       <tr>
   <th>MilliQ</th>
   <td>6 µL</td>
   <td>6 µL</td>
   <td>9 µL</td>
   <td>3 µL</td>
   <td>-</td>
   <td>4.5 µL</td>
   <td>3 µL</td>
   <td>-</td>
   </tr>
       <tr>
   <th>Ratio of concentration of 5ap_5B_16 to 5ap_M-3t4e_T0</th>
   <td>-</td>
   <td>-</td>
   <td>-</td>
   <td>1 %</td>
   <td>2 %</td>
   <td>5 %</td>
   <td>10 %</td>
   <td>20 %</td>    
   </tr>

</center> 

   </table>

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

   </ul>

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

<!--Reagent--> 

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

TACTCAGGGCACTGCAAGCAATTGTGGTCCCAATGGGCTGAGTACGCGACCTCATCTTTGACCCCCAGGCAGGGAG 

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

Biotin - TACTCAGCCCATTGGG 

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

   </table>
   <br>

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

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

   </table>


<!--Procedure--> 

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

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


   </ul>

   </article>
   <br>

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

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

<!--Reagent--> 

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

hybridization 

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

TACTCAGGGCACTGCAAGCAATTGTGGTCCCAATGGGCTGAGTACGCGACCTCATCTTTGACCCCCAGGCAGGGAG 

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

Biotin–TACTCAGCCCATTGGG–Cy3 

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

   </table>
   <br>

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

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

   </table>

5ap_tile 

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

<!--Procedure--> 

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

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

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

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

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

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

   </ul>

   </article>
   <br>

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

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

<!--Reagent--> 

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

<li>materials for the reaction</li> 

   <table>
   <tr>
   <th>10x tile buffer </th>
   <td>f. 1x tile buffer</td>
   </tr>

   <tr>
   <th>1 µM aptamer</th>
   <td>f. 0.3µM</td>
   </tr>

   <tr>
   <th>1 µM Ladder151515_1</th>
   <td>f. 0.3µM</td>
   </tr>

   <tr>
   <th>PDGF (dye 45nM)</th>
   <td>f. 20mM</td>
   </tr>

   </table>

<li>materials for the electrophoresis</li> 

   <gel for 10% Native-PAGE>
   <table>
   <tr>
   <th>MilliQ</th>
   <td>7.9 mL</td>
   </tr>

   <tr>
   <th>30% Acrylamide mix</th>
   <td>6.7 mL</td>
   </tr>

   <tr>
   <th>1.5 M Tris-HCl (pH 8.8)</th>
   <td>5 mL</td>
   </tr>

   <tr>
   <th>1 M MgCl2</th>
   <td>200 µL</td>
   </tr>

   <tr>
   <th>10 % APS</th>
   <td>80 µL</td>
   </tr>

   <tr>
   <th>TEMED</th>
   <td>80 µL</td>
   </tr>

   </table>

   <Electrophoresis buffer for 10 % Native-PAGE>
     1x TBE

   <materials for stain>
   <table>
   <tr>
   <th>Electrophoresis buffer for 10 % Native-PAGE</th>
   <td>50 mL</td>
   </tr>

   <tr>
   <th>SYBR Gold</th>
   <td>5 µL</td>
   </tr>

   </table>

   <others>
   <table>
   <tr>
   <th>Loading buffer</th>
   <th>20 % glycerol (as used 6x)</th>
   </tr>

   <tr>
   <th>Marker</th>
   <th>Cy5 38 mer</th>
   </tr>

   </table>


<!--Procedure--> 

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

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

   <tr>
   <th>Sample No.</th>
   <td>1</td>
   <td>2</td>
   <td>3</td>
   <td>4</td>
   <td>5</td>
   <td>6</td>
   </tr>

   <tr>
   <th>10x tile buffer</th>
   <td>1 µL</td>
   <td>1 µL</td>
   <td>1 µL</td>
   <td>1 µL</td>
   <td>1 µL</td>
   <td>1 µL</td>
   </tr>

       <tr>
   <th>1 µM aptamer</th>
   <td>3 µL</td>
   <td>-</td>
   <td>-</td>
   <td>3 µL</td>
   <td>-</td>
   <td>-</td>
   </tr>

   <tr>
   <th>1 µM Ladder151515_1</th>
   <td>-</td>
   <td>-</td>
   <td>-</td>
   <td>-</td>
   <td>3 µL</td>
   <td>3 µL</td>
   </tr>

   <tr>
   <th>PDGF (dye 45 nM)</th>
   <td>-</td>
   <td>6 µL</td>
   <td>-</td>
   <td>6 µL</td>
   <td>-</td>
   <td>6 µL</td>
   </tr>
       <tr>
   <th>MilliQ</th>
   <td>6 µL</td>
   <td>3 µL</td>
   <td>9 µL</td>
   <td>-</td>
   <td>6 µL</td>
   <td>-</td>
   </tr>

</center> 

   </table>

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

   </ul>

   </article>
   <br>


</article>

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

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

<!--Reagent--> 

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

hybridization 

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

TACTCAGGGCACTGCAAGCAATTGTGGTCCCAATGGGCTGAGTAGACCTCATCTTTGACCCCCAGGCAGGGAG 

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

TACTCAGCCCATTGGGttttttttttttttttttttttttttttttAAAACACTGCTCCATGTTACTTAACAAAGCT 

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

   </table>
   <br>

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

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

   </table>

5ap_tile 

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

<!--Procedure--> 

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

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

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

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

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

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

   </ul>

   </article>
   <br>


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

<!--◆◆SA dimer◆◆--> 

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

<!--Reagent--> 

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

   <table>
   <tr>
   <th>OCK (90 nM)</th>
   <td>8 µL</td>
   </tr>

   <tr>
   <th>Streptavidin (190 nM)</th>
   <td>2 µL</td>
   </tr>

   <tr>
   <th>CuSO4 aq (8 mM)</th>
   <td>1 µL</td>
   </tr>

   <tr>
   <th>THTA (32.5 mM)</th>
   <td>1 µL</td>
   </tr>

   <tr>
   <th>Sodium ascorbate (3.25 mM)</th>
   <td>1 µL</td>
   </tr>



   </table>
   <br>


<!--Procedure--> 

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

   </article>
   <br>


<!--◆◆Click_reaction◆◆--> 

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

<!--Reagent--> 

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

   <table>

   <tr>
   <th>azide solution (10μM)</th>
   <td>3μL</td>
   </tr>

   <tr>
   <th>alkyne solution (10μM)</th>
   <td>3μL</td>
   </tr>

   <tr>
   <th>CuSO<sub>4</sub> solution (50mM)</th>
   <td>1μL</td>
   </tr>

   <tr>
   <th>THTA solution (100mM)</th>
   <td>1μL</td>
   </tr>

   <tr>
   <th>sodium ascorbate solution (100mM)</th>
   <td>1μL</td>
   </tr>

   </table>
   <br>

<!--Procedure--> 

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

<!--◆◆Accelerated_Click_reaction◆◆--> 

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

<!--Reagent--> 

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

   <table>
   <tr>
   <th>2x barrel buffer</th>
   <td>6 µL</td>
   </tr>

   <tr>
   <th>alkyne oligo (carrying biotin) (15 µM)</th>
   <td>1 µL</td>
   </tr>

   <tr>
   <th>azide oligo (carrying biotin) (15 µM)</th>
   <td>1 µL</td>
   </tr>



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

   </table>
   <br>


<!--Procedure--> 

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

   </article>
   <br>

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

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

<!--Reagent--> 

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

   <table>
   <tr>
   <th>2x barrel buffer</th>
   <td>7 µL</td>
   </tr>

   <tr>
   <th>alkyne oligo (15 µM)</th>
   <td>1 µL</td>
   </tr>

   <tr>
   <th>azide oligo (15 µM)</th>
   <td>1 µL</td>
   </tr>



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

   </table>
   <br>


<!--Procedure--> 

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

<!--◆◆Click reaction cupper free◆◆--> 

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

<!--Reagent--> 

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

   <table>
   <tr>
   <th>2x barrel buffer</th>
   <td>7 µL</td>
   </tr>

   <tr>
   <th>alkyne oligo (15 µM)</th>
   <td>1 µL</td>
   </tr>

   <tr>
   <th>azide oligo (15 µM)</th>
   <td>1 µL</td>
   </tr>



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

   </table>
   <br>

<!--※10x OCK buffer (f. 100 µl)--> 

  <div class="zairyou-heading">[※※	2x barrel buffer]</div>
      <br>

   <table>
   <tr>
   <th>1M Tris (pH 7.5)</th>
   <td>5 µL</td>
   </tr>



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



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



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

   </table>
   <br>

<!--Procedure--> 

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

   </article>



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


<!--Procedure--> 

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

<!--Reference--> 

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

    <div>     
       <div class="reference-title">
       <a name="proref-1">
       [1] Folding DNA to create nanoscale shapes and patterns
       </a>
       </div>
          <div class="reference-author">
          Rothemund, P. W.
          </div>
             <div class="reference-journal">
             Nature 440, 297–302 (2006)
             </div>
    </div>

    <div>
       <div class="reference-title">
       <a name="proref-1">
       [2] Rapid Folding of DNA into Nanoscale Shapes at Constant Temperature
       </a>
       </div>
          <div class="reference-author">
           Jean-Philippe J. Sobczak, Thomas G. Martin, Thomas Gerling, Hendrik Dietz
          </div>
             <div class="reference-journal">
             Science, 2012, 338, 1458
             </div>
    </div>

    <div>     
       <div class="reference-title">
       <a name="proref-1">
       [3] Transcription Regulation System Mediated by Mechanical Operation of a DNA &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbs	p;Nanostructure
       </a>
       </div>
          <div class="reference-author">
          Masayuki Endo, Ryoji Miyazaki, Tomoko Emura, Kumi Hidaka, and Hiroshi Sugiyama
          </div>
             <div class="reference-journal">
             Journal of the American Chemical Society, 2012, 134, 2852-2855
             </div>
    </div>

    <div>     
       <div class="reference-title">
       <a name="proref-1">
       [4] the protocol of Jena Bioscience GmbH
       </a>
       </div>
         <div class="reference-journal">
          <a target="_blank" href="http://www.jenabioscience.com" style="color:#e00000">
          http://www.jenabioscience.com</a>
         </div>
    </div>

    <div>
       <div class="reference-title">
       <a name="proref-1">
       [5] Substrate-Assisted Assembly of Interconnected Single-Duplex DNA Nanostructures
       </a>
       </div>
          <div class="reference-author">
          Shogo Hamada, Satoshi Murata Prof.
          </div>
             <div class="reference-journal">
             Angewandte Chemie International Edition,2009,48(37),6820–6823
             </div>
    </div>
    <div>     
       <div class="reference-title">
       <a name="proref-1">
       [6] A primer to scaffolded DNA origami.</a>
       </div>
          <div class="reference-author">
          Castro CE, Kilchherr F, Kim DN, Shiao EL, Wauer T, Wortmann P, Bathe M

and Dietz H. 

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