Biomod/2013/Todai/Experiment: Difference between revisions

Revision as of 06:57, 25 October 2013

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   <div id="top">	
     <figure>
       <div  class="Logo">
         <img src="http://openwetware.org/images/9/9c/Logo-OCKver.png" width=730px height=128px>
       </div>
     </figure>
   </div>
   <br>

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

<li>

          <a href="http://openwetware.org/wiki/Biomod/2013/Todai/Experiment#Contents">
          Contents</a></li>
          <li>
          <a href="http://openwetware.org/wiki/Biomod/2013/Todai/Experiment#PilotStudy">
          Pilot Study</a></li>
          <li>
          <a href="http://openwetware.org/wiki/Biomod/2013/Todai/Experiment#Protocols">
          Protocols</a></li>
       </ul>
    </li>
    <li><a href="http://openwetware.org/wiki/Biomod/2013/Todai/Team">Team</a>
    </li>
    <li><a href="http://openwetware.org/wiki/Biomod/2013/Todai/Sponsors">Sponsors</a>
    </li>
 </ul>

</div>

 <a href="#TOP">
   <figure>
     <img src="http://openwetware.org/images/b/b1/Return-top-0828new.png" width:60px height:60px>
   </figure>
 </a>

</div>

</body> </html>

<html> <head> <title>Experiment-Todai nanORFEVRE-</title> <style>

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<body>

<h1 class="big-title"><a name="Experiment"> Experiment</a></h1>

   	<div id="Explist">
   	<ul>
      	<li><div class="mokuji"><a href="#Contents">Contents</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>

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

      	<article>
        <ul class="Contents-list">

        <li>
        <a name="#Assembling_of_DNA_structure">
        <b>Assembly of DNA nanostructure</b>
        </a>
        -->(See <a href="http://openwetware.org/wiki/Biomod/2013/Todai/Result" style="color:#E00000">Result</a>)
        <br>
        Optimize the assembly condition of the DNA nanostructure,"Cylinder in barrel"
        </li>
        <br>

        <li><a name="#flotation_assay"><b>Flotation assay</b></a>
        <ul class="Contents-list">
          <li><a href ="#Preparation_of_liposome">Preparation of liposome</a>
          <br>
          Making of liposome for floatation assay
        </li>
    
        <li>
        <a href ="#Flotation_assay_of_liposome_and_DNA_origami">
        Floatation assay of liposome and DNA origami
        </a>

<br>

        Assay to check the penetration of DNA origami

        </ul>
        <br>

        <li>
        <a href ="#Comparision_of_dimerization">
        <b>Comparision of dimerization method</b>
        </a>

        <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
            </li>
            <br>

          </ul>

          </li>

     	</ul>
     	</li>
     	</ul>
 		</article>
		</article>

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

  	<article>
  
  <h2 class="PS_title"><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 the
  <a href="http://openwetware.org/wiki/Biomod/2013/Todai/Result" style="color:#e00000">Result page</a>.
  <br>   
  <br>

  	<h2 class="PS_title"><a name="flotation_assay">&nbsp;2.Flotation assay</a></h2>
  	<div class="mini-title">
      <a name="Preparation_of_liposome">2-1. Preparation of liposome</a>
  	</div>

      <figure>
       <center>
        <img src="http://openwetware.org/images/b/b5/640px_liposomeDLS-Todai.png" width=640px height=360px >

<figcaption> <b>The result of DLS </b>

     </figcaption>
       </center>
      </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. Using Extruder device(Avanti), we prepared liposome of 120nm in radius.

    </p>
  </article>
  <br>

  <article>
  <div class="mini-title">
      <a name="Flotation_assay_of_liposome_and_DNA_origami">2-2. Floatation assay of liposome and DNA origami</a>
      <figure>
       <center>
        <img src="http://openwetware.org/images/1/1d/640pxflotationassay-Todai.jpg" width=300px height=300px >
	<figcaption> <b>Result of agarose gel electrophoresis of the sample of flotation assay</b> <br>
	The result of 1% agarose gel electrophoresis(100V,30min). In this measurement, the fluorescence of Cy5, which is

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

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

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

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

<br>

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

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

    </p>
  	<br>

</article>

  	<h2 class="PS_title"><a name="Comparision_of_dimerization">3. Comparision of dimerization method</a></h2>
  	<article>
  	<div class="mini-title">
      <a name="Click_reaction_via_(3+2)_cycloaddition">
      3-1. Click reaction via (3+2) cycloaddition<sup>[4]</sup>
      </a>
  	</div>
      <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>
  <br>

  	<article>
     <div class="mini-title">
     <a name ="Azobenzene">3-2. Azobenzene</a>
    </div>

    <div class="mini-title">
     <a name="Synthesis_of_Tube(Research_for_azobenzene)">&nbsp;&nbsp;3-2-1. Synthesis of tube<sup>[2],[3]</sup>(Resear	ch for azobenzene)</a>
    </div>

      <figure>
       <center>
        <img src="http://openwetware.org/images/b/b2/640x360px_tube_result-Todai.png" width=640px height=360px >
        <figcaption>
        <b>results of the electrophoresis of DNA-tube</b><br>
        </figcaption>
       </center>
      </figure>

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

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

   <sup>[2]</sup>. In the figure above, two bands derived from scaffold or DNA-tube were showed with cursors. At 56.4℃	, the scaffold

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- tube efficiently and improving the yield of DNA-tube.

    </p>
  <br>

  <article>
  <div class="mini-title">
      <a name="Synthesis_of_Motif(Research_for_azobenzene)">&nbsp;&nbsp;3-2-2. Synthesis of Motif<sup>[5]</sup></a>
  </div>

      <figure>
       <center>
        <img src="http://openwetware.org/images/a/a8/480px_electrophoresis_of_T-motif_improved-Todai.png" width=480px h	eight=360px >
        <figcaption>
         <b>Result of agarose gel electrophoresis of T-motif (wheel)</b>
         <br>
         The result of 1.5% agarose gel electrophoresis(100V) for 33min. 
        </figcaption>
       </center>
      </figure>

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

  <article>
  <h2 class="PS_title">
      <a name="Assembling_of_OCK">Assembly of OCK	<sup>[2]</sup></a>
      </h2>
   <br>

  <article>

     <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>

 *...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>

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

   </article>
   <br>

  	<article>
  	<h2>2.Flotation assay</h2>
  	<div class="mini-title">
      <a name="Preparation_of_liposome">2-1. Preparation of liposome</a>
  	</div>

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

   <table>
   <tr>
   <th>150mM aqueous 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>

  <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 aqueous 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>

  <article>
  <div class="mini-title">
      <a name="Flotation_assay_of_liposome">2-2. Flotation assay of liposome and DNA origami</a>
  </div>

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

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

   <tr>
   <th>KCl</th>
   <td>100mM</td>
   </tr>

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

   <tr>
   <th>Sucrose</th>
   <td>0.375M</td>
   </tr>
   </table>

   <div class="zairyou-heading-sub">b) 1.25M Sucrose buffer</div>
   <table>
   <tr>
   <th>HEPES-KOH(pH7.6)</th>
   <td>50mM</td>
   </tr>

   <tr>
   <th>KCl</th>
   <td>100mM</td>
   </tr>

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

   <tr>
   <th>Sucrose</th>
   <td>1.25M</td>
   </tr>
   </table>

   <div class="zairyou-heading-sub">c) 1.6M Sucrose buffer</div>
   <table>
   <tr>
   <th>HEPES-KOH(pH7.6)</th>
   <td>50mM</td>
   </tr>

   <tr>
   <th>KCl</th>
   <td>100mM</td>
   </tr>

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

   <tr>
   <th>Sucrose</th>
   <td>1.6M</td>
   </tr>
   </table>
   <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>
    <div style="font-size:110%;position:relative;left:60px;">~30nM</div>
   <br>

  <div class="zairyou-heading">[Procedure]</div>
   <ul class="procedure-list">
     <li>sample + sucrose buffer were layered in this order on centrifuge tubes.</li>
     <li>The tubes were centrifugalized.</li>
     <li>Samples were fructionized.</li>
     <li>Precipitation was resuspended with sucrose buffer.</li>
     <li>Samples were measured by the fluorescence of NIL(ex 500nm, em 550nm~700nm) and agarose gel electrophoresis.
     </li>
   </ul>
   </article>
   <br>

  <article>
  <h2>3. Comparision of dimerization method</h2>

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

     <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>

  <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>

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

     <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>

  <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>

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

     <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>

  <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>

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

     <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>

  <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>
   <br>

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

     <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>

  <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>

  	<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>
   <br>

  <article>
  <div class="mini-title">
      <a name="Research_for_azobenzene)">
      3-2. Research for azobenzene
      </a>
  </div>

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

    <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>
             
 </article>
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