Biomod/2014/Kashiwa/Protocol: Difference between revisions

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<body>
<font face="Century,Arial" font size="20pt">PROTOCOL</font>
<font face="Futura,Arial,Frutiger" font size="24px">PROTOCOL</font>
 
<br>
<br>
<br>
<br>
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   <div class="CollapsibleBoxBody" id="CBoxBody1">
   <div class="CollapsibleBoxBody" id="CBoxBody1">
       <ul style="list-style:none;">
       <ul style="list-style:none;">
         <li> 1. Polymerization
         <li> <a href=#1> 1. The Sensing System: The Receptor </a>
         <ul style="list-style:none;">
         <ul style="list-style:none;">
             <li> 1-1. Making the monomer </li>
             <li> <a href="#1-1">1-1. Preparing the components </a></li>
            <li> 1-2. Polymerization in solution
             <ul style="list-style:none;">
             <ul style="list-style:none;">
               <li> 1-2-1.  
               <li> <a href="#1-1-1">1-1(a). Folding the Wall </a></li>
               <li> 1-2-2.
              <li> <a href="#1-1-2">1-1(b). Producing MISTIC </a></li>
               <li> <a href="#1-1-3">1-1(c). Designing the Activator </a></li>
             </ul>
             </ul>
             </li>
             </li>
             <li> 1-3. Putting monomer into a liposome </li>
             <li> <a href="#1-2">1-2. Embedding the Wall into the liposome </a></li>
             <li> 1-4. Polymerize in a liposome </li>  
            <li> <a href="#1-3">1-3. Linking the Activator to the liposome </a></li>
             <li> 1-5. Deformation of a liposome </li>
             <li> <a href="#1-4">1-4. Combining the Wall and the Activator </a></li>
             <li> <a href="#1-5">1-5. Separating the Wall from the Activator </a></li>
         </ul>
         </ul>
         </li>
         </li>
         <li> 2. Receptor
       
         <li> <a href="#2">2. The Moving System: The Motor </a>
         <ul style="list-style:none">
         <ul style="list-style:none">
             <li> 2-1. Making the receptor </li>
             <li> <a href="#2-1">2-1. Producing the Motor-Monomer </a>
            <li> 2-2. Penetration into a liposome </li>
            <li> 2-3. Dimerization
             <ul style="list-style:none">
             <ul style="list-style:none">
              <li> 2-3-1. Dimerization in solution </li>
              <li> <a href="#2-1-1"> 2-1(a). Folding the Motor-Monomer body </a></li>
              <li> 2-3-2. Dimerization on a liposome </li>
              <li> <a href="#2-1-2"> 2-1(b). Synthesizing divalent SA </a></li>
            </ul>
              <li> <a href="#2-1-3"> 2-1(c). Equipping the Motor-Monomer with divalent SA </a></li>
            </li>
            <li> 2-4. Emission of polymerization initiator by dimerization
            <ul style="list-style:none">
              <li> 2-4-1. Emission by dimerization in solution </li>
              <li> 2-4-2. Emmision by dimerization on a liposome </li>
             </ul>
             </ul>
             </li>
             </li>
            <li> <a href="#2-2">2-2. Deactivating and reactivating the binding capacity of streptavidin </a></li>
            <li> <a href="#2-3">2-3. Putting the Motor-Monomers into the liposome </a></li>
         </ul>
         </ul>
        </li>
       </ul>
       </ul>
   </div>
   </div>
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<br>
<br>
<h1 class="title"><a name="contents">&nbsp;1. Polymerization</a></h1>
<a name="1-1">&nbsp;</a>
<br>
<br>
1-1. Making the monomer
<a name="1-1-1">&nbsp;</a>
 
<h1 class="title"><a name="contents">&nbsp;1. Developing the sensing system</a></h1>
<p class="title">1-1. The Receptor by itself</p>
 
<p class="subtitle">1-1-1. Design of the Receptor hetero-units</p>
<p class="headline">&bull; Make the hetero-units</p>
<table class="sample_01">
<table class="sample_01">
<caption>Reagents (f. 40.0 &micro;L)</caption>
<tbody>
<tbody>
<tr>
<tr>
<th>Reagent</th>
<th>M13mp18ss</th>
<th> </th>
<td>18.0 &micro;L</td>
</tr>
</tr>
<tr>
<tr>
<td>Staple mix</td>
<th>Staple mix</th>
<td>17.5 &micro;L</td>
<td>18.0 &micro;L</td>
</tr>
</tr>
<tr>
<tr>
<td>M13mp18ss</td>
<th>10 &times; Receptor buffer&sup1;</th>
<td>18.3 &micro;L</td>
<td>4.0 &micro;L</td>
</tr>
<tr>
<td>TE&sup1;</td>
<td>18.2 &micro;L</td>
<tr>
<td>10 &times; tile buffer&sup2;</td>
<td>6.0 &micro;L</td>
</tr>
</tr>
</tr>
</tbody>
</tbody>
</table>
</table>


<table class="sample_02">
 
<p class="mini">&sup1;10 &times; Receptor buffer &mdash; 50 mM Tris (HCl pH 7.5), 10 mM EDTA-Na (pH 8),<br> 100 mM MgCl<sub>2</sub>, 500 mM NaCl </p>
 
<a name="1-1-2">&nbsp;</a>
 
<p class="headline">Procedure</p>
<p class="line">1) The solution was mixed.</p>
<p class="line">2) The mixture was annealed at 47.5 °C for 4 hours.</p>
<p class="line">3) The mixture was purified by spin column.</p>
<p class="line">4) The mixture was analyzed by 1% agarose gel electrophoresis (100V, 40 min).</p>
<br>
<br>
 
<p class="subtitle">1-1-2. Dimerization mechanism of the Receptor</p>
<table class="sample_01">
<caption>Reagents (f. 20.0 &micro;L)</caption>
<tbody>
<tbody>
<tr>
<tr>
<th>&sup1;TE(f.50 mL)</th>
<th>10 &micro;M Aptamers</th>
<th> </th>
<td>3.5 &micro;L </td>
<th> </th>
</tr>
</tr>
<tr>
<tr>
<td>1M Tris-HCl(pH8.0)</td>
<th>1 &micro;M Alpha - thrombin</th>
<td>0.5 mL</td>
<td>3.5 &micro;L </td>
<td>(f.10 mM)
</tr>
</tr>
<tr>
<tr>
<td>0.5M EDTA</td>
<th>10 &times; physiological buffer&sup1;</th>
<td>0.1 mL</td>
<td>2.0 &micro; L</td>
<td>(f.1 mM)</td>
</tr>
</tr>
<tr>
<th>MQ</th>
<td>11.0 &micro; L</td>
</tr>
</tr>
</tbody>
</tbody>
</table>
</table>


<table class="sample_02">
<p class="mini">&sup1;10 &times; physiological buffer &mdash; 200  mM Tris - HCl (pH 7.5), 1.4 M NaCl, 50 mM KCl, <br>  10 mM CaCl <sub>2</sub>, 10 mM MgCl <sub>2</sub>, 5% (v/v) Glycerol</p>
 
<a name="1-1-3">&nbsp;</a>
 
<p class="headline">Procedure</p>
<p class="line">1) Aptamer solution was mixed.</p>
<p class="line">2) The solution was annealed from 90 °C to 24 °C, decrease by 0.4 °C / min.</p>
<p class="line">3) Thrombin-solution was added to the solution and incubated  at room temperature for 1 hour.</p>
<p class="line">4) The mixture was analyzed with Native-PAGE (200 V 25 min,  250 V 40 min).</p>
<p class="line">5) The gel was stained by EtBr for 30 minutes.
<p class="line">6) The gel was observed with UV and fluorescence by LAS - 4000.</p>
<br>
<br>
 
<p class="subtitle">1-1-3. Emission of the Initiator</p>
<table class="sample_01">
<caption>Reagents (f. 20.0 &micro;L 25 nM)</caption>
<tbody>
<tbody>
<tr>
<tr>
<th>&sup2;10 &times; tile buffer(f.500 mL)</th>
<th>100 nM Biotin modified single strand J’</th>
<th> </th>
<td>5.0 &micro;L</td>
<th> </th>
</tr>
</tr>
<tr>
<tr>
<td>Mg(OAc)<sub>2</sub></td>
<th>100 nM The Mismatch modified Strand J</th>
<td>10.73 g</td>
<td>5.0 &micro;L</td>
<td>f.100 mM
</tr>
</tr>
<tr>
<tr>
<td>1.0M Tris-HCl(pH7.5)</td>
<th>100 nM The Initiator</th>
<td>100 mL</td>
<td>5.0 &micro;L</td>
<td>f.200 mM</td>
</tr>
</tr>
<tr>
<tr>
<td>0.5M EDTA</td>
<th>Cy5-modified Streptavidin (SA)</th>
<td>10 mL</td>
<td>5.0 &micro;L</td>
<td>f.10 mM</td>
</tr>
</tr>
</tr>
</tbody>
</tbody>
</table>
</table>


Reagents (f. 60μL)


Staple mix 17.5 μL


M13mp18ss 18.3 μL
<p class="headline">Procedure</p>
<p class="line">1) Strand J and Initiator were mixed and annealed at 85 °C,67 °C,50 °C,25 °C for 5 minutes each (mismatch factor 20 %).</p>
<p class="line">2) Biotin modified single strand and SA were mixed and incubated at room temperature for 15 minutes.</p>
<p class="line">3) The reagents were mixed and incubated at room temperature for 1 hour.</p>
<p class="line">4) The mixture was analyzed with Native - PAGE (200 V 40 min, 250 V 40 min).</p>
<p class="line">5) The gel was stained by EtBr for 30 minutes</p>
<p class="line">6) The gel was observed with UV and fluorescence by LAS - 4000.</p>
<a name="1-2">&nbsp;</a>
<br>
<a name="1-2-1">&nbsp;</a>
<br>
<br>


TE*1 18.2 μL
<p class="title">1-2. The Receptor on the liposome</p>
<p class="subtitle">1-2-1. Penetration of the Receptor hetero-units to the liposome</p>


10 x tile buffer*2 6.0 μL
<p class="subtitle">Ⅰ. Preparation of GUVs</p>
<p class="line">GUV&nbsp;s were made as shown in <a href=“#2-2-1”>”Putting the Monomer into a liposome".</a></p>
<br>


*1 TE (f. 50 mL)
<p class="subtitle">Ⅱ. Preparation of LUVs</p>
<table class="sample_01">
<caption>Reagents (f. 1280 &micro;L)</caption>
<tbody>
<tr>
<th>10 mg/mL POPC</th>
<td>200.0 &micro;L</td>
</tr>
<tr>
<th>25 mg/mL POPG</th>
<td>80.0 &micro;L</td>
</tr>
<tr>
<th>150 mM KCl solution</th>
<td>1.0 mL</td>
</tr>
</tr>
</tbody>
</table>
<p class="headline">Procedure </p>
<p class="line">1) A lipid film was formed by evaporating POPC and POPG in a tube. </p>
<p class="line">2) The tube was kept under vacuum overnight to evaporate remaining chloroform. </p>
<p class="line">3) The lipid film was resuspended in 1 mL of 150 mM KCl solution. <br>Temperature was controlled to be 40 °C during suspension.  </p>
<p class="line">4) The solution was kept at 4 °C or -80°C and sample was sonicated before usage.</p>
<br>


1M Tris-HCl (pH 8.0) 0.5 mL (f. 10 mM)


0.5M EDTA 0.1 mL (f. 1 mM)
<p class="subtitle">Ⅲ. Penetration of the Receptor into liposomes</p>
<table class="sample_01">
<caption>Reagents (f. 100.0 &micro;L)</caption>
<tbody>
<tr>
<th>Liposomes (LUVs , GUVs)</th>
<td>50.0 &micro;L</td>
</tr>
<tr>
<th>Cholesterol hybridized Receptor&sup1;</th>
<td>50.0 &micro;L</td>
</tr>
</tr>
</tbody>
</table>
<p class="mini">&sup1;Cholesterol hybridized Receptor &mdash; x &micro;M Purified Receptor, 160 x &micro;M Cholesterol oligomer </p>


*2 10 x tile buffer (f. 500 mL)


Mg(OAc)2 10.73 g f. 100 mM
<p class="headline">Procedure</p>
<p class="line">1)Purified Receptor and cholesterol oligomer  were mixed and incubated at room temperature for 60 minutes.</p>
<p class="line">2)The reagents were mixed and incubated at room temperature for 30 minutes.</p>
<br>


1.0 M Tris-HCl (pH 7.5) 100 mL f. 200 mM


0.5 M EDTA 10mL f. 10 mM
<p class="subtitle">Ⅳ. Flotation assay</p>


1) Mix the reagents.
<table class="sample_01">
<caption>Reagents (f. 2.4 mL)</caption>
<tbody>
<tr>
<th>The Receptor</th>
<td>100.0 &micro;L</td>
</tr>
<tr>
<th>Cholesterol hybridized Receptor</th>
<td>100.0 &micro;L</td>
</tr>
<tr>
<th>Liposomes</th>
<td>100.0 &micro;L</td>
</tr>
<tr>
<th>2.25 M Sucrose buffer&sup1;</th>
<td>500.0 &micro;L</td>
</tr>
<tr>
<th>1.6 M Sucrose buffer&sup1;</th>
<td>900.0 &micro;L</td>
</tr>
<tr>
<th>150 mM KCl solution</th>
<td>100.0 &micro;L</td>
</tr>
<tr>
<th>1 &times; Flotation buffer&sup2;</td>
<td>600.0 &micro;L</td>
</tr>
</tr>
</tbody>
</table>


2) Anneal the mixture at XX °C for XX hours.
<p class="mini">&sup1;1.6, 2.25 M Sucrose buffer &mdash; 50 mM HEPES - KOH (pH 7.6), 100 mM KCl, 20 mM MgCl<sub>2</sub>, 1.6, 2.25 M Sucrose </p>
<p class="mini">&sup2;1 &times; Flotation buffer &mdash; 50 mM HEPES - KOH (pH 7.6), 100 mM KCl, 20 mM MgCl<sub>2</sub>


3) Analyze by 1 % agarose gel electrophoresis.


4) Stain the gel by EtBr for 30 minutes.
<p class="headline">Procedure</p>
<p class="line">1) Each sample was mixed as shown below　<a href="#table1">(table1)</a>.</p>
<p class="line">2) 1.6 M Sucrose buffer was overlaid with  sample mixture in centrifuge tubes (Beckman, cat#343778, 11 &times; 34 mm).</p>
<p class="line">3) Each sample was centrifuged for 16 minutes at 100 krpm at 4 °C using TLA 100.2 rotor (BECKMAN COULTER) with Ultracentrifuge (BECKMAN COULTER, Optima MAX-XP).</p>
<p class="line">4) Supernatant (each 150 &micro;L) was extracted from top to bottom for 3 times (Fraction 1 to 3) and the pellet was retrieved with 1 &times; Flotation buffer (Fraction 4).</p>
<p class="line">5) Fraction 1-4 of each sample were analyzed by 1 % agaraose gel electrophoresis (100V, 1 hour).</p>
<a name="table1"></a><p class="line">6)10 &micro;M Nile Red was added to each fraction of sample 1 and 3. (30 &micro;L) (6 &micro;L)  The Intensity of fluorescence of NileRed (liposomes) was measured with fluorescence spectrophotometer (JASCO, FP-6500) to investigate the existence of liposomes in each Fraction.</p>


5) Take photo of the gel by LAS-4000.
<br>
 
<table class="sample_04">
 
<caption>Table 1. Breakdown of Samples</caption>
 
2-3-1.Receptor monomers are assembled and heterodimerized by Thrombin.
 
【1:Assembly of receptor】(Barrel ver.)
<table class="sample_01">
<tbody>
<tbody>
<tr>
<tr>
<th>Reagent</th>
<th>sample No.</th>
<th> </th>
<td>1</td>
<td>2</td>
<td>3</td>
<td>4</td>
</tr>
</tr>
<tr>
<tr>
<td>M13mp18ss</td>
<th>Cholesterol hybridized Receptor</th>
<td> </td>
<td>50.0 &micro;L</td>
<td>50.0 &micro;L</td>
<td>&mdash;</td>
<td>&mdash;</td>
</tr>
</tr>
<tr>
<tr>
<td>Staple mix</td>
<th>Receptor</th>
<td> </td>
<td>&mdash;</td>
<td>&mdash;</td>
<td>50.0 &micro;L</td>
<td>50.0 &micro;L</td>
</tr>
</tr>
<tr>
<tr>
<td>10x barrel buffer</td>
<th>Liposome</th>
<td> </td>
<td>50.0 &micro;L</td>
<td>&mdash;</td>
<td>50.0 &micro;L</td>
<td>&mdash;</td>
</tr>
<tr>
<th>150 mM aqueous KCl solution</th>
<td>&mdash;</td>
<td>50.0 &micro;L</td>
<td>&mdash;</td>
<td>50.0 &micro;L</td>
</tr>
<tr>
<tr>
<td>MQ</td>
<th>2.25 M Sucrose buffer</th>
<td> </td>
<td>125.0 &micro;L</td>
<td>125.0 &micro;L</td>
<td>125.0 &micro;L</td>
<td>125.0 &micro;L</td>
</tr>
</tr>
</tr>
</tr>
</tbody>
</tbody>
</table>
</table>
<a name="1-2-2">&nbsp;</a>
<a name="1-2-3">&nbsp;</a>
<br>


*10x barrel buffer (f:10ml)


1M Tris Baric 0.5μl
<a name="1-2-2">&nbsp;</a>
<p class="subtitle">1-2-2. Dimerization mechanism of the Receptor on the liposome</p>


0.5M EDTA 200μl
<p class="subtitle">1-2-3. Emission of the initiator in the liposome </p>


1M MgCl2 2ml


5M NaCl 100μl


MQ 7.2ml
<a name="2">&nbsp;</a>
<br>
<a name="2-1">&nbsp;</a>
<br>
<a name="2-1-1">&nbsp;</a>
<br>


[Procedure]
<h1 class="title"><a name="contents">&nbsp;2. Developing the moving system</a></h1>


1. Mix the solutions.
<p class="title">2-1. The Motor by itself</p>


2. The mixture was annealed at 47.5oC for 4 hours.
<p class="subtitle">2-1-1. Design of the Motor-Monomer </p>
<p class="headline">&bull; Make the Moter-Monomer


3. Purified with spin column.
<table class="sample_01">
<caption>Reagents (f. 60.0 &micro;L)</caption>
<tbody>
<tr>
<th>Staple mix</th>
<td>17.5 &micro;L</td>
</tr>
<tr>
<th>M 13 mp 18 ss</th>
<td>18.3 &micro;L</td>
</tr>
<tr>
<th>TE&sup1;</th>
<td>18.2 &micro;L</td>
<tr>
<th>10 &times; tile buffer&sup2;</th>
<td> 6.0 &micro;L</td>
</tr>
</tr>
</tbody>
</table>
 
<p class="mini">&sup1;TE &mdash; 10 mM Tris - HCl (pH 8.0), 1 mM EDTA</p>
<p class="mini">&sup2;10 &times; tile buffer &mdash; 100 mM Mg (OAc) <sub>2</sub>, 200 mM Tris-HCl (pH 7.5), .10 mM EDTA</p>


4. Analyzed by 1% agarose gel electrophoresis (100V, 40 min).
<a name="2-1-2">&nbsp;</a>
 
<p class="headline">Procedure</p>
<p class="line">1) The reagents were mixed.</p>
<p class="line">2) The mixture was annealed at 55 °C for 3 hours.</p>
<p class="line">3) The mixture was analyzed by 1 % agarose gel electrophoresis.</p>
<p class="line">4) The gel was stained by EtBr for 30 minutes.</p>
<p class="line">5) A photo of the gel was taken  by LAS - 4000.</p>
<br>
<br>


【2:Dimerization of hetero receptor by Thrombin】
<p class="subtitle">2-1-2. Formation of the simple Polymer</p>
<table class="sample_01">
<caption>Reagents (f. 20.0 &micro;L)</caption>
<tbody>
<tr>
<th>Purified Monomer X</th>
<td>10.0 &micro;L</td>
</tr>
<tr>
<th>Purified Monomer Y</th>
<td>10.0 &micro;L</td>
</tr>
</tbody>
</table>


[Reagent]
<a name="2-1-3">&nbsp;</a>


Monomer A (purified)
<p class="headline">Procedure</p>
<p class="line">1) Monomers were made as shown in “Making DNA origami monomers”.</p>
<p class="line">2) Each Monomer solution was purified by spin column.</p>
<p class="line">3) The reagents were mixed.</p>
<p class="line">4) The mixture was incubated at room temperature for 24 hours.</p>
<p class="line">5) The mixture was analyzed by 1 % agarose gel electrophoresis (100 V, 80 min).</p>
<p class="line">6) The gel was stained by EtBr for 30 minutes.</p>
<p class="line">7) The gel was taken a photo by LAS - 4000.</p>
<br>
<br>


Monomer B (purified)


Thrombin (1.09μM)
<a name="2-2">&nbsp;</a>
<p class="subtitle">2-1-3. Controlling the ring - opening polymerization </p>
<a name="2-2-1">&nbsp;</a>
<br><br>


Loading-dye
<p class="title">2-2. The Motor in the liposome </p>


[Procedure]
<p class="subtitle">2-2-1. Putting the Motor - Monomers into the liposome</p>
<table class="sample_01">
<caption>Reagents (f.1420 &micro;L)</caption>
<tbody>
<tr>
<th>Lipid mix &sup1;</th>
<td>400.0 &micro;L</td>
</tr>
<tr>
<th>Glucose</th>
<td>500.0 &micro;L</td>
</tr>
<tr>
<th>Emulsion mix&sup2;</th>
<td>520.0 &micro;L</td>
</tr>
</tbody>
</table>


1. Mix the solutions, and incubated 1 hour at room temperature.
<p class="mini">&sup1;3 mM Lipid mix &mdash; POPC, Paraffin</p>
<p class="mini">&sup2;Emulsion mix &mdash; 0.5 mM Lipid mix, 50 mM HEPES, 10 mM EDTA,<br>400 mM Sucrose,Pyranine, The Motor-Monomers</p>


2. Analyzed by 1% agarose gel electrophoresis (100V, 80 min).
<a name="2-2-2">&nbsp;</a>
<p class="headline">Procedure</p>
<p class="line">1) Glucose (500 &micro;L) was taken into a tube as outer solution.</p>
<p class="line">2) Lipid mix (0.5 mM) was added on the glucose solution carefully.</p>
<p class="line">3) Emulsion mix was added on the top and rapidly centrifuged at 4 °C, 1800 rpm for 10 minutes.</p>
<p class="line">4) Centrifuged again at 4 °C, 5000 rpm for 10 minutes.</p>
<p class="line">5) The upper layer was removed and GUVs were collected by micropipette.</p>
<p class="line">6) The GUVs were stained by Nile red and observed with a confocal microscope.</p>
<br>
<br>


<h1 class="title"><a name="contents">&nbsp;2. Receptor</a></h1>


<p class="subtitle">2-2-2. Formation of the simple Polymer in the liposome </p>
<p class="subtitle">2-2-3. Controlling the ring opening polymerization in the liposome </p>


挫折。また明日。
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<body> <font face="Futura,Arial,Frutiger" font size="24px">PROTOCOL</font>

<br> <br> <div class="CollapsibleBox" id="CBoxCover1"> 

  <div class="CollapsibleBoxTitle">
     <p class="cBoxButtons">
        <a href="#" onclick="HideCBox('CBoxBody1');  return false;" title="折りたたみ／復元"><font color="white" font size="2">[show/hide]</font></a>
     </p>
     <p class="cBoxTitle">&nbsp; Contents：</p>
  </div>
  <div class="CollapsibleBoxBody" id="CBoxBody1">
     <ul style="list-style:none;">
        <li> <a href=#1> 1. The Sensing System: The Receptor </a>
        <ul style="list-style:none;">
           <li> <a href="#1-1">1-1. Preparing the components </a></li>
           <ul style="list-style:none;">
              <li> <a href="#1-1-1">1-1(a). Folding the Wall </a></li>
              <li> <a href="#1-1-2">1-1(b). Producing MISTIC </a></li>
              <li> <a href="#1-1-3">1-1(c). Designing the Activator </a></li>
           </ul>
           </li>
           <li> <a href="#1-2">1-2. Embedding the Wall into the liposome </a></li>
           <li> <a href="#1-3">1-3. Linking the Activator to the liposome </a></li>
           <li> <a href="#1-4">1-4. Combining the Wall and the Activator </a></li>
           <li> <a href="#1-5">1-5. Separating the Wall from the Activator </a></li>
        </ul>
        </li>
        
        <li> <a href="#2">2. The Moving System: The Motor </a>
        <ul style="list-style:none">
           <li> <a href="#2-1">2-1. Producing the Motor-Monomer </a>
           <ul style="list-style:none">
             <li> <a href="#2-1-1"> 2-1(a). Folding the Motor-Monomer body </a></li>
             <li> <a href="#2-1-2"> 2-1(b). Synthesizing divalent SA </a></li>
             <li> <a href="#2-1-3"> 2-1(c). Equipping the Motor-Monomer with divalent SA </a></li>
           </ul>
           </li>
           <li> <a href="#2-2">2-2. Deactivating and reactivating the binding capacity of streptavidin </a></li>
           <li> <a href="#2-3">2-3. Putting the Motor-Monomers into the liposome </a></li>
        </ul>
        </li>
     </ul>
  </div>

</div>

<br> <a name="1-1">&nbsp;</a> <br> <a name="1-1-1">&nbsp;</a>

<h1 class="title"><a name="contents">&nbsp;1. Developing the sensing system</a></h1> <p class="title">1-1. The Receptor by itself</p>

<p class="subtitle">1-1-1. Design of the Receptor hetero-units</p> <p class="headline">&bull; Make the hetero-units</p> <table class="sample_01"> <caption>Reagents (f. 40.0 &micro;L)</caption> <tbody> <tr> <th>M13mp18ss</th> <td>18.0 &micro;L</td> </tr> <tr> <th>Staple mix</th> <td>18.0 &micro;L</td> </tr> <tr> <th>10 &times; Receptor buffer&sup1;</th> <td>4.0 &micro;L</td> </tr> </tbody> </table>


<p class="mini">&sup1;10 &times; Receptor buffer &mdash; 50 mM Tris (HCl pH 7.5), 10 mM EDTA-Na (pH 8),<br> 100 mM MgCl<sub>2</sub>, 500 mM NaCl </p>

<a name="1-1-2">&nbsp;</a>

<p class="headline">Procedure</p> <p class="line">1) The solution was mixed.</p> <p class="line">2) The mixture was annealed at 47.5 °C for 4 hours.</p> <p class="line">3) The mixture was purified by spin column.</p> <p class="line">4) The mixture was analyzed by 1% agarose gel electrophoresis (100V, 40 min).</p> <br> <br>

<p class="subtitle">1-1-2. Dimerization mechanism of the Receptor</p> <table class="sample_01"> <caption>Reagents (f. 20.0 &micro;L)</caption> <tbody> <tr> <th>10 &micro;M Aptamers</th> <td>3.5 &micro;L </td> </tr> <tr> <th>1 &micro;M Alpha - thrombin</th> <td>3.5 &micro;L </td> </tr> <tr> <th>10 &times; physiological buffer&sup1;</th> <td>2.0 &micro; L</td> </tr> <tr> <th>MQ</th> <td>11.0 &micro; L</td> </tr> </tbody> </table>

<p class="mini">&sup1;10 &times; physiological buffer &mdash; 200 mM Tris - HCl (pH 7.5), 1.4 M NaCl, 50 mM KCl, <br> 10 mM CaCl <sub>2</sub>, 10 mM MgCl <sub>2</sub>, 5% (v/v) Glycerol</p>

<a name="1-1-3">&nbsp;</a>

<p class="headline">Procedure</p> <p class="line">1) Aptamer solution was mixed.</p> <p class="line">2) The solution was annealed from 90 °C to 24 °C, decrease by 0.4 °C / min.</p> <p class="line">3) Thrombin-solution was added to the solution and incubated at room temperature for 1 hour.</p> <p class="line">4) The mixture was analyzed with Native-PAGE (200 V 25 min, 250 V 40 min).</p> <p class="line">5) The gel was stained by EtBr for 30 minutes. <p class="line">6) The gel was observed with UV and fluorescence by LAS - 4000.</p> <br> <br>

<p class="subtitle">1-1-3. Emission of the Initiator</p> <table class="sample_01"> <caption>Reagents (f. 20.0 &micro;L 25 nM)</caption> <tbody> <tr> <th>100 nM Biotin modified single strand J’</th> <td>5.0 &micro;L</td> </tr> <tr> <th>100 nM The Mismatch modified Strand J</th> <td>5.0 &micro;L</td> </tr> <tr> <th>100 nM The Initiator</th> <td>5.0 &micro;L</td> </tr> <tr> <th>Cy5-modified Streptavidin (SA)</th> <td>5.0 &micro;L</td> </tr> </tbody> </table>


<p class="headline">Procedure</p> <p class="line">1) Strand J and Initiator were mixed and annealed at 85 °C,67 °C,50 °C,25 °C for 5 minutes each (mismatch factor 20 %).</p> <p class="line">2) Biotin modified single strand and SA were mixed and incubated at room temperature for 15 minutes.</p> <p class="line">3) The reagents were mixed and incubated at room temperature for 1 hour.</p> <p class="line">4) The mixture was analyzed with Native - PAGE (200 V 40 min, 250 V 40 min).</p> <p class="line">5) The gel was stained by EtBr for 30 minutes</p> <p class="line">6) The gel was observed with UV and fluorescence by LAS - 4000.</p> <a name="1-2">&nbsp;</a> <br> <a name="1-2-1">&nbsp;</a> <br> <br>

<p class="title">1-2. The Receptor on the liposome</p> <p class="subtitle">1-2-1. Penetration of the Receptor hetero-units to the liposome</p>

<p class="subtitle">Ⅰ. Preparation of GUVs</p> <p class="line">GUV&nbsp;s were made as shown in <a href=“#2-2-1”>”Putting the Monomer into a liposome".</a></p> <br>

<p class="subtitle">Ⅱ. Preparation of LUVs</p> <table class="sample_01"> <caption>Reagents (f. 1280 &micro;L)</caption> <tbody> <tr> <th>10 mg/mL POPC</th> <td>200.0 &micro;L</td> </tr> <tr> <th>25 mg/mL POPG</th> <td>80.0 &micro;L</td> </tr> <tr> <th>150 mM KCl solution</th> <td>1.0 mL</td> </tr> </tr> </tbody> </table> <p class="headline">Procedure </p> <p class="line">1) A lipid film was formed by evaporating POPC and POPG in a tube. </p> <p class="line">2) The tube was kept under vacuum overnight to evaporate remaining chloroform. </p> <p class="line">3) The lipid film was resuspended in 1 mL of 150 mM KCl solution. <br>Temperature was controlled to be 40 °C during suspension. </p> <p class="line">4) The solution was kept at 4 °C or -80°C and sample was sonicated before usage.</p> <br>


<p class="subtitle">Ⅲ. Penetration of the Receptor into liposomes</p> <table class="sample_01"> <caption>Reagents (f. 100.0 &micro;L)</caption> <tbody> <tr> <th>Liposomes (LUVs , GUVs)</th> <td>50.0 &micro;L</td> </tr> <tr> <th>Cholesterol hybridized Receptor&sup1;</th> <td>50.0 &micro;L</td> </tr> </tr> </tbody> </table> <p class="mini">&sup1;Cholesterol hybridized Receptor &mdash; x &micro;M Purified Receptor, 160 x &micro;M Cholesterol oligomer </p>


<p class="headline">Procedure</p> <p class="line">1)Purified Receptor and cholesterol oligomer were mixed and incubated at room temperature for 60 minutes.</p> <p class="line">2)The reagents were mixed and incubated at room temperature for 30 minutes.</p> <br>


<p class="subtitle">Ⅳ. Flotation assay</p>

<table class="sample_01"> <caption>Reagents (f. 2.4 mL)</caption> <tbody> <tr> <th>The Receptor</th> <td>100.0 &micro;L</td> </tr> <tr> <th>Cholesterol hybridized Receptor</th> <td>100.0 &micro;L</td> </tr> <tr> <th>Liposomes</th> <td>100.0 &micro;L</td> </tr> <tr> <th>2.25 M Sucrose buffer&sup1;</th> <td>500.0 &micro;L</td> </tr> <tr> <th>1.6 M Sucrose buffer&sup1;</th> <td>900.0 &micro;L</td> </tr> <tr> <th>150 mM KCl solution</th> <td>100.0 &micro;L</td> </tr> <tr> <th>1 &times; Flotation buffer&sup2;</td> <td>600.0 &micro;L</td> </tr> </tr> </tbody> </table>

<p class="mini">&sup1;1.6, 2.25 M Sucrose buffer &mdash; 50 mM HEPES - KOH (pH 7.6), 100 mM KCl, 20 mM MgCl<sub>2</sub>, 1.6, 2.25 M Sucrose </p> <p class="mini">&sup2;1 &times; Flotation buffer &mdash; 50 mM HEPES - KOH (pH 7.6), 100 mM KCl, 20 mM MgCl<sub>2</sub>


<p class="headline">Procedure</p> <p class="line">1) Each sample was mixed as shown below　<a href="#table1">(table1)</a>.</p> <p class="line">2) 1.6 M Sucrose buffer was overlaid with sample mixture in centrifuge tubes (Beckman, cat#343778, 11 &times; 34 mm).</p> <p class="line">3) Each sample was centrifuged for 16 minutes at 100 krpm at 4 °C using TLA 100.2 rotor (BECKMAN COULTER) with Ultracentrifuge (BECKMAN COULTER, Optima MAX-XP).</p> <p class="line">4) Supernatant (each 150 &micro;L) was extracted from top to bottom for 3 times (Fraction 1 to 3) and the pellet was retrieved with 1 &times; Flotation buffer (Fraction 4).</p> <p class="line">5) Fraction 1-4 of each sample were analyzed by 1 % agaraose gel electrophoresis (100V, 1 hour).</p> <a name="table1"></a><p class="line">6)10 &micro;M Nile Red was added to each fraction of sample 1 and 3. (30 &micro;L) (6 &micro;L) The Intensity of fluorescence of NileRed (liposomes) was measured with fluorescence spectrophotometer (JASCO, FP-6500) to investigate the existence of liposomes in each Fraction.</p>

<br> <table class="sample_04"> <caption>Table 1. Breakdown of Samples</caption> <tbody> <tr> <th>sample No.</th> <td>1</td> <td>2</td> <td>3</td> <td>4</td> </tr> <tr> <th>Cholesterol hybridized Receptor</th> <td>50.0 &micro;L</td> <td>50.0 &micro;L</td> <td>&mdash;</td> <td>&mdash;</td> </tr> <tr> <th>Receptor</th> <td>&mdash;</td> <td>&mdash;</td> <td>50.0 &micro;L</td> <td>50.0 &micro;L</td> </tr> <tr> <th>Liposome</th> <td>50.0 &micro;L</td> <td>&mdash;</td> <td>50.0 &micro;L</td> <td>&mdash;</td> </tr> <tr> <th>150 mM aqueous KCl solution</th> <td>&mdash;</td> <td>50.0 &micro;L</td> <td>&mdash;</td> <td>50.0 &micro;L</td> </tr> <tr> <th>2.25 M Sucrose buffer</th> <td>125.0 &micro;L</td> <td>125.0 &micro;L</td> <td>125.0 &micro;L</td> <td>125.0 &micro;L</td> </tr> </tr> </tbody> </table> <a name="1-2-2">&nbsp;</a> <a name="1-2-3">&nbsp;</a> <br>


<a name="1-2-2">&nbsp;</a> <p class="subtitle">1-2-2. Dimerization mechanism of the Receptor on the liposome</p>

<p class="subtitle">1-2-3. Emission of the initiator in the liposome </p>


<a name="2">&nbsp;</a> <br> <a name="2-1">&nbsp;</a> <br> <a name="2-1-1">&nbsp;</a> <br>

<h1 class="title"><a name="contents">&nbsp;2. Developing the moving system</a></h1>

<p class="title">2-1. The Motor by itself</p>

<p class="subtitle">2-1-1. Design of the Motor-Monomer </p> <p class="headline">&bull; Make the Moter-Monomer

<table class="sample_01"> <caption>Reagents (f. 60.0 &micro;L)</caption> <tbody> <tr> <th>Staple mix</th> <td>17.5 &micro;L</td> </tr> <tr> <th>M 13 mp 18 ss</th> <td>18.3 &micro;L</td> </tr> <tr> <th>TE&sup1;</th> <td>18.2 &micro;L</td> <tr> <th>10 &times; tile buffer&sup2;</th> <td> 6.0 &micro;L</td> </tr> </tr> </tbody> </table>

<p class="mini">&sup1;TE &mdash; 10 mM Tris - HCl (pH 8.0), 1 mM EDTA</p> <p class="mini">&sup2;10 &times; tile buffer &mdash; 100 mM Mg (OAc) <sub>2</sub>, 200 mM Tris-HCl (pH 7.5), .10 mM EDTA</p>

<a name="2-1-2">&nbsp;</a>

<p class="headline">Procedure</p> <p class="line">1) The reagents were mixed.</p> <p class="line">2) The mixture was annealed at 55 °C for 3 hours.</p> <p class="line">3) The mixture was analyzed by 1 % agarose gel electrophoresis.</p> <p class="line">4) The gel was stained by EtBr for 30 minutes.</p> <p class="line">5) A photo of the gel was taken by LAS - 4000.</p> <br> <br>

<p class="subtitle">2-1-2. Formation of the simple Polymer</p> <table class="sample_01"> <caption>Reagents (f. 20.0 &micro;L)</caption> <tbody> <tr> <th>Purified Monomer X</th> <td>10.0 &micro;L</td> </tr> <tr> <th>Purified Monomer Y</th> <td>10.0 &micro;L</td> </tr> </tbody> </table>

<a name="2-1-3">&nbsp;</a>

<p class="headline">Procedure</p> <p class="line">1) Monomers were made as shown in “Making DNA origami monomers”.</p> <p class="line">2) Each Monomer solution was purified by spin column.</p> <p class="line">3) The reagents were mixed.</p> <p class="line">4) The mixture was incubated at room temperature for 24 hours.</p> <p class="line">5) The mixture was analyzed by 1 % agarose gel electrophoresis (100 V, 80 min).</p> <p class="line">6) The gel was stained by EtBr for 30 minutes.</p> <p class="line">7) The gel was taken a photo by LAS - 4000.</p> <br> <br>


<a name="2-2">&nbsp;</a> <p class="subtitle">2-1-3. Controlling the ring - opening polymerization </p> <a name="2-2-1">&nbsp;</a> <br><br>

<p class="title">2-2. The Motor in the liposome </p>

<p class="subtitle">2-2-1. Putting the Motor - Monomers into the liposome</p> <table class="sample_01"> <caption>Reagents (f.1420 &micro;L)</caption> <tbody> <tr> <th>Lipid mix &sup1;</th> <td>400.0 &micro;L</td> </tr> <tr> <th>Glucose</th> <td>500.0 &micro;L</td> </tr> <tr> <th>Emulsion mix&sup2;</th> <td>520.0 &micro;L</td> </tr> </tbody> </table>

<p class="mini">&sup1;3 mM Lipid mix &mdash; POPC, Paraffin</p> <p class="mini">&sup2;Emulsion mix &mdash; 0.5 mM Lipid mix, 50 mM HEPES, 10 mM EDTA,<br>400 mM Sucrose,Pyranine, The Motor-Monomers</p>

<a name="2-2-2">&nbsp;</a> <p class="headline">Procedure</p> <p class="line">1) Glucose (500 &micro;L) was taken into a tube as outer solution.</p> <p class="line">2) Lipid mix (0.5 mM) was added on the glucose solution carefully.</p> <p class="line">3) Emulsion mix was added on the top and rapidly centrifuged at 4 °C, 1800 rpm for 10 minutes.</p> <p class="line">4) Centrifuged again at 4 °C, 5000 rpm for 10 minutes.</p> <p class="line">5) The upper layer was removed and GUVs were collected by micropipette.</p> <p class="line">6) The GUVs were stained by Nile red and observed with a confocal microscope.</p> <br> <br>


<p class="subtitle">2-2-2. Formation of the simple Polymer in the liposome </p>

<p class="subtitle">2-2-3. Controlling the ring opening polymerization in the liposome </p>


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