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            <a href="http://openwetware.org/wiki/Biomod/2013/Sendai"><h1 style="color:white;" ><b>Biomod<span>2013<br>&emsp; Team</span>Sendai</b></h1></a>

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       <article>

<h2>Protocol</h2>

<table id="toc" class="toc" summary="Contents"><tr><td><div id="toctitle"><h2>Contents</h2></div> <ul> <li class="toclevel-1"><a href="#chain"> <span class="tocnumber">1</span> <span class="toctext">Step1 Disruption of temperature sensitive liposomes</span></a></li> <ul> <li class="toclevel-2"><a href="#bending"> <span class="tocnumber">1-1</span> <span class="toctext">Disruption of temperature sensitive liposomes</span></a></li> </ul> <li class="toclevel-1"><a href="#Flower"> <span class="tocnumber">2</span> <span class="toctext">Step2 Liposome disruption induced by attachment of key DNA with anchor DNA</span></a></li> <ul> <li class="toclevel-2"><a href="#sensing"> <span class="tocnumber">2-1</span> <span class="toctext">DNA Origami approach</span></a></li> <ul> <li class="toclevel-2"><a href="#5"> <span class="tocnumber">2-1-1</span> <span class="toctext">Making DNA Origami</span></a></li> <li class="toclevel-2"><a href="#6"> <span class="tocnumber">2-1-2</span> <span class="toctext">Labeling DNA Origami with fluorescent-tagged DNA</span></a></li>

<li class="toclevel-2"><a href="#7"> <span class="tocnumber">2-1-3</span> <span class="toctext">Disruption of liposomes by DNA Origami</span></a></li> <li class="toclevel-2"><a href="#8"> <span class="tocnumber">2-1-4</span> <span class="toctext">Confirming sequence specificity of DNA</span></a></li> </ul> <li class="toclevel-1"><a href="#9"> <span class="tocnumber">2-2</span> <span class="toctext">Flower DNA approach</span></a></li> <ul> <li class="toclevel-2"><a href="#10"> <span class="tocnumber">2-2-1</span> <span class="toctext">Confirming the formation of the loop structure by SPR</span></a></li> <li class="toclevel-2"><a href="#11"> <span class="tocnumber">2-2-2</span> <span class="toctext">Disruption of liposomes by Flower DNA</span></a></li> <li class="toclevel-2"><a href="#12"> <span class="tocnumber">2-2-3</span> <span class="toctext">Confirming sequence specificity of DNA</span></a></li>

</li>

</ul> </li> </ul> </td></tr></table>

<h3 id=chain>1 Step1 Disruption of temperature sensitive liposomes</h3> <h4 id=bending>1-1 Disruption of temperature sensitive liposomes</h4> <h5> Structure of NIPAM</h5> <h5> Making liposome</h5> <table border cellspacing="3" bgcolor="lightyellow"> <tr bgcolor="moccasin"> <td> Egg York PC(10mM)</td> <td> 10µl </td> </tr> <tr bgcolor="moccasin"> <td> Cholesterol(10mM)</td> <td> 1µl</td> </tr> <tr bgcolor="moccasin"> <td> CHCl<sub>3</sub></td> <td> 90µl</td> </tr> <tr bgcolor="moccasin"> <td> TXR</td> <td> 0.01µl </td> </tr> </table> Table.1 Making liposomes<br><br>

1. Drying the liposomes above with argon gas and letting them stand for a night<br> 2. Adding L paraffin 100µl to 1 and sonicating them for an hour<br> 3. Picking up 10µl from 2, adding 25μl NIPAM2mg/ml to them and vibrating them with Vortex<br> <h3 id=Flower>2 Step2 Liposome disruption induced by attachment of key DNA with anchor DNA</h5> <h4 id=sensing>2-1 DNA Origami approach</h4> <h5 id=5>2-1-1 Making DNA Origami</h5> <h5>Making DNA origami</h5> <h6>DNA origami recipe</h6> We designed DNA origami by <A Href="http://cadnano.org/">caDNAno2</A>, software for designing 2D and 3D DNA origami.<br> Our DNA origami has 141 staples that have 30nt free single-stranded parts outside the DNA origami. The sequence of the parts is <i>“<font color="#00a0c0">each DNA origami staple</font>-TTTTTTTTTTTTTTT<font color="red">CTGTCGCATCGAGAG</font>”</i>.<br> Between the staple and unique (<i><font color="red">CTGTCGCATCGAGAG</font></i>) sequences, 15 T bases are inserted. They are to make a T loop. Thanks to this T loop, single-stranded DNA complementary to the unique sequences (such as Anchored DNA) are expected to easily hybridize with the unique sequence.<br> The 30nt single-stranded parts are stable till 37 degrees, according to <A Href="http://www.nupack.org/">NUPACK</A>).<br> The 141 staples have the same length so that they may be present at the same intervals in the DNA origami.<br> Each side of our origami is not fully covered with staples, and single-stranded M13 remains. This is for preventing π-π interaction and stacking by hydrophobic interaction between base pairs of double-stranded DNA.<br> This design enables each DNA origami to exist individually.<br> <br> <h6>The list of strands</h6> The other strands exept DNA origami staples used in our experiment are shown in Table1.<br> The sequence of cholesterol-conjugated DNA (in the rest of this document, referred to as Anchored DNA) is shown below (at the first sequence in Table1). For labeling, we also attached fluorescent tagged DNA (at the second in Table1) to our DNA origami.<br> To hybridize different strands of Anchored DNA and fluorescent tagged DNA with the same unique single-stranded parts of our origami, we arranged two kinds of adaptor DNA (at the third and fourth in Table1). One adaptor has complementary sequences to both the unique sequence and Anchored DNA. The other has complementary sequences to both the unique sequence and the fluorescent tagged DNA. Thanks to these two adaptors, two different strands can bind to the same unique sequence. <br> <br> <table border cellspacing="3" bgcolor="lightyellow"> <tr bgcolor="lightyellow"> <td> The kinds of DNAtrands </td> <td> Its sequence </td> </tr> <tr bgcolor="moccasin"> <td> Cholesterol-conjugated DNA (Anchored DNA)</td> <td> CCAGAAGACG </td> </tr> <tr bgcolor="moccasin"> <td> Fluorescent tagged DNA </td> <td> ACTAGTGAGTGCAGCAGTCGTACCA </td> </tr> <tr bgcolor="moccasin"> <td> Adaptor strand for Anchored DNA and the unique sequence in DNA origami </td> <td> CGTCTTCTGGCTCTCGATGCGACAG </td> </tr> <tr bgcolor="moccasin"> <td> Adaptor strand for fluorescent tagged DNA and the unique sequence in DNA origami </td> <td> TGGTACGACTGCTGCACTCACTAGTCTCTCGATGCGACAG </td> </tr> </table> Table.1 The sequence of the strands used in our experiment<br> <br> <h6>Annealing of DNA origami</h6> The annealing solution is shown in Table2. The annealing was conducted for 2 hours and 51minutes (from 95 to 25 degrees: lower 1 degree per 2 minutes).<br> <br> <ur><li>Annealing solution with fluorescent tagged DNA 50µl<br> <table border cellspacing="3" bgcolor="lightyellow"> <tr bgcolor="moccasin"> <td>84nM M13mp18</td> <td>2.38µl</td> </tr> <tr bgcolor="moccasin"> <td>Staples</td> <td></td> </tr> <tr> <td>1µM migihaji</td> <td>1µl</td> </tr> <tr> <td>1µM hidarihaji</td> <td>1µl</td> </tr> <tr> <td>1µM ashibatemae</td> <td>1µl</td> </tr> <tr> <td>200nM ashiba</td> <td>5µl</td> </tr> <tr bgcolor="moccasin"> <td>1µM cholesterol-hybridizing ssDNA</td> <td>3µl</td> </tr> <tr bgcolor="moccasin"> <td>1µM fluorescent-tagged DNA-hybridizing ssDNA</td> <td>3µl</td> </tr> <tr bgcolor="moccasin"> <td>5xTAE Mg2+</td> <td>10µl</td> </tr> <tr bgcolor="moccasin"> <td>mQ</td> <td>20.62µl</td> </tr> <tr bgcolor="moccasin"> <td>1µM fluorescent-tagged DNA</td> <td>3µM</td> </tr> </table> </li> Table.2 Annealing solution with fluorescent tagged DNA<br> <br> <li>Annealing solution with no fluorescent tagged DNA (control) 50µl<br> We changed 3µl fluorescent tagged DNA in the above solution into the same quantity of mQ.</li><br> <br> <h5>AFM observation</h5> As we thought excess staples produced more aggregation and made AFM observation difficult, control annealing solution was used for AFM observation.<br> <br> <h5 id=6>2-1-2 Labeling DNA Origami with fluorescent-tagged DNA</h5> <h5>Electrophoresis </h5> We confirmed that our DNA origami was fluorescently labeled by electrophoresis.<br> <br> 50µl of Annealing solution with fluorescent tagged DNA (used in 1-1)Making DNA origami) contains 3µl of 1µM fluorescent tagged DNA. <br> To see if the origami binds to the fluorescent tagged DNA in shorter time, we added 0.6µl of 1µM fluorescent tagged DNA into 10 µl control annealing solution, and left it for 40 minutes.<br> <br> Agarose gel recipe: 0.4g agarose, 0.8ml 50xTAE, 39.2ml mQ<br> <br> The electrophoresis was conducted with 1% agarose gel, CV 100V, for 50 minutes.<br> <br> <h5 id=7>2-1-3 Disruption of liposomes by DNA Origami</h5> <h5>Concentration of Anchored DNA</h5> To float Anchored DNA on the surface of liposome, we added Anchored DNA into liposomes at the final concentration of 0.018, 0.069, 1.8, and 6.9µM. Each sample was as follows.<br> <ur><li>Liposome with 0.018µM Anchored DNA: 1µl 0.1µM Anchored DNA and 2.5µl liposome</li> <li>Liposome with 0.069µM Anchored DNA: 10µl 0.1µM DNAs and 2.5µl liposome</li> <li>Liposome with 1.8µM Anchored DNA: 1µl 10µM DNAs and 2.5µl liposome</li> <li>Liposome with 6.9µM Anchored DNA: 10µl 10µM DNAs and 2.5µl liposome</li> <br> <h5>Observation by phase and fluorescent microscope </h5> We observed each sample with a phase microscope.<br> <br> Then we added 2µl DNA origami into each sample and saw if some change would happen with a fluorescent microscope.<br> The DNA origami for fluorescent microscope observation was made according to Table3 annealing solution. It contained more cholesterol-hybridizing ssDNAs and fluorescent-tagged DNA-hybridizing ssDNAs than Annealing solution used in 1-1), because we considered a sample with more fluorescent molecules was suitable for observation. <br> <br>

<table border cellspacing="3" bgcolor="lightyellow"> <tr bgcolor="moccasin"> <td>84nM M13mp18</td> <td>2.38µl</td> </tr> <tr bgcolor="moccasin"> <td>Staples</td> <td></td> </tr> <tr> <td>1µM migihaji</td> <td>1µl</td> </tr> <tr> <td>1µM hidarihaji</td> <td>1µl</td> </tr> <tr> <td>1µM ashibatemae</td> <td>1µl</td> </tr> <tr> <td>200nM ashiba</td> <td>5µl</td> </tr> <tr bgcolor="moccasin"> <td>100µM cholesterol-hybridizing ssDNA</td> <td>4.23µl</td> </tr> <tr bgcolor="moccasin"> <td>100µM fluorescent-tagged DNA-hybridizing ssDNA</td> <td>4.23µl</td> </tr> <tr bgcolor="moccasin"> <td>5xTAE Mg2+</td> <td>10µl</td> </tr> <tr bgcolor="moccasin"> <td>mQ</td> <td>23.54µl</td> </tr> </table>

Table.3 50µl Annealing solution for fluorescent microscope observation<br> <br> After annealing, we added 4.23µl 100µM fluorescent-tagged DNA (the same quantity of fluorescent-tagged DNA-hybridizing ssDNA).<br> <br>

<h5 id=8>2-1-4 Confirming sequence specificity of DNA</h5> <h5>Making liposome</h5> We made liposomes in a spontaneous-transfer way. They were divided into two types: liposomes A of GFP, Green Fluorescent Protein, and liposomes B of Red Fluorescent Protein. These two kinds of liposomes have the same Outer Buffer but different Inner Buffer. Composition of these two buffers is as follows.<br><br>

<table border cellspacing="3" bgcolor="lightyellow"> <tr bgcolor="moccasin"> <td>Outer Buffer </td> <td>STE（as substitute for GFP）</td> <td>10µl</td> </tr> <tr bgcolor="moccasin"> <td></td> <td>glucose(1M)</td> <td>250µl </td> </tr> <tr bgcolor="moccasin"> <td></td> <td>25×TAE</td> <td>20µl </td> </tr> <tr bgcolor="moccasin"> <td></td> <td>25×TAE</td> <td>20µl </td> </tr>

<tr bgcolor="SpringGreen"> <td>LiposomeA Inner Buffer</td> <td>GFP</td> <td>5µl</td> </tr> <tr bgcolor="SpringGreen"> <td> </td> <td>sucrose(1M)</td> <td>125µl</td> </tr> <tr bgcolor="SpringGreen"> <td> </td> <td>25×TAE Mg<sup>2+</sup></td> <td>10µl</td> </tr> <tr bgcolor="SpringGreen"> <td> </td> <td>mQ</td> <td>110µl</td> </tr>

<tr bgcolor="#FF6699 "> <td>LiposomeB Inner Buffer</td> <td>Rhodamine</td> <td>0.5µl</td> </tr> <tr bgcolor="#FF6699"> <td> </td> <td>sucrose(1M)</td> <td>12.5µl</td> </tr> <tr bgcolor=" #FF6699"> <td> </td> <td>25×TAE Mg<sup>2+</sup></td> <td>10µl</td> </tr> <tr bgcolor=" #FF6699"> <td> </td> <td>mQ</td> <td>110µl</td> </tr> </table>

1. Tapping of inner 2 and lipid paraffin 50<br> 2. Putting paraffin 50 on outer 50<br> 3. Putting 1 on 2<br> 4. Centrifuging 3 for 5 minutes<br> 5. Observing leak of liposomes from the bottom of tubes by needles<br>

<h4 id=9>2-2 Flower DNA approach</h4> <h5 id=10> 2-2-1 Confirming the formation of the loop structure by SPR</h5> <h5> Making liposome</h5> <h5> </h5>

<h5 id=11>2-2-2 Disruption of liposomes by Flower DNA</h5> <h5> Making liposome</h5>

<h5 id=12>2-2-3 Confirming sequence specificity of DNA</h5> <h5>Making liposome</h5>

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Biomod/2013/Sendai/protocol: Difference between revisions

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@@ Line 53: / Line 53: @@
 <ul>
 <li class="toclevel-1"><a href="#chain">
-<span class="tocnumber">1</span> <span class="toctext">Step1 温度感受性リポソームの破壊</span></a></li>
+<span class="tocnumber">1</span> <span class="toctext">Step1 Disruption of temperature sensitive liposomes</span></a></li>
 <ul>
 <li class="toclevel-2"><a href="#bending">
-<span class="tocnumber">1-1</span> <span class="toctext">温度感受性リポソームを破壊する実験</span></a></li>
+<span class="tocnumber">1-1</span> <span class="toctext">Disruption of temperature sensitive liposomes</span></a></li>
 </ul>
 <li class="toclevel-1"><a href="#Flower">
-<span class="tocnumber">2</span> <span class="toctext">Step2 DNAによる連鎖的リポソームの破壊</span></a></li>
+<span class="tocnumber">2</span> <span class="toctext">Step2 Liposome disruption induced by attachment of key DNA with anchor DNA</span></a></li>
 <ul>
 <li class="toclevel-2"><a href="#sensing">
-<span class="tocnumber">2-1</span> <span class="toctext">DNAオリガミによるアプローチ</span></a></li>
+<span class="tocnumber">2-1</span> <span class="toctext">DNA Origami approach</span></a></li>
 <ul>
 <li class="toclevel-2"><a href="#5">
-<span class="tocnumber">2-1-1</span> <span class="toctext">デザインしたDNAオリガミの作製</span></a></li>
+<span class="tocnumber">2-1-1</span> <span class="toctext">Making DNA Origami</span></a></li>
 <li class="toclevel-2"><a href="#6">
-<span class="tocnumber">2-1-2</span> <span class="toctext">DNAオリガミに蛍光付きDNAがハイブリしていることの確認実験</span></a></li>
+<span class="tocnumber">2-1-2</span> <span class="toctext">Labeling DNA Origami with fluorescent-tagged DNA</span></a></li>
 <li class="toclevel-2"><a href="#7">
-<span class="tocnumber">2-1-3</span> <span class="toctext">DNAオリガミによりリポソームを破壊する実験</span></a></li>
+<span class="tocnumber">2-1-3</span> <span class="toctext">Disruption of liposomes by DNA Origami</span></a></li>
 <li class="toclevel-2"><a href="#8">
-<span class="tocnumber">2-1-4</span> <span class="toctext">DNAによる配列特異性を証明する実験</span></a></li>
+<span class="tocnumber">2-1-4</span> <span class="toctext">Confirming sequence specificity of DNA</span></a></li>
 </ul>
 <li class="toclevel-1"><a href="#9">
-<span class="tocnumber">2-2</span> <span class="toctext">フラワーミセルによるアプローチ</span></a></li>
+<span class="tocnumber">2-2</span> <span class="toctext">Flower DNA approach</span></a></li>
 <ul>
 <li class="toclevel-2"><a href="#10">
-<span class="tocnumber">2-2-1</span> <span class="toctext">SPRによるループ構造の確認</span></a></li>
+<span class="tocnumber">2-2-1</span> <span class="toctext">Confirming the formation of the loop structure by SPR</span></a></li>
 <li class="toclevel-2"><a href="#11">
-<span class="tocnumber">2-2-2</span> <span class="toctext">フラワーミセルによりリポソームを破壊する実験</span></a></li>
+<span class="tocnumber">2-2-2</span> <span class="toctext">Disruption of liposomes by Flower DNA</span></a></li>
 <li class="toclevel-2"><a href="#12">
-<span class="tocnumber">2-2-3</span> <span class="toctext">DNAによる配列特異性を証明する実験</span></a></li>
+<span class="tocnumber">2-2-3</span> <span class="toctext">Confirming sequence specificity of DNA</span></a></li>
@@ Line 93: / Line 93: @@
 </td></tr></table>
-<h3 id=chain>1 Step1 温度感受性リポソームの破壊</h3>
+<h3 id=chain>1 Step1 Disruption of temperature sensitive liposomes</h3>
-<h4 id=bending>1-1温度感受性リポソームを破壊する実験</h4>
+<h4 id=bending>1-1 Disruption of temperature sensitive liposomes</h4>
 <h5> Structure of NIPAM</h5>
 <h5> Making liposome</h5>
@@ Line 121: / Line 121: @@
 .	Adding L paraffin 100µl to 1 and sonicating them for an hour<br>
 .	Picking up 10µl from 2, adding 25μl NIPAM2mg/ml to them and vibrating them with Vortex<br>
-<h3 id=Flower>2 Step2 DNAによる連鎖的リポソームの破壊</h5>
+<h3 id=Flower>2 Step2 Liposome disruption induced by attachment of key DNA with anchor DNA</h5>
-<h4 id=sensing>2-1 DNAオリガミによるアプローチ</h4>
+<h4 id=sensing>2-1 DNA Origami approach</h4>
-<h5 id=5>2-1-1 デザインしたDNAオリガミの作製</h5>
+<h5 id=5>2-1-1 Making DNA Origami</h5>
 <h5>Making DNA origami</h5>
 <h6>DNA origami recipe</h6>
@@ Line 223: / Line 223: @@
 As we thought excess staples produced more aggregation and made AFM observation difficult, control annealing solution was used for AFM observation.<br>
 <br>
-<h5 id=6>2-1-2 DNAオリガミに蛍光付きDNAがハイブリしていることの確認実験</h5>
+<h5 id=6>2-1-2 Labeling DNA Origami with fluorescent-tagged DNA</h5>
 <h5>Electrophoresis </h5>
 We confirmed that our DNA origami was fluorescently labeled by electrophoresis.<br>
@@ Line 234: / Line 234: @@
 The electrophoresis was conducted with 1% agarose gel, CV 100V, for 50 minutes.<br>
 <br>
-<h5 id=7>2-1-3 DNAオリガミによりリポソームを破壊する実験</h5>
+<h5 id=7>2-1-3 Disruption of liposomes by DNA Origami</h5>
 <h5>Concentration of Anchored DNA</h5>
 To float Anchored DNA on the surface of liposome, we added Anchored DNA into liposomes at the final concentration of 0.018, 0.069, 1.8, and 6.9µM. Each sample was as follows.<br>
@@ Line 297: / Line 297: @@
 <br>
-<h5 id=8>2-1-4 DNAによる配列特異性を証明する実験</h5>
+<h5 id=8>2-1-4 Confirming sequence specificity of DNA</h5>
 <h5>Making liposome</h5>
 We made liposomes in a spontaneous-transfer way. They were divided into two types: liposomes A of GFP, Green Fluorescent Protein, and liposomes B of Red Fluorescent Protein. These two kinds of liposomes have the same Outer Buffer but different Inner Buffer. Composition of these two buffers is as follows.<br><br>
@@ Line 304: / Line 304: @@
 <tr bgcolor="moccasin">
 <td>Outer Buffer </td>
-<td>STE（GFPの代わり）</td>
+<td>STE（as substitute for GFP）</td>
 <td>10µl</td>
 </tr>
 <tr bgcolor="moccasin">
 <td></td>
-<td>グルコース(1M)</td>
+<td>glucose(1M)</td>
 <td>250µl </td>
 </tr>
@@ Line 330: / Line 330: @@
 <tr bgcolor="SpringGreen">
 <td> </td>
-<td>スクロース(1M)</td>
+<td>sucrose(1M)</td>
 <td>125µl</td>
 </tr>
@@ Line 346: / Line 346: @@
 <tr bgcolor="#FF6699 ">
 <td>LiposomeB Inner Buffer</td>
-<td>ローダミン</td>
+<td>Rhodamine</td>
 <td>0.5µl</td>
 </tr>
 <tr bgcolor="#FF6699">
 <td> </td>
-<td>スクロース(1M)</td>
+<td>sucrose(1M)</td>
 <td>12.5µl</td>
 </tr>
@@ Line 372: / Line 372: @@
 .	Observing leak of liposomes from the bottom of tubes by needles<br>
-<h4 id=9>2-2 フラワーミセルによるアプローチ</h4>
+<h4 id=9>2-2 Flower DNA approach</h4>
-<h5 id=10> 2-2-1 SPRによるループ構造の確認</h5>
+<h5 id=10> 2-2-1 Confirming the formation of the loop structure by SPR</h5>
 <h5> Making liposome</h5>
 <h5> </h5>
-<h5 id=11>2-2-2フラワーミセルによりリポソームを破壊する実験</h5>
+<h5 id=11>2-2-2 Disruption of liposomes by Flower DNA</h5>
 <h5> Making liposome</h5>
-<h5 id=12>2-2-3 DNAによる配列特異性を証明する実験</h5>
+<h5 id=12>2-2-3 Confirming sequence specificity of DNA</h5>
 <h5>Making liposome</h5>