Biomod/2013/Sendai/protocol

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<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">1st stage: Sensing system</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">2nd stage: Amplification system</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(microscopic analysis)</span></a></li> <li class="toclevel-2"><a href="#13"> <span class="tocnumber">2-1-4</span> <span class="toctext">>Disruption of liposomes by DNA Origami(quantitative analysis)</span></a></li>

<li class="toclevel-2"><a href="#8"> <span class="tocnumber">2-1-5</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="#11"> <span class="tocnumber">2-2-1</span> <span class="toctext">Disruption of liposomes by Flower DNA</span></a></li> <li class="toclevel-2"><a href="#12"> <span class="tocnumber">2-2-2</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> <img src="http://openwetware.org/images/f/f5/Nipam.png"width="180"height="210"><br> poly-N-isopropyl acrylamide <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> 1µl </td> </tr> </table> Table.1 Materials 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(microscopic analysis)</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=13>2-1-3 Disruption of liposomes by DNA Origami(quantitative analysis)</h5> マイクロリットルの記述統一<br> 分量は表で書く<br> 数字の箇条書きで<br> 他のプロトコルとかぶっていると思われる箇所あり。参考にしてかく<br> <h5>Making liposome</h5>

Liposomeは界面通過法により作成した。<br> 1.以下の脂質を混合したあとクロロホルム260ulを加える。アルゴンガスで乾燥させた後乾燥機で一晩乾燥させてlipid filmを作る。<br>

DOPC(10mM) 20ul DPPC(10mM) 20ul Cholesterol(10mM) 20ul DOPE(10mM) 20ul<br><br>

2.(以下、箇条書きで)<br>

Inner buffer(250ul) <br> ・GFP(濃度不明、藤原さんに聞いとく) 5ul ・スクロース(1M) 125ul ・25xTAE Mg2+ 10ul ・mQ 110ul<br><br>

Outer buffer(250ul) <br> ・STE(0.3mM)5 ul ・グルコース(1M) 125ul ・25xTAE Mg2+ 10ul ・mQ 110ul<br><br>

Lipid filmにミネラルオイル260ulを加えて超音波(43Hz, 2時間, 60度)にかける。<br> 1.5mlチューブにOuter buffer(下層)50ul、lipid溶液(中間層)50ulの順番に加えて2層にする。<br> 0.2mlチューブにlipid溶液50ulとInner buffer 2ulを入れてタッピングで混ぜ、この溶液52ulを上層になるように1.5mlチューブにそっと加える。<br> 30秒間遠心にかけて、下層のみを取り出す。<br> サンプル1 リポソーム+フラワーアンカーDNA+KeyDNA<br> ・リポソーム(GFP)(4mM) 10ul ・Chol-leg A (10uM) 25ul ・Origami A (5nM) 20ul ・1xTAE Mg2+ 55ul<br><br> サンプル2 リポソーム+フラワーアンカーDNA<br> ・リポソーム(GFP)(4mM) 10ul ・Chol-leg A (10uM) 25ul ・1xTAE Mg2+ 75ul<br><br> サンプル3 リポソーム+フラワーアンカーDNA+界面活性剤(2%NP)<br> ・リポソーム(GFP)(4mM) 10ul ・Chol-leg A (10uM) 25ul ・1xTAE Mg2+ 75ul ・界面活性剤 2%NP 2ul<br><br>

リポソームはChol-legを加えた後30分放置、オリガミを加えた後10分放置した。<br><br>

一回目サンプル1とサンプル2<br> 二回目サンプル2とサンプル3<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=11>2-2-1 Disruption of liposomes by Flower DNA</h5> 2-2 Flower DNA approach

リポソームを3μlでループ100μ 50かけサイバーゴールド トリガー(100μM)4μl<br>

(対応するプロトコルへのリンク)<br>

500µl outer buffer STE 10µl 1M glucose 250µl 1M HEPES 5µl 1M MgCl2 6.25µl mQ 228.8µl

Inner buffer (green) GFP 10µl 1M glucose 250µl 1M HEPES 5µl 1M MgCl2 6.25µl mQ 228.8µl

Inner buffer (red) デキストリンTXR 20µl 1M glucose 250µl 1M HEPES 5µl 1M MgCl2 6.25µl mQ 218.8µl Phase-separated liposome 10mM DOPC 20µl 10mM DPPC 20µl 10mM cholesterol 20µl

1.Tapping of inner 2マイクロリットル and lipid paraffin 50 マイクロリットル<br> 2.Putting 流動 paraffin 50 マイクロリットル on outer 50 マイクロリットル<br> 3.Putting 1(inner + lipid paraffin) on 2(流動paraffin+outer) <br> 4.Centrifuging 3のサンプル for 5minutes<br> 5.Observing leak of liposomes from the bottom of tubes by needles<br>

<h5 id="12">Confirming sequence specificity of DNA</h5>


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