Difference between revisions of "Biomod/2013/Sendai/protocol"

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We designed DNA origami by <A Href="http://cadnano.org/">caDNAno2</A>, software for designing 2D and 3D DNA origami.<br>
 
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>
 
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 DNAs complementary to the unique sequence are expected to easily hybridize with the unique sequence.<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 DNAs complementary to the unique sequences (such as aptamers) 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 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 place at the same intervals in the DNA origami.<br>
 
The 141 staples have the same length so that they may place at the same intervals in the DNA origami.<br>
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<h5>The list of strands</h5>
 
<h5>The list of strands</h5>
 
The other strands exept DNA origami staples used in our experiment are shown in Table1.<br>
 
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 ccDNA) 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>
+
The sequence of cholesterol-conjugated DNA (in the rest of this document, referred to as aptamer) 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 cc DNA and fluorescent tagged DNA with the same unique single-stranded parts of our origami, we arranged two kinds of adaptor DNAs (at the third and fourth in Table1). One adaptor has complementary sequences to both the unique sequence and cc 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>  
+
To hybridize different strands of aptamer and fluorescent tagged DNA with the same unique single-stranded parts of our origami, we arranged two kinds of adaptor DNAs (at the third and fourth in Table1). One adaptor has complementary sequences to both the unique sequence and aptamer. 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>
 
<br>
 
<table border cellspacing="3" bgcolor="lightyellow">
 
<table border cellspacing="3" bgcolor="lightyellow">
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</tr>
 
</tr>
 
<tr bgcolor="moccasin">
 
<tr bgcolor="moccasin">
<td> Cholesterol-conjugated DNA (ccDNA)</td>
+
<td> Cholesterol-conjugated DNA (aptamer)</td>
 
<td> CCAGAAGACG
 
<td> CCAGAAGACG
 
</td>
 
</td>
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</tr>
 
</tr>
 
<tr bgcolor="moccasin">
 
<tr bgcolor="moccasin">
<td> Adaptor strand for cc DNA and the unique sequence in DNA origami </td>
+
<td> Adaptor strand for aptamer and the unique sequence in DNA origami </td>
 
<td> CGTCTTCTGGCTCTCGATGCGACAG </td>
 
<td> CGTCTTCTGGCTCTCGATGCGACAG </td>
 
</tr>
 
</tr>
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<br>
 
<br>
 
<h4>2-2) Investigating the interaction of DNA origami and liposomes</h4>
 
<h4>2-2) Investigating the interaction of DNA origami and liposomes</h4>
To float cc DNAs on the surface of liposome, we added cc DNAs into liposomes at the final concentration of 0.018, 0.069, 1.8, and 6.9µM. Each sample was as follows.<br>
+
To float aptamers on the surface of liposome, we added aptamers 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 cc DNAs: 1µl 0.1µM cc DNAs and 2.5µl liposome</li>
+
<ur><li>Liposome with 0.018µM aptamers: 1µl 0.1µM aptamers and 2.5µl liposome</li>
<li>Liposome with 0.069µM cc DNAs: 10µl 0.1µM DNAs and 2.5µl liposome</li>
+
<li>Liposome with 0.069µM aptamers: 10µl 0.1µM DNAs and 2.5µl liposome</li>
<li>Liposome with 1.8µM cc DNAs: 1µl 10µM DNAs and 2.5µl liposome</li>
+
<li>Liposome with 1.8µM aptamers: 1µl 10µM DNAs and 2.5µl liposome</li>
<li>Liposome with 6.9µM cc DNAs: 10µl 10µM DNAs and 2.5µl liposome</li>
+
<li>Liposome with 6.9µM aptamers: 10µl 10µM DNAs and 2.5µl liposome</li>
 
<br>
 
<br>
 
We observed each sample with a phase microscope.<br>
 
We observed each sample with a phase microscope.<br>
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For the sake of observation convenience, we mixed 1µl 1µM TR-DHPE (red fluorescent dye) with 1µl lipid (10mM DOPC) and 98µl solvate (CHCl3) in a microtube, and desiccate it with Argon gas. Then we left it for one night in a vacuum dryer. After drying, we added 100µl 1xTAE Mg2+ into the sample and heat it in warm water (about 90 degrees) for a few hours.<br>
 
For the sake of observation convenience, we mixed 1µl 1µM TR-DHPE (red fluorescent dye) with 1µl lipid (10mM DOPC) and 98µl solvate (CHCl3) in a microtube, and desiccate it with Argon gas. Then we left it for one night in a vacuum dryer. After drying, we added 100µl 1xTAE Mg2+ into the sample and heat it in warm water (about 90 degrees) for a few hours.<br>
 
<br>
 
<br>
After liposome was made, we added 1µl 10µM cc DNA into 2.5µl liposome (the final concentration of cc DNA was 1.8 µl). We counted the number of liposomes with a fluorescent microscope. <br>
+
After liposome was made, we added 1µl 10µM aptamer into 2.5µl liposome (the final concentration of aptamer was 1.8 µl). We counted the number of liposomes with a fluorescent microscope. <br>
 
After counting, we added 2µl DNA origami and counted the number of liposomes again. For control, we changed 2µl DNA origami into 2µl 1xTAE Mg2+ buffer. <br>
 
After counting, we added 2µl DNA origami and counted the number of liposomes again. For control, we changed 2µl DNA origami into 2µl 1xTAE Mg2+ buffer. <br>
 
<br>
 
<br>

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<h2>Protocol</h2>

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     <article data-title="Egg type molecular robbot">

<h3 id="experimentsubproject1">内側からアルギン酸膜を破壊するサブプロジェクト</h3></br>

<h4>1-1リポソームの作製とそれをバッファーに入れてアルギン酸ゲルビーズ内に入れる実験</h4></br>

グルコース100μℓにoil70μℓを加えてouterバッファーを作製した。次に、oil40μℓにBSG(?)(蛍光物質)を1μℓ加えて、ピペッティングとタッピングで白く濁るまで混ぜて、innerバッファーを作製した。そのinnerバッファーをouterバッファーの上に注いで70秒遠心にかけて、リポソームを作製した。</br> 遠心した後、チューブの底にたまったベシクルを取り出し、1.5% アルギン酸ナトリウムに加え、それをキャピラリー中に入れ、400mM 塩化カルシウム 140μL中に滴下してFig1のような装置で2~3分遠心した。</br>

<img src="http://openwetware.org/images/9/9f/Ensin.png"></br>

    Fig1 アルギン酸ゲルビーズの作製</br></br>


<h4>1-2内部にバッファーの入ったアルギン酸膜の作製</h4></br>


Fig3のように外側の太いキャピラリーと内側の細いキャピラリーを作成する。 キャピラリーは外径1mmのものを熱加工した。</br> </br> 外管には1.5%アルギン酸ナトリウム溶液を、内管には蛍光物質+mQをいれた</br>

2重キャピラリーを用いて2~3分遠心にかけ0.4M塩化カルシウム水溶液に滴下する。</br>

<img src="http://openwetware.org/images/1/1d/Image_%E4%BB%AE.png"></br>

    Fig3 二重ノズルの構造</br></br>


<h4>2ニッパムの効果でリポソームが割れることの確認実験</h4></br>


ニッパム分子は32度以下では水和していて親水性だが、32度以上では収縮して疎水性になる。ニッパムがリポソームに修飾されると、32度以下ではニッパム分子の水和により安定な状態になるが、32度以上ではニッパム分子が疎水性になって不安定な状態になるので、32度以上になった時にリポソームが割れることになる。</br>

参考</br> http://www.sigmaaldrich.com/etc/medialib/docs/SAJ/Brochure/1/j_recipedds2.Par.0001.File.tmp/j_recipedds2.pdf</br></br>


まず、PNIPAMに脂質を修飾する。まず、PNIPAMにCHCl3とAr存在下でジシクロヘキシルカーボジイミド(DCC)と、N-ヒドロキシ-シアナミド(NHS)を反応させる。(下図の1ができる)次に、CHCl3とAr存在下でジミリストイルホスファチジルエタノールアミン(dimyristoylphosphatidylethanolamine、DMPE)を反応させる。(下図の2ができる)このようにしてPNIPAMに脂質を修飾して、GUVによりニッパム修飾されたリポソームを作製した。</br></br>

<img src="http://openwetware.org/images/e/e8/NIPAMgousei.png"></br>

    Fig4 PNIPAMへの脂質の修飾</br></br>


※W/Oエマルション法を用いたGUV(Giant Unilamellar Vesicle)によるリポソームの作製</br></br>

10mM DOPCのストック溶液をArガスで乾燥させた脂質フィルムをミクロチューブ内に作り真空デシケータ内でさらに乾燥させた。リン脂質フィルムに流動パラフィン 500μLを加える。超音波洗浄機を用いて60℃で60分間リン脂質をオイルに溶かした。インナー溶液を150mM スクロース、350mM グルコース、100mM EGTAとする。オイルに溶かしたリン脂質にインナー溶液 50μLを加え、遠心分離し、エマルション溶液を作製した。アウター溶液を600mM グルコースとする。アウター溶液 100μLにエマルション溶液 70μLを加える。</br></br>


参考</br> Thermoresponsive Nanostructures by Self-Assembly of a Poly(N-isopropylacrylamide)−Lipid Conjugate Daniel N. T. Hay ,† Paul G. Rickert ,‡ Sönke Seifert ,§ and Millicent A. Firestone *† J. Am. Chem. Soc., 2004, 126 (8), pp 2290–229 Publication Date (Web): February 3, 2004</br></br>


<h4>3アルギン酸ゲルビーズを溶かすのに必要なEGTAの濃度と時間の測定</h4></br>

<h4>4尿素アニーリングによるDNAオリガミの作製とその作製にかかる時間の測定</h4></br>


M13pm18(7249nt)と226個のステプルを100μℓの12.5mMの酢酸マグネシウム入りの10×TAEの中に入れ、95℃から20℃に1℃/分でアニーリングしたものとM13pm18と226個のステイプルを300μℓの12.5mMの酢酸マグネシウムと85%のホルムアルデヒド入りの10×TAEの中に入れ、透析装置で0.2mℓ/分の割合で次第にホルムアルデヒドの濃度を薄くしていったものとをAFMや電気泳動で調べた。</br>


<h4>5温度を上げればアルギン酸膜が破壊されることの確認</h4></br>


<h4>6アルギン酸膜内で尿素アニーリングができていることの確認</h4></br>


<h4>7全体のシステムの機能確認</h4></br>


     </article>

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     <article data-title="Chain Reaction">

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<h3>Chain-reactive burst</h3> <h4>i)Bending approach</h4> <h4>1)Making DNA origami</h4> <h5>DNA origami recipe</h5> 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 DNAs complementary to the unique sequences (such as aptamers) 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 place 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 DNAs.<br> This design enables each DNA origami to exist individually.<br> <br> <h5>The list of strands</h5> 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 aptamer) 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 aptamer and fluorescent tagged DNA with the same unique single-stranded parts of our origami, we arranged two kinds of adaptor DNAs (at the third and fourth in Table1). One adaptor has complementary sequences to both the unique sequence and aptamer. 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 DNA strands </td> <td> Its sequence </td> </tr> <tr bgcolor="moccasin"> <td> Cholesterol-conjugated DNA (aptamer)</td> <td> CCAGAAGACG </td> </tr> <tr bgcolor="moccasin"> <td> Fluorescent tagged DNA </td> <td> ACTAGTGAGTGCAGCAGTCGTACCA </td> </tr> <tr bgcolor="moccasin"> <td> Adaptor strand for aptamer 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> <h5>Annealing</h5> 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 DNAs 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 DNAs<br> <br> <li>Annealing solution with no fluorescent tagged DNAs (control) 50µl<br> We changed 3µl fluorescent tagged DNAs in the above solution into the same quantity of mQ.</li><br> <br> <h4>1-1)AFM observation</h4> As we thought excess staples produced more aggregation and made AFM observation difficult, control annealing solution was used for AFM observation.<br> <br> <h4>1-2)Labeling DNA origami</h4> We confirmed that our DNA origami was fluorescently labeled by electrophoresis.<br> <br> 50µl of Annealing solution with fluorescent tagged DNAs (used in 1-1)Making DNA origami) contains 3µl of 1µM fluorescent tagged DNAs. <br> To see if the origami binds to the fluorescent tagged DNA in shorter time, we added 0.6µl of 1µM fluorescent tagged DNAs 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> <h4>2)Destroying liposomes</h4> <h4>2-1) Making liposomes</h4> We made liposome that was to be broken by DNA origami.<br> First we mixed 1µl lipid (10mM DOPC) and 99µl solvent (CHCl3) in a microtube, and desiccate it with Argon gas. Then we left it for one night in a vacuum dryer. After drying, we added 100µl of the same buffer as that of DNA origami (1xTAE Mg2+) into the sample and heat it in warm water (about 90 degrees) for a few hours.<br> <br> <h4>2-2) Investigating the interaction of DNA origami and liposomes</h4> To float aptamers on the surface of liposome, we added aptamers 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 aptamers: 1µl 0.1µM aptamers and 2.5µl liposome</li> <li>Liposome with 0.069µM aptamers: 10µl 0.1µM DNAs and 2.5µl liposome</li> <li>Liposome with 1.8µM aptamers: 1µl 10µM DNAs and 2.5µl liposome</li> <li>Liposome with 6.9µM aptamers: 10µl 10µM DNAs and 2.5µl liposome</li> <br> 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> <h4>2-3)Counting liposomes</h4> For the sake of observation convenience, we mixed 1µl 1µM TR-DHPE (red fluorescent dye) with 1µl lipid (10mM DOPC) and 98µl solvate (CHCl3) in a microtube, and desiccate it with Argon gas. Then we left it for one night in a vacuum dryer. After drying, we added 100µl 1xTAE Mg2+ into the sample and heat it in warm water (about 90 degrees) for a few hours.<br> <br> After liposome was made, we added 1µl 10µM aptamer into 2.5µl liposome (the final concentration of aptamer was 1.8 µl). We counted the number of liposomes with a fluorescent microscope. <br> After counting, we added 2µl DNA origami and counted the number of liposomes again. For control, we changed 2µl DNA origami into 2µl 1xTAE Mg2+ buffer. <br> <br> <br> <h4>ii)Flower micelle approach</h4> <h4>2)Confirming the hybridization of trigger and loop DNA</h4> We checked whether trigger DNA hybridizes with loop DNA at normal temperature by electrophoresis.<br> <br> <table border cellspacing="3" bgcolor="lightyellow"> <tr bgcolor="moccasin"> <td>30% Acryl amide</td> <td>3.3ml</td> </tr> <tr bgcolor="moccasin"> <td>mQ</td> <td>5.59ml</td> </tr> <tr bgcolor="moccasin"> <td>10xTAE</td> <td>1ml</td> </tr> <tr bgcolor="moccasin"> <td>TEMED</td> <td>10µl</td> </tr> <tr bgcolor="moccasin"> <td>10xAPS</td> <td>100µl</td> </tr> </table> Table.4 10% Acrylic amide gel recipe<br>

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