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   <title>Biomod2013 Sendai ver2.0</title>
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Protocol

<a href="#5"> 2-1-1 デザインしたDNAオリガミの作製</a>
<a href="#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</a>
<a href="#7"> 2-1-3 DNAオリガミによりリポソームを破壊する実験</a>
<a href="#8"> 2-1-4 DNAによる配列特異性を証明する実験</a>

<a href="#9"> 2-2 フラワーミセルによるアプローチ</a>

<a href="#10"> 2-2-1 SPRによるループ構造の確認</a>
<a href="#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</a>
<a href="#12"> 2-2-3 DNAによる配列特異性を証明する実験</a>

1 Step1 温度感受性リポソームの破壊

1-1温度感受性リポソームを破壊する実験

Structure of NIPAM

Making liposome

Lipidの組成は以下の通りである

Egg York PC(10mM)	10µl
Cholesterol(10mM)	1µl
CHCl₃	90µl
TXR	0.01µl

Table.1 Making liposomes

1. Drying the liposomes above with argon gas and letting them stand for a night
2. Adding L paraffin 100µl to 1 and sonicating them for an hour
3. Picking up 10µl from 2, adding NIPAM2mg/ml to them and vibrating them with Vortex

上記のリポソームをArガスで乾燥させて一晩置いた。
Lパラフィン100µlを加えて超音波を1時間かけた。
そのうち10µlを取り出してNIPAM2mg/mlを加え、それをVortexにかけてリポソームを作製した

2 Step2 DNAによる連鎖的リポソームの破壊

2-1 DNAオリガミによるアプローチ

2-1-1 デザインしたDNAオリガミの作製

Making DNA origami

DNA origami recipe

We designed DNA origami by <A Href="http://cadnano.org/">caDNAno2</A>, software for designing 2D and 3D DNA origami.
Our DNA origami has 141 staples that have 30nt free single-stranded parts outside the DNA origami. The sequence of the parts is “each DNA origami staple-TTTTTTTTTTTTTTTCTGTCGCATCGAGAG”.
Between the staple and unique (CTGTCGCATCGAGAG) 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.
The 30nt single-stranded parts are stable till 37 degrees, according to <A Href="http://www.nupack.org/">NUPACK</A>).
The 141 staples have the same length so that they may be present at the same intervals in the DNA origami.
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.
This design enables each DNA origami to exist individually.

The list of strands

The other strands exept DNA origami staples used in our experiment are shown in Table1.
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.
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.

The kinds of DNAtrands	Its sequence
Cholesterol-conjugated DNA (Anchored DNA)	CCAGAAGACG
Fluorescent tagged DNA	ACTAGTGAGTGCAGCAGTCGTACCA
Adaptor strand for Anchored DNA and the unique sequence in DNA origami	CGTCTTCTGGCTCTCGATGCGACAG
Adaptor strand for fluorescent tagged DNA and the unique sequence in DNA origami	TGGTACGACTGCTGCACTCACTAGTCTCTCGATGCGACAG

Table.1 The sequence of the strands used in our experiment

Annealing of DNA origami

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).

<ur>

Annealing solution with fluorescent tagged DNA 50µl

84nM M13mp18	2.38µl
Staples
1µM migihaji	1µl
1µM hidarihaji	1µl
1µM ashibatemae	1µl
200nM ashiba	5µl
1µM cholesterol-hybridizing ssDNA	3µl
1µM fluorescent-tagged DNA-hybridizing ssDNA	3µl
5xTAE Mg2+	10µl
mQ	20.62µl
1µM fluorescent-tagged DNA	3µM

Table.2 Annealing solution with fluorescent tagged DNA

Annealing solution with no fluorescent tagged DNA (control) 50µl
We changed 3µl fluorescent tagged DNA in the above solution into the same quantity of mQ.

AFM observation

As we thought excess staples produced more aggregation and made AFM observation difficult, control annealing solution was used for AFM observation.

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

Electrophoresis

We confirmed that our DNA origami was fluorescently labeled by electrophoresis.

50µl of Annealing solution with fluorescent tagged DNA (used in 1-1)Making DNA origami) contains 3µl of 1µM fluorescent tagged DNA.
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.

Agarose gel recipe: 0.4g agarose, 0.8ml 50xTAE, 39.2ml mQ

The electrophoresis was conducted with 1% agarose gel, CV 100V, for 50 minutes.

2-1-3 DNAオリガミによりリポソームを破壊する実験

Concentration of Anchored DNA

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.

<ur>

Liposome with 0.018µM Anchored DNA: 1µl 0.1µM Anchored DNA and 2.5µl liposome

Liposome with 0.069µM Anchored DNA: 10µl 0.1µM DNAs and 2.5µl liposome

Liposome with 1.8µM Anchored DNA: 1µl 10µM DNAs and 2.5µl liposome

Liposome with 6.9µM Anchored DNA: 10µl 10µM DNAs and 2.5µl liposome

Observation by phase and fluorescent microscope

We observed each sample with a phase microscope.

Then we added 2µl DNA origami into each sample and saw if some change would happen with a fluorescent microscope.
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.

84nM M13mp18	2.38µl
Staples
1µM migihaji	1µl
1µM hidarihaji	1µl
1µM ashibatemae	1µl
200nM ashiba	5µl
100µM cholesterol-hybridizing ssDNA	4.23µl
100µM fluorescent-tagged DNA-hybridizing ssDNA	4.23µl
5xTAE Mg2+	10µl
mQ	23.54µl

Table.3 50µl Annealing solution for fluorescent microscope observation

After annealing, we added 4.23µl 100µM fluorescent-tagged DNA (the same quantity of fluorescent-tagged DNA-hybridizing ssDNA).

2-1-4 DNAによる配列特異性を証明する実験

Making liposome

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.

リポソームは界面通過法により作製した。GFPの緑の蛍光を持つリポソームAとローダミンの赤の蛍光を持つリポソームBの二種類を作製した。この二種類のリポソームはOuter Bufferは共通でInner Bufferは異なる。各バッファーの組成は以下のとおりである。

Outer Buffer	STE（GFPの代わり）	10µl
	グルコース(1M)	250µl
	25×TAE	20µl
	25×TAE	20µl
LiposomeA Inner Buffer	GFP	5µl
	スクロース(1M)	125µl
	25×TAE Mg²⁺	10µl
	mQ	110µl
LiposomeB Inner Buffer	ローダミン	0.5µl
	スクロース(1M)	12.5µl
	25×TAE Mg²⁺	10µl
	mQ	110µl

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

１Inner 2+lipidパラフィン50 たっぴんぐ
２パラフィン50をouter 50に乗せる
３１を2の上に乗せる
４遠心5分間
５針でチューブの底のリポを取り出して観察する

2-2 フラワーミセルによるアプローチ

2-2-1 SPRによるループ構造の確認

Making liposome

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

Making liposome

2-2-3 DNAによる配列特異性を証明する実験

Making liposome

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@@ Line 113: / Line 113: @@
 </tr>
 </table>
-Table.1 １－１の実験のリポソームの組成<br><br>
+Table.1 Making liposomes<br><br>
+.	Drying the liposomes above with argon gas and letting them stand for a night<br>
+.	Adding L paraffin 100µl to 1 and sonicating them for an hour<br>
+.	Picking up 10µl from 2, adding NIPAM2mg/ml to them and vibrating them with Vortex<br>
 上記のリポソームをArガスで乾燥させて一晩置いた。<br>
@@ Line 297: / Line 301: @@
 <h5 id=8>2-1-4 DNAによる配列特異性を証明する実験</h5>
 <h5>Making liposome</h5>
-リポソームは界面通過法により作製した。GFPの緑の蛍光を持つリポソームAとローダミンの赤の蛍光を持つリポソームBの二種類を作製した。この二種類のリポソームはOuter Bufferは共通でInner Bufferは異なる。各バッファーの組成は以下のとおりである。
+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>
+リポソームは界面通過法により作製した。GFPの緑の蛍光を持つリポソームAとローダミンの赤の蛍光を持つリポソームBの二種類を作製した。この二種類のリポソームはOuter Bufferは共通でInner Bufferは異なる。各バッファーの組成は以下のとおりである。<br><br>
 <table border cellspacing="3" bgcolor="lightyellow">
@@ Line 342: / Line 348: @@
 </tr>
-<tr bgcolor="Crimson">
+<tr bgcolor="#FF6699 ">
 <td>LiposomeB Inner Buffer</td>
 <td>ローダミン</td>
 <td>0.5µl</td>
 </tr>
-<tr bgcolor="Crimson">
+<tr bgcolor="#FF6699">
 <td> </td>
 <td>スクロース(1M)</td>
 <td>12.5µl</td>
 </tr>
-<tr bgcolor=" Crimson ">
+<tr bgcolor=" #FF6699">
 <td> </td>
 <td>25×TAE Mg<sup>2+</sup></td>
 <td>10µl</td>
 </tr>
-<tr bgcolor=" Crimson ">
+<tr bgcolor=" #FF6699">
 <td> </td>
 <td>mQ</td>
@@ Line 363: / Line 369: @@
 </tr>
 </table>
+.	Tapping of inner 2 and lipid paraffin 50<br>
+.	Putting paraffin 50 on outer 50<br>
+.	Putting 1 on 2<br>
+.	Centrifuging 3 for 5 minutes<br>
+.	Observing leak of liposomes from the bottom of tubes by needles<br>
 １Inner 2+lipidパラフィン50 たっぴんぐ<br>

Biomod/2013/Sendai/protocol: Difference between revisions

Revision as of 13:16, 24 October 2013

Biomod2013
TeamSendai

Protocol

Contents

1 Step1 温度感受性リポソームの破壊

1-1温度感受性リポソームを破壊する実験

Structure of NIPAM

Making liposome

2 Step2 DNAによる連鎖的リポソームの破壊

2-1 DNAオリガミによるアプローチ

2-1-1 デザインしたDNAオリガミの作製

Making DNA origami

DNA origami recipe

The list of strands

Annealing of DNA origami

AFM observation

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

Electrophoresis

2-1-3 DNAオリガミによりリポソームを破壊する実験

Concentration of Anchored DNA

Observation by phase and fluorescent microscope

2-1-4 DNAによる配列特異性を証明する実験

Making liposome

2-2 フラワーミセルによるアプローチ

2-2-1 SPRによるループ構造の確認

Making liposome

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

Making liposome

2-2-3 DNAによる配列特異性を証明する実験

Making liposome

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Biomod/2013/Sendai/protocol: Difference between revisions

Revision as of 13:16, 24 October 2013

Biomod2013 TeamSendai

Protocol

Contents

1 Step1 温度感受性リポソームの破壊

1-1温度感受性リポソームを破壊する実験

Structure of NIPAM

Making liposome

2 Step2 DNAによる連鎖的リポソームの破壊

2-1 DNAオリガミによるアプローチ

2-1-1 デザインしたDNAオリガミの作製

Making DNA origami

DNA origami recipe

The list of strands

Annealing of DNA origami

AFM observation

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

Electrophoresis

2-1-3 DNAオリガミによりリポソームを破壊する実験

Concentration of Anchored DNA

Observation by phase and fluorescent microscope

2-1-4 DNAによる配列特異性を証明する実験

Making liposome

2-2 フラワーミセルによるアプローチ

2-2-1 SPRによるループ構造の確認

Making liposome

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

Making liposome

2-2-3 DNAによる配列特異性を証明する実験

Making liposome

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