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Protocol

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 Labeling DNA Origami with fluorescent-tagged DNA

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 Disruption of liposomes by DNA Origami

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).

2-1-3 Disruption of liposomes by DNA Origami その2

マイクロリットルの記述統一
分量は表で書く
数字の箇条書きで
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

Making liposome

Liposomeは界面通過法により作成した。
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。

DOPC(10mM) 20ul DPPC(10mM) 20ul Cholesterol(10mM) 20ul DOPE(10mM) 20ul

2.（以下、箇条書きで）

Inner buffer(250ul)
・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

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

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。
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。
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。
３０秒間遠心にかけて、下層のみを取り出す。
サンプル１リポソーム＋フラワーアンカーDNA＋KeyDNA
・リポソーム(GFP)(4mM) 10ul ・Chol-leg A (10uM) 25ul ・Origami A (5nM) 20ul ・1xTAE Mg2+ 55ul

サンプル２　リポソーム＋フラワーアンカーDNA
・リポソーム(GFP)(4mM) 10ul ・Chol-leg A (10uM) 25ul ・1xTAE Mg2+ 75ul

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）
・リポソーム(GFP)(4mM) 10ul ・Chol-leg A (10uM) 25ul ・1xTAE Mg2+ 75ul ・界面活性剤　2%NP 2ul

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。

一回目サンプル１とサンプル２
二回目サンプル２とサンプル３

2-1-4 Confirming sequence specificity of DNA

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.

Outer Buffer	STE（as substitute for GFP）	10µl
	glucose(1M)	250µl
	25×TAE	20µl
	25×TAE	20µl
LiposomeA Inner Buffer	GFP	5µl
	sucrose(1M)	125µl
	25×TAE Mg²⁺	10µl
	mQ	110µl
LiposomeB Inner Buffer	Rhodamine	0.5µl
	sucrose(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

2-2 Flower DNA approach

2-2-2 Disruption of liposomes by Flower DNA

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

（対応するプロトコルへのリンク）

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
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
3.Putting 1(inner + lipid paraffin) on 2(流動paraffin+outer)
4.Centrifuging 3のサンプル for 5minutes
5.Observing leak of liposomes from the bottom of tubes by needles

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@@ Line 96: / Line 96: @@
 <h4 id=bending>1-1 Disruption of temperature sensitive liposomes</h4>
 <h5> Structure of NIPAM</h5>
+poly-N-isopropyl acrylamide
 <h5> Making liposome</h5>
 <table border cellspacing="3" bgcolor="lightyellow">
@@ Line 113: / Line 114: @@
 <tr bgcolor="moccasin">
 <td> TXR</td>
-<td> 0.01µl </td>
+<td> 1µl </td>
 </tr>
 </table>
-Table.1 Making liposomes<br><br>
+Table.1 Materials Making liposomes<br><br>
 .	Drying the liposomes above with argon gas and letting them stand for a night<br>

Biomod/2013/Sendai/protocol: Difference between revisions

Revision as of 10:59, 26 October 2013

Biomod2013
TeamSendai

Protocol

Contents

1 Step1 Disruption of temperature sensitive liposomes

1-1 Disruption of temperature sensitive liposomes

Structure of NIPAM

Making liposome

2 Step2 Liposome disruption induced by attachment of key DNA with anchor DNA

2-1 DNA Origami approach

2-1-1 Making DNA Origami

Making DNA origami

DNA origami recipe

The list of strands

Annealing of DNA origami

AFM observation

2-1-2 Labeling DNA Origami with fluorescent-tagged DNA

Electrophoresis

2-1-3 Disruption of liposomes by DNA Origami

Concentration of Anchored DNA

Observation by phase and fluorescent microscope

2-1-3 Disruption of liposomes by DNA Origami その2

Making liposome

2-1-4 Confirming sequence specificity of DNA

Making liposome

2-2 Flower DNA approach

2-2-2 Disruption of liposomes by Flower DNA

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

Biomod/2013/Sendai/protocol: Difference between revisions

Revision as of 10:59, 26 October 2013

Biomod2013 TeamSendai

Protocol

Contents

1 Step1 Disruption of temperature sensitive liposomes

1-1 Disruption of temperature sensitive liposomes

Structure of NIPAM

Making liposome

2 Step2 Liposome disruption induced by attachment of key DNA with anchor DNA

2-1 DNA Origami approach

2-1-1 Making DNA Origami

Making DNA origami

DNA origami recipe

The list of strands

Annealing of DNA origami

AFM observation

2-1-2 Labeling DNA Origami with fluorescent-tagged DNA

Electrophoresis

2-1-3 Disruption of liposomes by DNA Origami

Concentration of Anchored DNA

Observation by phase and fluorescent microscope

2-1-3 Disruption of liposomes by DNA Origami その2

Making liposome

2-1-4 Confirming sequence specificity of DNA

Making liposome

2-2 Flower DNA approach

2-2-2 Disruption of liposomes by Flower DNA

Navigation menu

Search

Biomod2013
TeamSendai