Biomod/2013/Sendai/protocol

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Protocol

Contents

  • <a href="#chain"> 1 Step1 Disruption of temperature sensitive liposomes</a>
    • <a href="#bending"> 1-1 Disruption of temperature sensitive liposomes</a>
  • <a href="#Flower"> 2 Step2 Liposome disruption induced by attachment of key DNA with anchor DNA</a>
    • <a href="#sensing"> 2-1 DNA Origami approach</a>
      • <a href="#5"> 2-1-1 Making DNA Origami</a>
      • <a href="#6"> 2-1-2 Labeling DNA Origami with fluorescent-tagged DNA</a>
      • <a href="#7"> 2-1-3 Disruption of liposomes by DNA Origami</a>
      • <a href="#8"> 2-1-4 Confirming sequence specificity of DNA</a>
    • <a href="#9"> 2-2 Flower DNA approach</a>
      • <a href="#10"> 2-2-1 Confirming the formation of the loop structure by SPR</a>
      • <a href="#11"> 2-2-2 Disruption of liposomes by Flower DNA</a>
      • <a href="#12"> 2-2-3 Confirming sequence specificity of DNA</a>


    </td></tr></table>

    1 Step1 Disruption of temperature sensitive liposomes

    1-1 Disruption of temperature sensitive liposomes

    Structure of NIPAM
    Making liposome
    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 25μl NIPAM2mg/ml to them and vibrating them with Vortex

    2 Step2 Liposome disruption induced by attachment of key DNA with anchor DNA</h5>

    2-1 DNA Origami approach

    2-1-1 Making DNA Origami
    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 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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