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
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 1-1の実験のリポソームの組成

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 caDNAno2, 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 NUPACK).
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).

  • 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.
  • 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
    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
    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