Experiment

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

Purpose
私たちのプロジェクトでは外部刺激を感知するイニシエーターの一例としてニッパム修飾したリポソーム(温度感受性リポソーム)を使用する。そのため、温度上昇によりニッパム修飾のリポソームが割れること実験により確認する。
In our project, liposome collapses by temperature shift is a crucial step. Thus, we should confirm the temperature sensitivity of PNIPAM lipids-based liposome.(訳途中)
Method
脂質にはEggPC、バッファーにはLパラフィン?を使用し、ボルテックス法によりリポソームを作製した。
リポソームにNIPAM (in クロロホルム)を質量比???の割合で加えた。
スライドガラスを作製し、位相差顕微鏡でリポソームがあることを確認した。
リポソームが確認できたら、スライドガラスの上にお湯を乗せて温度を上げた。
Protocol
(対応するプロトコルへのリンク)
Result
温度を上げる前のリポソームの状態は図1のようになった。

図1 温度を上げる前のニッパム付きのリポソーム
スライドガラスにお湯を乗せて温度を上昇させた後のリポソームの様子は以下図2、図3のようになった。観察しているリポソームは図1のものと同じである。まず、図2のようにバックグラウンドがザラザラになって、しばらくすると図3のようにリポソームが確認できなくなった。ピントを調節してもリポソームは確認できなかった。

図2 温度上昇後のニッパム付きのリポソーム

図3 温度を上昇させた後のニッパム付きのリポソームが消えた様子
Discussion
図1で見えていたリポソームが、図2,3のように消えてしまったのでニッパム付きのリポソームが割れたと考えられる。しかし、リポソームによっては温度を上げた後も残っているものがいくつか確認できた。これはリポソームがマルチラメラ(脂質二重膜が複数重なっているもの)になっているものと考えられ、脂質二重膜が単一層のユニラメラよりも割れにくいからだと考えられる。上記図1,2,3で定点観察したリポソームはユニラメラであると考えられる。

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
Purpose
In our project, we used DNA origami as triggers for collapsing liposomes. We designed a rectangular DNA origami with a chipped edge and tried to make it.
Method
We mixed M13mp18, staples, 5xTAE Mg2+, and mQ in a microtube and annealed it for 2.5 hours.
Protocol

Result
We confirmed that our DNA origami was well formed by AFM (Atomic Force Microscope) (Fig.1).

Fig.1 AFM image of DNA origami (M13: 4nM, staples:20nM)

Discussion
Just like our design, rectanglar origamis with chipped edges were observed.
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
Purpose
If the origami is fluorescently labeled, it is much easier to observe the effect of DNA origami on liposomes. So we labeled our origami by hybridizing it with fluorescent tagged DNAtrands.

Method
Our DNA origami has many staples that can bind to fluorescent tagged DNA for labeling. We mixed fluorescent tagged DNA together with DNA origami staples in annealing solution.
In addition, to see if the origami binds to the fluorescent tagged DNA in shorter time, we added the fluorescent tagged DNA into control annealing solution, which contained no fluorescent tagged DNA, and left it for 40 minutes.
To see the origami was well labeled with fluorescent molecules, we used electrophoresis.
Electrophoresis was conducted with a 1% agarose gel, CV100V for 50 minutes.
Protocol

By scanning a gel before staining, we can see only the bands of DNAtructures with fluorescent molecules; scanning a gel after staining, we can see the bands of all DNAtructures. So we scanned a gel before and after staining (we scanned both a non-stained and a stained gel).
First we saw the bands of our origami in a non-stained gel. Then, we compared the bands with those in a stained gel. If the bands of origami in a non-stained gel were at the same height as those in a stained gel, we can say that our origami is successfully fluorescently labeled.

Result
In a non-stained gel (Fig.2), only bands in lane 3 and 4 from the left (*Ori, **Ori) can be seen. They are fluorescent labeled structures. In addition, as they gave the same result, 40 minutes is long enough for fluorescent labeling.

Fig.2 Non-stained gel image: only bands in two lanes can be seen. From the left, they are DNA origami with fluorescent molecules in pre-annealing (Ori*), and DNA origami with fluorescent molecules in post-annealing (Ori**)

In a stained gel (Fig.3), marker (lane 1) had the longest DNAtrand of 20kb. Comparing this and M13mp18 (lane 2) with annealed DNA origamis (lane 3,4,5), the bands of the origamis are at the higher position. Therefore, we concluded that in lane3~5, DNA origami structure made of M13 and staples were made as we had expected.
We considered that the bands in lane3~5 are seen as if they were diffused, just because our origami has many staples binding to the fluorescent tagged DNA, and each origami attaches to different number of them, and its molecular weight varies.

Fig.3 Stained gel image: from the left, marker, M13mp18, Ori*, Ori**, and DNA origami with no fluorescent molecule (Ori)

Discussion
Combining the results of Fig.2 and 3, the fluorescent labeled bands in lane3 and 4 in Fig.2 are at the same height as those of DNA origami in Fig.3. Thus, we concluded our origami was successfully fluorescently labeled.

2-1-3 DNAオリガミによりリポソームを破壊する実験
Purpose
To collapse liposome with our origami, first we investigated how our DNA origami affected liposomes.

Principle
To collapse liposomes with our origami, many origamis have to hybridize with the surface of liposomes.
To begin with, we added cholesterol-conjugated single-stranded DNA (in the rest of this document, referred to as Anchored DNA) into liposomes, and made them float on the surface. If the Anchored DNA have a complementary part to our origami, the origami is expected to hybridize with the surface. In this way, many origamis would hybridize with liposome via Anchored DNA.

Method
We added Anchored DNA into liposomes at the final concentration of 0.018, 0.069, 1.8, and 6.9µM. Then we observed the samples with a phase microscope. Next, adding fluorescently labeled DNA origamis into the above liposomes, we saw if some change would happen with a fluorescent microscope.
Protocol

Result
In all four conditions, liposomes were observed with a phase microscope. We confirmed the formation of multilamella liposomes (Fig.4~7).


Fig.4 Phase microscope image of liposomes (cholesterol-conjugated DNA: 0.018µM)


Fig.5 Phase microscope image of liposomes (cholesterol-conjugated DNA: 0.069µM)


Fig.6 Phase microscope image of liposomes (cholesterol-conjugated DNA: 1.8µM)


Fig.7 Phase microscope image of liposomes (cholesterol-conjugated DNA: 6.9µM)

Adding fluorescently labeled DNA origamis into the above liposomes, we saw if some change would happen with a fluorescent microscope.
When the concentration of Anchored DNA was 0.018, 0.069µM, many gleaming (in green color) liposomes were observed. We confirmed that the fluorescently labeled origamis well hybridized with the liposomal surface (Fig.8,9,10).
Fig.8,9 fluorescent microscope image of liposomes (cholesterol-conjugated DNA: 0.018µM)

Fig.10 fluorescent microscope image of liposomes (cholesterol-conjugated DNA: 0.069µM)

On the other hand, when the concentration of Anchored DNA was 1.8µM, few gleaming liposomes could be seen with a fluorescent microscope (Fig.11). This result indicates the possibility that liposomes were collapsed.

Fig.11 fluorescent microscope image of liposomes (cholesterol-conjugated DNA: 1.8µM)

When the concentration of Anchored DNA is 6.9µM, some liposomes were gleaming and others distorted, forming networks (Fig.12).

Fig.12 fluorescent microscope image of liposomes (cholesterol-conjugated DNA: 6.9µM)

Discussion
From these results, we put forward the following hypothesis about the interaction of DNA origami and liposomes.
When the concentration of Anchored DNA is low (0.018, 0.069µM), DNA origamis hybridize with the surface of the liposomes relatively stablely. When the concentration is middle (1.8µM), more DNA origamis hybridizes with the surface and place stress on it. Then, liposomes become fragile and easy to be collapsed. When the concentration is high (6.9µM), some liposomes exist individually, and others form networks via Anchored DNA and DNA origami complexes.


According to this hypothesis, when the concentration of Anchored DNA is 1.8µM, DNA origami collapses liposomes. Therefore, in the following experiment, we checked if DNA origami would collapse liposomes at this concentration of Anchored DNA.


2-1-4 DNAによる配列特異性を証明する実験
Purpose
このプロジェクトにおいてリポソームをDNAで割る理由は、DNAの配列特異性を利用して割れるリポソーム間に関係性を持たせるためである。そこで配列の異なる2種類のDNAを生やしたリポソームを用意して片方だけに相補なDNAを加え、2種類のリポソームのうちDNAが相補になっている片方のリポソームだけが破壊されることを確かめた。
Method
内部にGFP(緑の蛍光)を含んだリポソームAとローダミン(赤の蛍光)を含んだリポソームBの2種類のリポソームを、界面通過法により作製する。どちらのリポソームも
リポソームAには5'-CCAGAAGACG-chol-3'の配列を持つコレステロール付きのDNAをリポソームBには配列5'-TCCACTAACG-chol-3'をもつコレステロール付きのDNAを振り掛けた。
1分間遠心分離器にかけて、リポソームにくっつかなったコレステロール付きDNAとリポソームを分離する。
リポソームAとリポソームBが入っているリポソームを1μℓずつ混合し、位相差顕微鏡で観察した。
精製したDNAオリガミ を4μℓをリポソームA,Bの混合サンプルに加え、位相差顕微鏡で観察した。
Protocol
(対応するプロトコルへのリンク)
Result
トリガーのDNA折り紙を入れていないリポソームA、Bの混合サンプルを位相差顕微鏡で観察すると、図17のようになった

図17 GFPとローダミン染色した2種類のリポソーム混合サンプルの位相差顕微鏡画像
Discussion

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
Purpose
To collapse liposomes by flower micelle method, we aim to attach many loop strands to the surface of liposomes.
To achieve this, we adopt the same hybridization method via Anchored DNA as used in i)Bending approach into liposomes: the Anchored DNA has a complementary part to our loop strand and the loop strand is expected to hybridize with liposomes.
We checked the hybridization of liposomes and Anchored DNA, and that of Anchored DNA and our loop strands.

Principle
As our loop strand is too small to observe with an AFM or a fluorescent microscope, we used an apparatus called SPR.
SPR is a Surface Plasmon Resonance assay that estimates the weight of molecules attached to membrane surface, by the change of the reflection of the laser beam.
If Anchored DNA attaches to a liposome, and then loop strand attaches to it, SPR value increases after each step.
We measured SPR value after each step of adding DOPC into liposomes, and loop DNA into it.

Method
  • 1. Inject 45µl DOPC (100mM) into SPR
  • 2. Inject 5µl NAOH to SPR in order to stabilize the point
  • 3. Inject 10µl Anchored DNA (0.1µM) to SPR
  • 4. Inject 10µl loop DNA of 40 bp (0.1µM) to SPR
  • 5. Inject 10 µl trigger DNA of 40 bp (0.1µM) to SPR


  • Result
    The result was shown in Fig.15 below.

    Fig.15 The transition of SPR value

    As the first injection of Anchored DNA caused no change of SPR value, we injected Anchored DNA for two times.
    Fig 15 shows that SPR value increased after injecting Anchored DNA and loop DNA. Moreover, we should note that after injecting trigger DNAome changes of SPR value were observed.

    Discussion
    Fig.15 shows the behavior of materials on the surface of liposomes. The increase of SPR value after injecting Anchored DNA indicates that Anchored DNA successfully combined with liposomes. Similarly, it is considered that loop DNA combined with Anchored DNA.
    Thus, we confirmed the formation of the loop structures on liposomes.

    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
    Purpose
    フラワーミセルアプローチでは鍵DNAストランドがリポソーム表面に生えているアンカーDNAにハイブリしてリポソームが割れる必要がある。それを確かめるために。
    Method
    DNAオリガミに蛍光をハイブリさせたものをアニーリングにより作製する 膜染色(テキサスレッド)したリポソームをつくる リポソームのみを蛍光顕微鏡で観察する。 リポソームにDNAオリガミを加えてその後の様子を観察する (対応するプロトコルへのリンク)
    Result
    Discussion
    2-2-3 DNAによる配列特異性を証明する実験
    Purpose
    Method
    (対応するプロトコルへのリンク)
    Result
    Discussion