Experiment

Contents

1 Step1 Disruption of temperature sensitive liposomes

1-1Disruption of temperature sensitive liposomes

Purpose
In our project, we adopt liposomes conjugated with NIPAM polymer (temperature sensitive liposomes) as initiators that sense environmental change (temperature increase).
We confirm that the initial liposomes with NIPAM break with temperature increase.

Method
We used Egg PC for the lipids and L paraffin for the buffer. Vortex process was applied to make liposomes.
Then we added NIPAM (dissolved in chloroform) into the liposomes.
The liposomes were observed on the slide glass with a phase-contrast microscopy.
After confirming the formation of the liposomes, we put a petri dish with hot water inside on the slide glass to increase the temperature.
Protocol
(対応するプロトコルへのリンク)
Result
Fig.1 shows liposomes before the temperature increase.

Fig.1 Liposomes with NIPAM before the temperature increase

Figure 2 and 3 shows the state after the temperature increase by putting a petri dish with hot water inside on the slide glass. The view sight in Fig.2 and 3 was the same as that in Figure 1.
Only a rough background and no liposome were observed in Fig.3. Even after focus shifts, no liposome was seen.

Fig.2 the state after the temperature increase

Fig.3 the state after the temperature increase
Discussion
As liposomes present in Fig.1 disappeared in Fig.3, liposomes with NIPAM were likely to have burst.
On the other hand, some liposomes were still present even after the temperature increase. This is probably because they are multi-lamella liposomes (liposomes that consist of many lipid bilayers). Multi-lamella liposomes are more difficult to break than uni-lamella ones. Therefore, we suppose that liposomes that were present in Fig.1 but disappeared in Fig.3 were uni-lamella ones.

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

2-1 DNA Origami approach

2-1-1 Making DNA Origami
Purpose
In our project, we use DNA Origami as Key DNA to break liposomes. We design rectangular DNA Origami with a chipped edge and try 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.4).

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

Discussion
Just like our design, rectangular DNA Origami with a chipped edge was observed.
2-1-2 Labeling DNA Origami with fluorescent-tagged DNA
Purpose
If 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 DNA strand.

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 a shorter time, we added the fluorescent-tagged DNA into the control annealing solution, which had 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 DNA structures with fluorescent molecules; scanning a gel after staining, we can see the bands of all DNA structures. 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 that in a stained gel, we can say that our Origami was successfully fluorescently labeled.

Result
In a non-stained gel (Fig.5), only bands in lane 3 and 4 from the left (*Ori, **Ori) can be seen. They are fluorescently labeled structures. In addition, as they gave the same result, 40 minutes is long enough for fluorescently labeling.

Fig.5 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.6), marker lane (lane 1) had the longest DNA strand of 20kb. Comparing this band and the band of M13mp18 (lane 2) with annealed DNA Origami (lane 3,4,5), the bands of the Origami are at the higher position. Therefore, we concluded that in lane3~5, DNA Origami structure was 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.6 Stained gel image: from the left, marker, M13mp18, Ori*, Ori**, and DNA Origami with no fluorescent molecule (Ori)

Discussion
Combining the results of Fig.5 and 6, the fluorescently labeled bands in lane3 and 4 in Fig.5 are at the same height as those of DNA Origami in Fig.6. Thus, we concluded our Origami was successfully fluorescently labeled.

2-1-3 Disrupting liposomes by DNA Origami
Purpose
To break liposomes with our Origami, first we investigate how our DNA Origami affect liposomes.

Principle
To break liposomes with our Origami, a lot of Origami has to hybridize to the surface of the liposomes.
To begin with, we added cholesterol-conjugated single-stranded DNA (in the rest of this document, referred to as Origami-anchor DNA) into liposomes, and made it float on the surface. The Origami-anchor DNA has a complementary part to our Origami, so the Origami is expected to hybridize to Origami-anchor DNA on the liposomes. In this way, lots of Origami would hybridize to liposomes via Origami-anchor DNA.

Method
We added Origami-anchor 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 Origami 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 multi-lamella liposomes (Fig.7~10).


Fig.7 Phase microscope image of liposomes (Origami-anchor DNA: 0.018µM)


Fig.8 Phase microscope image of liposomes (Origami-anchor DNA: 0.069µM)


Fig.9 Phase microscope image of liposomes (Origami-anchor DNA: 1.8µM)


Fig.10 Phase microscope image of liposomes (Orgami-anchor DNA: 6.9µM)

Adding fluorescently labeled DNA Origami into the above liposomes, we saw if some change would happen with a fluorescent microscope.
When the concentration of Origami-anchor DNA was 0.018, 0.069µM, many gleaming (in green color) liposomes were observed. We confirmed that the fluorescently labeled Origami well hybridized to the liposomal surface (Fig.11,12,13).
Fig.11,12 fluorescent microscope image of liposomes (Origami-anchor DNA: 0.018µM)

Fig.13 fluorescent microscope image of liposomes (Origami-anchor DNA: 0.069µM)

On the other hand, when the concentration of Origami-anchor DNA was 1.8µM, few gleaming liposomes could be seen with a fluorescent microscope (Fig.14). This result indicates the possibility that liposomes have broken.

Fig.14 fluorescent microscope image of liposomes (Origami-anchor DNA: 1.8µM)

When the concentration of Origami-anchor DNA is 6.9µM, some liposomes were gleaming and others distorted, forming networks (Fig.15).

Fig.15 fluorescent microscope image of liposomes (Origami-anchor 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 Origami-anchor DNA is low (0.018, 0.069µM), DNA Origami hybridizes to the surface of liposomes relatively stablely. When the concentration is middle (1.8µM), more DNA Origami hybridizes to the surface and loads on it. The liposomes become fragile and easy to break. When the concentration is high (6.9µM), some liposomes exist individually, and others form networks via Origami-anchor DNA and DNA Origami complex.


According to this hypothesis, when the concentration of Origami-anchor DNA is 1.8µM, DNA Origami breaks liposomes.


2-1-4 Confirming sequence specificity of DNA
Purpose
In this project, we adopt DNA for the Key of chain reaction because DNA has a significant characteristic: sequence specificity. Utilizing this sequence specificity, we aim to select liposomes that will be broken by Key DNA, induce chain-reactive liposomal disruption by some Key DNA, and make connections between liposomes.
Corresponding to the liposomes, we arrange two kinds of Origami-anchor DNA of different sequences and attach the anchor to the liposomes. Then we mix both kinds of liposomes together.
Into the mixture, Key DNA for just one kind of liposomes is added. We confirm that the Key DNA breaks only the corresponding kind of liposomes.

Method
We made two kinds of liposomes: liposomeA and liposomeB by water-in-oil emulsion process. LiposomeA contains GFP (Green Fluorescent Protein) inside, and liposomeB has Rhodamine (red fluorescent dye) in itself.

Origami-anchor DNA for liposome A has the sequence of 5'-CCAGAAGACG-chol-3'. The anchor for liposome B has the sequence of 5'-TCCACTAACG-chol-3'. Both Origami-anchor DNA was mixed with the corresponding liposomes.
Each liposome was centrifuged for one minute to remove the excess Origami- anchor DNA.
Then we mixed 1µl of each liposome and observed it with a phase-contrast microscope. Next, 4µl refined DNA Origami was added to the mixture (of liposomeA and B). The sample was also observed with a phase-contrast microscope.
Protocol
(対応するプロトコルへのリンク)
Result
Fig.16 is the phase-contrast microscope image of the mixture of liposome A and B before the addition of Key DNA Origami.

Fig.16 Phase contrast microscope image of the mixture of liposome A (Green) and B (Red)
Discussion
(要加筆)

2-2 Flower DNA approach

2-2-1 Confirming the formation of the loop structure by SPR
Purpose
To break liposomes by flower DNA 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 to 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 Key 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 Key DNA some 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
    In flower DNA approach, the Key DNA strand attaches to Flower-anchor DNA on liposomes, load on the liposomes, and break them. We confirm the disruption of liposomes 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
    We annealed DNA Origami with fluorescently labeled staples. Liposomes dyed with Texas Red (fluorescent dye) were prepared, and observed with a fluorescent microscope. Then we added DNA Origami into the liposomes. They were also observed with a fluorescent microscope. 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