Experiment

Contents

1st stage: Sensing system

1-1Disruption of temperature sensitive liposomes

Purpose
In our project, we planned to use liposomes conjugated with NIPAM polymer as a chain reaction initiator. NIPAM (poly-N-isopropyl acrylamide) is a temperature sensitive molecule that has a unique critical temperature (Tc: 32~40˚C ).
When the temperature increased over than Tc, the hydrophilic polymer changes its property hydrophobic. It is expected that the change should disrupt the membrane lipid alignment. Here we confirm that the possibility of breaking liposomes with NIPAM by increasing temperature.
NIPAM was purchesed from Sigma Aldrich

Method
The liposomes were prepared by natural swelling method. Obtained sample included a mixture of unilamellar and multilamellar liposomes.
Then we added NIPAM-conjugated lipids (dissolved in ultra pure water (Milli-Q)) to the liposomes solution.
The liposomes were observed on the slide glass by phase-contrast microscopy.
After confirming the formation of the liposomes, a petri dish with hot water (~90˚C) was put on the sample slide glass to increase the temperature.
Detailed Protocol
Protocol
Result

Fig.1 Phase contrast images of liposomes in NIPAM solution. Temperature increased from RT to enough over than Tc (left to right).

Fig.1 shows the continuous images before and after the temperature increase. The view sight was the same position.
NIPAM polymer turned into globular states with increasing temperature. Liposomes disappeared by increasing temperature (> Tc).

Discussion
Thermosensitive polymer NIPAM can disrupt the coexisting liposomes by the polymers phase transition.
On the other hand, some liposomes still present even at the high temperature. In this experiment, some fractions were multi-lamellar liposomes. Since globular states of NIPAM (hydrophobic) at high temperature attack the liposome membrane from the outside, it is not surprising that the multi-lamellar liposomes consist of many lipid bilayers are more difficult to disrupt. Therefore, we suppose that liposomes disrupted by temperature shift in Fig.1 were uni-lamella ones. These results confirmed that triggering by heat disrupted the liposomes.

2nd stage: Amplification system

2-1 DNA Origami approach

2-1-1 Making DNA Origami
Purpose
In our project, to use DNA Origami as the Key DNA to break liposomes, we design the rectangular DNA Origami with a chipped edge.
Method
Mixing M13mp18, staples, 5xTAE Mg2+, and mQ in a microtube and annealing for 2.5 hours.
Protocol
Result
We obtain DNA Origami same as our design. The result was confirmed by AFM (Atomic Force Microscope.)

Fig.2 AFM image of DNA Origami (M13: 4nM, staples:20nM)

Discussion
As shown in Fig. 2, DNA Origami was well-formed.
2-1-2 Labeling DNA Origami with fluorescent-tagged DNA
Purpose
To observe the fluorescent effect of DNA Origami on liposomes by microscope, we labeled our Origami by hybridizing with the fluorescent-tagged DNA strand.

Method
Our DNA Origami is composed of many staples that can bind to the same fluorescent-tagged DNA for labeling. We mixed fluorescent-tagged DNA with DNA Origami staples before annealing, and after annealing solution. Labeling of the DNA Origami was confirmed by gel-electrophoresis. Gel-electrophoresis was conducted with a 1% Agarose gel, 100V for 50 minutes.
Protocol

Result
Figure 3 shows florescence detection of the gel before staining (Left) and after SYBR Gold staining (Right). In a non-stained gel, only lane 3 and 4 (*Ori, **Ori) was found. The fluorescent bands of Origami in a non-stained gel were at the same height as that in a stained gel, we conclude that our Origami was successfully fluorescently labeled irrespective of the timing of adding the fluorescent DNA.

Fig.3 Labeling of DNA origami by fluorescence tagged DNA. Left panel shows non-stained gels, and right panel shows the same gels after SYBR Gold stain. From the left, marker, M13mp18, DNA Origami with fluorescent molecules added in pre-annealing (Ori*), DNA Origami with fluorescent molecules added in post-annealing (Ori**), and DNA Origami with no fluorescent molecule (Ori).


Discussion
The results indicate we succeeded to label our Origami by the fluorescence DNA.

2-1-3 Disrupting liposomes by DNA Origami (microscopic analysis)
Purpose
To break liposomes with our Origami, first we investigate how our DNA Origami affects 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
To begin with, we mixed 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. Origami-anchor DNA has a complementary part to our Origami. We should note that the anchor DNA was added after liposome formation to avoid the anchor DNA are inserted on inner side of liposome.
We added Origami-anchor DNA into liposomes at the final concentration of 0.018, 0.069, 1.8, and 6.9µM (NOTE: the concentration of DNA origami is constant, only the concentrations of the anchor DNA varied). Then we observed the samples with a phase microscope. Next, adding the fluorescently labeled DNA Origami into the above liposomes.
Protocol

Result
In all four conditions, liposomes were observed with a phase microscope. We used the mixture of uni- and multi-lamella liposomes (Fig.4~7).

Fig.4 Phase microscope image of liposomes (Origami-anchor DNA: 0.018µM)

Fig.5 Phase microscope image of liposomes (Origami-anchor DNA: 0.069µM)

Fig.6 Phase microscope image of liposomes (Origami-anchor DNA: 1.8µM)

Fig.7 Phase microscope image of liposomes (Orgami-anchor DNA: 6.9µM)

After Addition of the DNA Origami into the above liposomes, we observed with a fluorescent microscope. When the concentration of Origami-anchor DNA was 0.018, 0.069µM, many gleaming (in green color) liposomes were observed. These results confirmed that the fluorescently labeled Origami well hybridized to the liposomal surface but that did not disrupt (Fig.8, 9,10).

Fig.8,9 fluorescent microscope image of liposomes (Origami-anchor DNA: 0.018µM)
Fig.10 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.11). This result indicates the possibility that liposomes have broken.

Fig.11 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.12).
Fig.12 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 the anchor DNA is low (0.018, 0.069µM), liposomes was still stable. When the concentration is middle (1.8µM), more DNA Origami hybridizes to the surface and loads on it. This loading made liposomes become fragile and easy to break. When the concentration is high (6.9µM), disrupted liposomes were connected with others, and consequently, form networks via Origami-anchor DNA and DNA Origami complex.
Anyway, these data strongly indicted the designed DNA origami disrupted liposomes with high concentration of the anchor DNA.


2-1-4 Disrupting liposomes by DNA Origami (quantitative analysis)
Purpose
The above experiments in 2-1-3 microscopic analysis suggest that our DNA Origami disrupted liposomes. Thus, we performed more quantitative analysis.

Method
Fig. 13 Threshold cutting in
the flow cytometer analysis
by EV-SS plot (Sample 3)
We did the experiment using Flow cytometer (Cell Lab Quanta SC Flow Cytometer) in the same way as experiment 2-1-4. Only 7-13 μm diameter liposomes were analyzed (cut off by EV value) (Fig. 13). Liposomes showing over 100 SS value (the indicator of sample complexity) were also omitted because of reliability of the data. Liposomes encapsulating GFP molecules were used in this experiment.

Following 50 μL of samples(Fig.なんとか) were examined with the Flow cytometer. Protocol

Fig.なんとか