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 3 types of samples

Result
As figure below, we were able to observe liposomes containing GFP, by the confocal microscope.

Fig.なんとか Liposomes conteaining GFP(confocal microscope)


Fig.なんとか Fig.14. Quantitative analysis by flow cytometer

The x axis of the following graph is the fluorescence intensity of only liposomes, and the y axis represents the number of liposomes.
Without addition, liposomes having high fluorescence intensity (over 100 a.u.) were mainly observed. When a surfactant NP40, which must disrupt liposomes, were added, the fraction of the high fluorescence liposomes decreased. When the key DNA origami was added, the fraction of the high fluorescence liposomes were completely diminished.

Discussion
These results confirmed that our designed DNA origami actually disrupted liposomes. (The efficiency was higher than 2% surfactant!)

2-1-5 Confirming sequence specificity of DNA flow cytometer
Purpose
To confirm the selectivity of Key DNAs to the anchor DNA, we compared the effect of the complementary Key DNA and the no-binding Key DNA.

Method
Experimental conditions were the same in 2-1-4 except samples.

Sample 1 (Complement). Liposomes + Origami-anchor DNA(A) + Key DNA(A)
Sample 2 (no binding pair). Liposomes + Orgiami-anchor DNA(A) + Key DNA(B)

Protocol
Protocol
Result
The results were shown in figures 14.
以下も前の節同様に1つのfigureにする!
Fig.1 Sample1(complement)
Fig.2 Sample2(No binding pair)
Fig.3 Adding Complementary key DNA
Fig.4 Adding no binding Key DNA

Fig.1 Sample1(complement)
Fig.2 Sample2(No binding pair)

Fig.3 Adding Complementary key DNA
Fig.4 Adding no binding Key DNA

In the sample1, Origami-anchor DNA and Key DNA are complementary each other. In the sample B, the Key DNA has a different sequence that does not hybridize with anchor DNA. The X axis in the figures shows fluorescent intensity. Y axis indicate the number of count. High fluorescence (>100) means liposome with GFP, low fluorescence means that GFP inside liposomes were leaked. The non-binding key DNA does not affect liposome with anchor DNA. On the other hands, the complement key DNA disrupt liposomes.

Discussion
These results demonstrated the selectivity of the Key DNA sequence, and strongly supported our designed DNA actually disrupted liposomes via hybridization of the Key DNA and the anchor (keyhole) DNAs.

2-2 Flower DNA approach

2-2-1 Liposome disruption by flower DNA approach
Purpose
We evaluated whether flower DNA approach works to disrupt liposomes.

Method
We made phase-separated liposomes (DOPC: DPPC: cholesterol= 1: 1: 1) with encapsulating texas-Red (conjugated with dextran) dye inside by the water-in-oil emulsion method. Because of the difference of solid-liquid temperature between critical DOPC and DPPC, these two lipids were phase separated at room temperature. Cholesterol, which is also used in the anchor DNA, presents in the both phases. We expected that the anchor DNA in the solid phase (DPPC domain) provide strong stress on liposomes by hybridizing with the Key DNA.

First, the Flower-anchor DNA (stained with SYBR Gold) was added into the liposomes. Next, we added the Key DNA that stretches the Flower anchored DNA into the mixture. The liposomes were observed in a chamber on a slide glass with a fluorescent microscope.
Protocol
Result



We observed shrinking of liposomes (detected by red filter) by adding the Key DNA (Figure XXXXX). Shifting to green filter showed that the flower-anchor DNA (dyed with SYBR Gold) presented around shrunk liposomes (Figure XXXX). We should note that such aggregation of the flower anchor DNA was not observed before the addition of the key DNA.


Next, we made solution mixing chamber. In this chamber, we can observe the contact surface between Key DNA solution and liposomes solution. The right side of the boundary is Key DNA solution and the left side is liposome solution. Network structures (Green, which indicate the anchor DNA) were observed on the boundary of solutions. This result strongly supported the key DNA is the factor to form the network structure.



When zoom up in the networks, liposomes that lost Texas-Red dextran were observed (Figure XXXXX).

Discussion
In this experiment, three different changes were observed by adding the Key DNA: i) shrinking, ii) network structure formation, and iii) leak of fluorescence. These results suggest that we achieved disruption of liposomes by our flower DNA approach.

2-2-2 Confirming sequence specificity of DNA
Purpose
We demonstrate the selectivity of our Key DNA: the Key DNA only affects the corresponding Flower-anchor DNA and liposomes.

Method
Two types of phase separated liposomes were prepared by droplet transfer methods. One type is liposomes with GFP inside (Green liposome); the other type is liposomes with Texas-Red dextran inside (Red liposome).
The anchor DNA for Green liposome is named “A-flower-anchor DNA”, and the anchor DNA for Red liposome is named “B-flower-anchor DNA”. Each flower-anchor DNA can bind only the complementary Key DNA. This time, only Key DNA for Red liposomes (complementary to B-flower-anchor DNA) is added.
After adding B-Key DNA, the number of each color liposomes is counted to confirm the selectivity.
As a control, only buffer is added instead of B-Key DNA.
Protocol

Result
Fig1 shows fluorescent microscope image of liposomes added B-Key DNA. Only Green liposomes (marked with green rectangles) and no Red liposomes can be seen.

Fig.1 fluorescent microscope image of liposomes added B-Key DNA
(Green rectangles represent Green liposomes)

Fig.2 is fluorescent microscope image of liposomes added buffer (control). In this figure, almost the same number of Green and Red liposomes are seen.
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Fig.2 fluorescent microscope image of liposomes added Buffer
(Green rectangle represent Green liposomes; Red, Red liposomes)

Additives B-key DNA Buffer
Green:Red 17:2 (n = 19) 16:17 (n= 33)
Table1 Ratio of Green and Red liposomes
Table1 shows the Ratio of Green and Red liposomes.
When the control buffer is added, the number of Green and Red liposomes are almost the same. On the other hand, when B-Key DNA is added, much less number of red liposomes is seen compared to the number of Green liposomes.

Discussion
Comparing Fig.1 and 2, the ratio of Red to Green liposomes decreases due to the addition of B-Key DNA. This means that B-Key DNA only breaks Red liposomes and it has little effect on Green liposomes.
The selectivity of Key DNA has been successfully demonstrated.