Biomod/2012/TeamSendai/Experiment
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Gate formation
We made the Gate and observed whether it is successfully made or not by 3 steps: annealing, electrophoresis, and AFM.
Electrophoresis & AFM
We mixed M13mp18 and staples of tube and annealed them. We tried two annealing method. The one is annealing with boiled water, and the other is annealing based on ”A Logic-Gated Nanorobot for Targeted Transport of Molecular Payloads”. And we did electrophoresis using this sample. This results is following.
As a result of the experiment, we got that the Shawn annealing was better than annealing with boiled water. And the tube structure made on Shawn annealing was observed by AFM. This result is following.
This object whose shape is rectangular object may be the tube because we consider that the shape of the tube is transformed to rectangle when it is crushed by cantilever. Then, we tried various patterns of annealing condition; the concentration of Mg and the speed of cooling. We tried three condititons of annealing.
i ) 20h annealing (80℃ to 70℃→2min/-1℃,70℃ to 30℃→30min/-1℃,30℃ to 4℃→2min/-1℃)
ii ) 40h annealing （80℃ to 70℃→2min/-1℃,70℃ to 30℃→60min/-1℃,30℃ to 4℃→2min/-1℃）
iii) annealing based on the article by Dr. Shawn Douglas [1].
For the result, the GATE annealed by 20h annealing and 40h annealing could been seen as a band when we did electrophoresis but could not been seen when we observed by AFM. On the other hand, when we observed the GATE annealed by the condition based on the article proposed by Dr. Shown Douglas [1] by AFM, we could find several structural object. In general, we considered the best condition of annealing is next; the condition of Mg is 8 mM the speed of annealing is based on the article proposed by Dr. Shawn.
Next,we tried to create the GATE which can be decorated with cholesterol to connect it with liposome. We call this tube “connect-able GATE”.
When we annealed the connect-able GATE and did electrophoresis, the band could be seen at the similar position compared with normal GATE. For this, we consider the connect-able GATE also may be created and we could find many granular objects when we observed the connect-able GATE by AFM.
[1] A Logic-Gated Nanorobot for Targeted Transport of Molecular Payloads
(Shawn M. Douglas,* Ido Bachelet,* George M. Church 2012)
Porter
We carried out electrophoresis to see if the target moved and hybridized to Porter of higher bonding energy and if the target was successfully transported through the three Porters.
Electrophoresis
DNA sequences
Transportation of Target by Porter
Protocol
First, we mixed Porter1 and the target for reaction.
Second, we added Porter2, expecting that the target would be passed from Porter1 to Porter2.
Finally, we added Porter3, expecting that the target would be passed from Porter2 to Porter3.We waited for 15 minutes at each step, letting the target attach to a Porter of higher bonding energy.
The final concentration of samples were follows.
The target DNA: 0.5 μM
Porter 1,2,3: 1.0 μM
Dilution was performed with 1 x TAE Mg2+. The total volume was 5 μM.
Electrophoresis were performed with:
20% acrylamide gel
1×TAE as running buffers
Constant volt 100 V, 3h
Results
If the target has passed well through Porters, bands of the target hybridizing with Porter1, 2, and 3 should appear. In the gel, bands above each Porter can be seen as the Porter hybridizing with the target.
In the first 15 minutes, Porter1 attached to the target (fig.1: lane 3). After another 15 minutes, the band of Porter1 and Porter2 hybridizing with the target were seen. In the last 15 minutes, the band of Porter1 and Porter2 and Porter3 were seen.
So the result shows that target hybridized with available Porter during the 15 minutes, and then it moved to the Porter that has higher bonding energy during another 15 minutes. Therefore, this indicates that Porters successfully work as “porter” of the target.
We also saw the gel after staining all the samples (fig.2)
Comparison of Porter and Toehold
We saw that Porter catches the target more effectively than the toehold structure by our simulations. In addition, we actually confirmed this idea through experiments.
Protocol
We compared the carrier set of the target as below.
1. Porter1 and Porter2
2. Toehold A and toehold B
First, we mixed the target and Porter1. At the same time, we also mixed the target and toehold A.Second, we added Porter2 to the sample of Porter1 and the target. At the same time, we added toehold B to the sample of toehold B and the target. We waited for 15 minutes at each step, for the sample to react.
The final concentration of samples were follows.
The target DNA: 0.5 μM
Porter 1,2,3: 1.0 μM
Toehold A,B: 1.0 μM
Dilution was performed with 1 x TAE Mg2+. The total volume was 5 μM.
Electrophoresis were performed with:
20% acrylamide gel
1×TAE as running buffers
Constant volt 100 V, 3h
Results
Similar to Transportation of Target by Porter, Porters and the target, Porter1 and Porter2 attached to the target respectively. Also, the target was passed between Porters (figure3: lane 7).
As for the toehold structure, the target hybridized with toehold B. On the other hand, in the lane of the target and toehold A, the band of the target was still strong (lane 9). So, the target didn’t hybridize with toehold A.
It follows that toehold A cannot catch the target only in 15 minutes, and that the target cannot be delivered by using toehold structures.
Therefore, we concluded that Porter structure, which has some loops, is more effective and efficient than toehold in terms of catching the target.
Membrane
Cholesterol tag modification
Cholesterol legs were attached to the GATE by mixing Cholesterol conjugated DNA. The GATE has a DNA strand around the side-surface, which has complementary sequence of the Cholesterol conjugated DNA. Hybridization of the cholesterol DNA was performed by mixing with the GATE after annealing at room temperature.
Electrophoresis
M13 single strand DNA, the GATE, and the GATE mixed with Cholesterol conjugated DNA was loaded on agarose gel. After 1 h electrophoresis at 50 V, DNA were stained by SYBR Gold. By mixing Cholesterol conjugated DNA, a slower migration band appeared. Such a slower migration band were not observed without mixing Cholesterol conjugated DNA. Thus, we concluded, Cholesterol conjugated DNA was introduced to the GATE.
Making mini-gate
We prepared a preliminary step to that cell gate insert into the liposome. We designed a smaller tube and attempted to insert into liposomes using it.
Similar to the cell gate, we stretched single-stranded DNA of 10 bases that can be modified cholesterol from the side of this tube.
We attached cholesterol to the single-stranded DNA, and confirmed by electrophoresis.
We expected that these enter into the hydrophobic portion of the liposome. Then, it is likely to that the tube stick to the liposome.
Making liposome
We examined the appropriate composition of the liposome. And we decided the composition; DOPC:DSPE-PEG2000: Fructose = 100:1:1000.
General methods for formation and functional analysis of liposome with DNA
At first, the lipids mixture as follows was prepared.
DOPC 5mM 10μL
DSPE-PEG2000 0.5mM 1μL
Fructose 10mM 50μL
Tube 20μL
Chloroform 70μL
We dry a sample of that composition under argon gas condition.
For liposome formation, 125μL of 1×TAE Mg2+ were added and incubate for 1 h at room temperature.
When indicated, 1 mM Lucifer Yellow fluorescein was added 1/50 fold of total volume (20 μM final). For DNA staining 1/100 fold Hoechst was added after liposome formations. Fluorescence of DNA by Hoechst was observed by a fluorescence microscope.
As this picture, we observed liposome. So there seems to be some liposome.
Confirming insertion of the Gate s into the membrane
We use fluoresce moelcules to confirm that the tube insert into the liposome. If the mini-gate were inserted into membrane and make a hole on membrane, inner solution without negative charges should be leaked. Lucifer Yellow fluorescein were encapsulated into giant liposome. Leaking was measured by comparing fluorescent intensity between inside and outside of liposome.As a positive control of the leaking assay, alpha- Hemolysin protein was used. alpha- Hemolysin protein is known to make a hole in liposomes. The following figure summarizes the leaking assay.
And, the result of the leaking assay with positive control, negative control, and with the mini-gate, are shown in the following figures.
This is the image of the vesicle which putting alpha-hemolysin. Dotted yellow line is the position of vesicle.And yellow line area is a domain of a graph expressed by "FigureA2'"'
This is the gray scale graph using ImageJ soft.The area of this graph is expressed by FigureA1's yellow line. Z-axis's gray scale is separated to 256 gradation.The differences between maximum and minimum of the gray scale are about 0 gradation.
This is the image only vesicles. Yellow line area is a domain of a graph expressed by "FigureB2'"'
This is the gray scale graph using ImageJ soft.The area of this graph is expressed by FigureB1's yellow line. Z-axis's gray scale is separated to 256 gradation.The difference between maximum and minimum of the gray scale is about 10 gradation.]]
This is the image of the vesicle which putting the cholesterol modified mini-gate. Yellow line area is a domain of a graph expressed by "FigureC2'"'
This is the gray scale graph using ImageJ soft.The area of this graph is expressed by FigureC1's yellow line. Z-axis's gray scale is separated to 256 gradation.The difference between maximum and minimum of the gray scale is about 5 gradation.
Figure A1 is a image of positive control, vesicles with many hole by alpha-hemolysin. If our mini-gate modified cholesterol insert into liposomes, fluorescein is expected to be similar to this positive control. Figure B1 is negative control image existing only vesicle. Figure C1 is the result of adding mini-gate modified cholesterol to condition of existing only vesicle. Gray scale is separated to 256 gradation. 'ImageJ tool’was used for analysis. In Figure B2, the difference of between maximum and minimum of the gray scale is about 10 gradation. But in Figure C2, the difference between maximum and minimum of the gray scale is about 5 gradation. These result weakly suggest the mini-gate with modified cholesterol are inserted into liposome, because the gray scale graph of FigureC2 is medium between negative and positive controls. We should obtain more data to analysis for statistics.
SPR analysis
Purpose
Confirming whether min-gate with cholesterol-leg sticks onto liposome by using surface plasmon resonance (SPR).
What's SPR analysis?
SPR is a optical phenomena that can monitor the interaction between the biological molecules and the surface in real time, without any labeling. We used Biacore-X(GE) for analysis the attaching event. To study an interaction, one of the interaction partners is immobilized onto the sensor surface of a Biacore sensor chip. Immobilization occurs by direct coupling to the surface or via a suitable molecule already coupled to the surface. It is necessary to choose the most suitable sensor tip by contents of the kind of molecules to immobilize and the analysis.
We used sensor tip L1. The matrix is that: lipophilic groups are covalently attached to carboxymethylated dextran, making the surface suitable for direct attachment of lipid membrane vesicles such as liposomes. After attachment, the lipid bilayer structure is retained, facilitating the study of interactions involving transmembrane receptors in membrane-like environments.
Immobilization of lipid
1. Set a sensor tip L1 and running buffer on the SPR sensor.
2. Inject 5% Triton X-100 25 μL when the sensor gram becomes steady.
3. Inject lipid suspended in running buffer 100 μL when the sensor gram becomes steady again.
4. Inject 10mM NaOH 5 μL.
5. Inject the lipid 50 μL.
6. Inject 10mM NaOH 5 μL.
7. Repeat 5-6 until the baseline of the sensor gram does not rise.
8. Check the rise in RU levels is 100 RU or under after injection of 100 μg/mL BSA 25 μL.
Running buffer: 10 mM HEPES 150 mM NaCl pH =7.0
BSA: Bovine Serum Albumin
Measurements of samples
Inject sample 10 μL and start measurement. After end of measurement, inject 10 mM NaOH 5 μL. It is necessary to unify measurement time and RU levels when inject sample.
Sample 1:
min-gate (4.2 nM) 50 μL
Cholesterol 12 μL
Running buffer 38 μL
Sample2:
min-gate 50 (4.2 nM) μL
Running buffer 50 μL
Results
From the graph, we confirmed that min-gate with cholesterol-leg had higher RU levels than only min-gate. It shows that min-gate with cholesterol-leg has higher strength of binding. Therefore, we can insert min-gate into liposome by attaching cholesterol-leg.
References
Biacore Life Sciences https://www.biacore.com/lifesciences/index.html
