Biomod/2011/TeamJapan/Tokyo/Project/Results: Difference between revisions
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:[[Image:Tokyo 1μEDAC.jpg|thumb|left| | :[[Image:Tokyo 1μEDAC.jpg|thumb|left|The result of denaturing PAGE of φ1 μm polystyrene beads using EDAC. | ||
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:Lanes of 5 to 8 are needed for checking deoxyribozyme activity of DNA ciliate body. Lane 5 and 6 are lanes for checking to polystyrene beads. If polystyrene beads had deoxyribozyme activity, the cleaved band would be appeared. Lanes of 7 and 8 are needed for checking that DNA ciliates body have deoxyribozyme activity. If DNA ciliate has normal deoxyribozyme activity, the cleaved band is appeared in lane 8 because metal ions are needed for deoxyribribozyme activity. | :Lanes of 5 to 8 are needed for checking deoxyribozyme activity of DNA ciliate body. Lane 5 and 6 are lanes for checking to polystyrene beads. If polystyrene beads had deoxyribozyme activity, the cleaved band would be appeared. Lanes of 7 and 8 are needed for checking that DNA ciliates body have deoxyribozyme activity. If DNA ciliate has normal deoxyribozyme activity, the cleaved band is appeared in lane 8 because metal ions are needed for deoxyribribozyme activity. | ||
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==Three independent modes of the DNA ciliate== | ==Three independent modes of the DNA ciliate== |
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Experimental Results
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<td align="center" width="200px"><a href="/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Results#The_body_of_the_DNA_ciliate"><img src="http://openwetware.org/images/4/4a/BIOMOD_Tokyo20111031Result_figure_ciliate.png" border=0 width=200 height=200></a></td>
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<td align="center" width="200px"><a href="/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Results#1._Free_moving_mode"><img src="http://openwetware.org/images/a/ac/BIOMOD_Tokyo20111031Result_figure1.png" border=0 width=200 height=200></a></td>
<td align="center" width="200px"><a href="/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Results#2._The_track_walking_mode"><img src="http://openwetware.org/images/0/05/BIOMOD_Tokyo20111031Result_figure2.png" border=0 width=200 height=200></a></td>
<td align="center" width="200px"><a href="/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Results#3._Light-irradiated_gathering_mode"><img src="http://openwetware.org/images/b/bf/BIOMOD_Tokyo20111031Result_figure3.png" border=0 width=200 height=200></a></td>
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The construction of the body of the DNA ciliate
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Method
- The deoxyribozymes were attached to the polystyrene body of the DNA ciliate using the following chemical reaction by 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDAC). The polystyrene bead for the DNA ciliate body has carboxyl groups on its surface. The EDAC reacts with the carboxyl group and forms a reactive group (see the following figure). An amino-modified DNA reacts with the reactive group on the bead surface, and then the DNA is immobilized on the surface of the polystyrene bead body. We carried out this reaction using a chemical reagent kit, PolyLink Protein Coupling Kit for COOH Microspheres (Polyscience) (see Protocols).
- After the above reaction, we investigated whether the deoxyribozymes were actually attached on the surface of the polystyrene body of the DNA ciliate, using a deoxyribozyme activity assays because the deoxyribozyme cannot be recognized through an optical microscope. The deoxyribozyme activity is an RNA cleaving activity in a solution with a divalent ion, Zn2+.
The results of the investigation of the deoxyribozyme activity on the DNA ciliate
In these experimentations, we could confirm two things. First was the deoxyribozyme activity of DNA ciliate body. Second was the firmness of the bond between polystyrene beads and deoxyribozyme. The deoxyribozyme activity of DNA ciliate body can be confirmed. If there is deoxyribozyme activity of DNA ciliate body, the cleaved substrate band is appeared. Furthermore, DNA ciliate body is removed before loading to polyacrylamide gel by centrifuge, so if deoxyribozyme can’t be attached to polystyrene beads firmly, the leg band appeared. In all pictures of gels, there are cleaved substrate bands in lane 8, so it is confirmed that all polystyrene beads are attached deoxyribozyme. Furthermore, there are not deoxyribozyme bands in lane 8, so it is confirmed that all polystyrene beads are attached deoxyribozyme firmly and the deoxyribozyme activity is not because of dislocated deoxyribozyme. In conclusion, we succeeded in making DNA ciliate body. Comparing four results, about both 200 nm and 1um polystyrene beads in diameter, the cleaved substrate band of EDAC method is stronger than EDC method, so DNA ciliate body made by EDAC method is better than EDC method. We decided using DNA ciliate body bodymade by EDAC method. |
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- Lanes of 5 to 8 are needed for checking deoxyribozyme activity of DNA ciliate body. Lane 5 and 6 are lanes for checking to polystyrene beads. If polystyrene beads had deoxyribozyme activity, the cleaved band would be appeared. Lanes of 7 and 8 are needed for checking that DNA ciliates body have deoxyribozyme activity. If DNA ciliate has normal deoxyribozyme activity, the cleaved band is appeared in lane 8 because metal ions are needed for deoxyribribozyme activity.
Three independent modes of the DNA ciliate
1. Free moving mode
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Method
- In the observation of the DNA ciliate, we used 1× saline-sodium citrate (SSC) buffer with 3% bovine serum albumin (BSA) (the materials for experiments are listed in Protocols). The sizes of DNA ciliate bodies were 200 nm and 1 μm. We put the solution including the DNA ciliates on a glass slide and covered by a cover slip. The solutions including the DNA ciliates were observed by a phase-contrast microscope and took videos (the equipment for experiments is also listed in Protocols).
Results
- Video (A1) (the left two videos) shows the observation results of the DNA ciliates with a diameter of 200 nm under the optical microscope. Video (B1) (the right two videos) shows the observation results of the DNA ciliates with a diameter of 1 μm. The lower videos are enlarged views of the upper videos.
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<td>Video (B1)
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- From these videos, we observed that the DNA ciliates were freely and randomly moving in the solution. By comparing the motion of the DNA ciliates with a diameter of 200 nm and 1 μm, the smaller DNA ciliates (200 nm in diameter) moved more strongly than the larger DNA ciliates (1 μm in diameter). We observed a very slow directional flow of the solution as an experimental artifact but the motion of the DNA ciliate was random independently of the flow. In addition, we observed some DNA ciliates that did not move at all; the DNA ciliates were probably crystalized one another or sticking at the surface of the glass slide.
- In conclusion, we achieved the free moving mode of the DNA ciliate. The motion of the DNA ciliate was based on the Brownian motion of the DNA ciliates. The random motion of the smaller DNA ciliates was stronger than that of the larger DNA ciliate. This result consistent with the theory of the Brownian motion described in Project page.
2. The track walking mode
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Confirmation of Deoxyribozyme activity
- We confirmed the cleaving activity of the deoxyribozyme for cilia attached on the DNA ciliate body using the polyacrylamide gel electrophoresis. Actually, the results have already been shown in the section of the confirmation of constructing the DNA ciliate body above (see the results) From the results, we conclude that the deoxyribozyme activity for the substrate DNA worked as we designed.
Construction of DNA tracks
Method of the construction of DNA tracks
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Results of the construction of DNA tracks
- Figure 1 shows a picture of the polyacetal resin mold (the mold for microchannels are shown by broken lines). This is a part of large microchannels we used to array DNAs. Figure 2 shows a fluorescence microscope image of the DNA tracks. In Figure 3, we observed the fluorescence of the DNA hybridizing with the DNA track arrayed on the glass plate. In addition, Figure 6 shows the whole picture of the large DNA tracks with a human like shape (observed similarly).
- In conclusion, we successfully constructed DNA tracks using microchannels, and we confirmed the ability of hybridization between the immobilized DNA as the tracks and its complementary DNA. Thus, we believe that the deoxyribozyme on the DNA ciliate also hybridizes with the track DNA, which has a complementary DNA sequence of the deoxyribozyme.
Investigation of the directional walking by simulations
- We investigated the directional walking of the DNA ciliate by numerical simulations (see Simulations)
3. Light-irradiated gathering mode
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UV-switching system
- The UV-switching DNA has a stem-loop structure and short blocking DNA, which blocks hybridization of deoxyribozyme. After UV irradiation, this loop becomes open, and hybridize with the deoxyribozyme. (more detail...)
1. Confirmation of UV-switching
Results
- Non-denaturing 20% PAGE result is here.
- U…UV-switching-trap-DNA
- B…Blocking DNA
- D…Deoxyribozyme DNA
- Reaction solution…A 0.225uM and B 0.45uM and D 0.225uM
- All solutions are in 5x SSC (sodium citrate 75mM)
- From left, these bands mean followings.
- U 0.225uM and Mg2+ 80mM
- B 0.45uM and Mg2+ 80mM
- D 0.225uM and Mg2+ 80mM
- U 0.225uM and B 0.45uM and Mg2+ 80mM
- U 0.225uM and D 0.225uM and Mg2+ 80mM (UV isn’t spotted)
- U 0.225uM and D 0.225uM and Mg2+ 80mM (UV is spotted for 60 min.)
- Reaction solution (UV isn’t spotted)
- Reaction solution (UV is spotted for 15 min.)
- Reaction solution (UV is spotted for 60 min.)
- Reaction solution and Mg2+ 80mM (UV isn’t spotted)
- Reaction solution and Mg2+ 80mM (UV is spotted for 15 min.)
- Reaction solution and Mg2+ 80mM (UV is spotted for 60 min.)
- The control bands were appeared in lane 1 to 6. Lane 4 (U 0.225uM and B 0.45uM and Mg2+ 80mM) means the bands when the loop is stable and hybridization U and B (band U-B). Lane 5 (U 0.225uM and D 0.225uM and Mg2+ 80mM (UV isn’t spotted)) means the bands when the loop is open and hybridization of U and D. Lane 6 (U 0.225uM and D 0.225uM and Mg2+ 80mM (UV is spotted for 60 min.)) means the bands when the loop is open and spotted UV (band U-D).
- In the presence of Mg2+, the switching was caused clearly (lane 10 to 12) because of the stable effect of Mg2+.
- Before UV irradiation (lane 10), the UV-switching DNA was closed state (band U-B). After UV irradiation (lane 11, 12), the band shifted to the position of hybridized state (band U-D). Thus, the UV-switching device we designed worked successfully as we intended.
2. Gathering at the specific area
method
- Attaching complementary DNA of deoxyriboazyme on a glass plate
- Making the situation which deoxyribozymes hybridize with complementary DNA on the glass plate
- How to make the situation for hybridization is here
- Putting DNA ciliates on the glass plate
- Waiting for 2 hours
- Observing the DNA ciliates under an fluorescent microscope
result
- A fluorescent image of the DNA ciliates gathering at the spot of complementary DNA is here.
- Complementary DNA was attached on upper-right area in this image.
- There was no DNA in lower-left area in this image.
- DNA ciliates gathered at the spot of complementary DNA, and didn't gather at another area. Following this result, it was confirmed that DNA ciliates can gather at the specific area after UV irradiation.