Biomod/2011/TeamJapan/Tokyo/Project/Results

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Experimental Results

  • We constructed the micrometer-sized body of the DNA ciliate by surface modification.
    Shown in detail below






  • We confirmed that DNA ciliate can move freely and randomly alomost all area based on Brownian motion.
    Shown in detail below
  • We confirmed that substrate DNA are cut by deoxyribozyme legs of DNA ciliate.
  • We constructed DNA tracks by using the microchannel.
  • We confirmed that DNA ciliates move directionally in simulation.
    Shown in detail below
  • We designed the DNA which react.
  • When we irradiated UV, we succeeded to gather beads at the spot where we irradiate UV.
    Shown in detail below

The construction of the body of the DNA ciliate

The DNA ciliate has a micrometer-sized body with DNAs as cilia. We constructed the micrometer-sized body of the DNA ciliate using a polystyrene bead. The DNAs as cilia on the body was made of deoxyribozyme DNAs. To attach the deoxyribozyme DNAs to the polystyrene body, a chemical covalent bond, the amide bond, was utilized. Here, we show the construction method of the DNA ciliate and the investigation of the constructed DNA ciliate based on deoxyribozyme activity assays.

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).
The scheme of the reaction of EDAC
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


From the right pictures, the cleaved band appears clearly in lane 8, so we confirmed this DNA ciliate body has strong deoxyribozyme activity. In lane 8, there is not deoxyribozyme band, so substrate is cleaved by DNA ciliate body, not cleaved by deoxyribozyme.


In lane 8, the density of the cleaved band is varies between four results. By Comparing density between substrate and cleaved substrate, it is thought the deoxyribozyme activity of the DNA ciliate which 1um in diameter is stronger than the DNA ciliate which 200nm in diameter. However, the mass of DNA ciliate is difficult to make uniform, so we cannot determine the quantity of deoxyribozyme of each result.


In conclusion, in two DNA ciliate bodies, there is deoxyribozyme activity. Furthermore, deoxyribozyme is not dissociated from DNA ciliate body. In conclusion, we confirmed deoxyribozyme is attached to DNA ciliate body firmly.
The denaturing PAGE image for the investigation of the deoxyribozyme activity on the DNA ciliate which is 200nm in diameter
The denaturing PAGE image for the investigation of the deoxyribozyme activity on the DNA ciliate which is 200nm in diameter


The denaturing PAGE image for the investigation of the deoxyribozyme activity on the DNA ciliate which is 1um in diameter
The denaturing PAGE image for the investigation of the deoxyribozyme activity on the DNA ciliate which is 1um in diameter



Three independent modes of the DNA ciliate

1. Free moving mode

In the free moving mode, the DNA ciliates freely and randomly move around in a broad range of space. Here, we confirmed the free moving mode by the observation of the DNA ciliate that exhibited the Brownian motion in an aqueous solution. (see below)

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 (A) (the left two videos) shows the observation results of the DNA ciliates with a diameter of 200 nm under the optical microscope. Video (B) (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.

Video (A) Video (B)

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

The DNA ciliates walk directionally along the DNA tracks using deoxyribozyme cleving activity as described in Project page. We designed a deoxyribozyme and a substrate DNA for the track (see DNA design).
As already mentioned in the project page, we used "Deoxyribozyme-substrate reaction "
Here, we show the results of (1) conformation of deoxyribozyme activity, (2) construction of DNA tracks, and (3) investigation of the directional walking of the DNA ciliate.

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

To construct the DNA tracks, we used microchannels to immobilize the substrate DNA at a specific region we designed. The construction of the microchannel is as follows. A mold of a microchannel was constructed with polyacetal resin by cutting the polystyrene resin as we designed with an endmill controlled by micro-machining system.
The poly(dimethylsiloxane) (PDMS) and its hardener were mixed at the ratio of 10:1, and bubbles in the mixed PDMS solution was cleaned with a vacuum desiccator. The PDMS solution was poured over the mold and cured and hardened the PDMS solution at 75 degree Celsius for 1 hour. Then, the hardened PDMS was peeled off from the mold. The PDMS has a microchannel transferred from the mold.
Next, we constructed a DNA track using the PDMS microchannel. To make DNA tracks, we used microchannel and arrayed DNAs on a glass plate. To attach DNAs on a glass plate, we used disuccinimidyl suberate (DSS) as a linker between amino-modified DNA and an amino-modified glass plate (MAS-coated glass, Matsunami) . The DSS linker reacts with amino groups that are exposed on the surface of the MAS-coated glass . By the DSS linker, DNAs were attached on the glass plate by a covalent bond. We poured an amino-modified DNA solution into the PDMS microchannel on the MAS-coated glass plate treated with DSS; thus, the DNAs were arrayed as the shape of the microchannel we designed. We can design the shapes of microchannels freely, so we can make DNA tracks with arbitrary shapes. Finally, we observed the arrayed DNA track by hybridizing a fluorescence-labeled DNA complementary to the DNA strands of the DNA track.
Construction of DNA track

A series of attaching aminated DNA to glass reaction

Confirmed by DNA hybridization

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. We observed the fluorescence of the DNA hybridizing with the DNA track arrayed on the glass plate. In addition, Figure 3 shows the whole picture of the large DNA tracks with a human like shape (observed similarly).
figure 1. The image of microchannel in PDMS-mold. This figure was observed by phase contrast.
figure 1. The image of microchannel in PDMS-mold. This figure was observed by phase contrast.
Figure 2. the result of arrayed DNAs on glass plate using microchannel of Figure 1 and hybridized with their complementary fluorescent labeling DNA strands. This figure was observed by fluorescent phase contrast.
Figure 2. the result of arrayed DNAs on glass plate using microchannel of Figure 1 and hybridized with their complementary fluorescent labeling DNA strands. This figure was observed by fluorescent phase contrast.
Figure 3. The microchannel of human form The left image is the design drawing. The right figure is the result of hybridization. Because camera view was too narrow to observe total image, we stuck together the part of pictures.
Figure 3. The microchannel of human form The left image is the design drawing. The right figure is the result of hybridization. Because camera view was too narrow to observe total image, we stuck together the part of pictures.


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.
Figure 4. Fluorescent image of DNA ciliate gathering at the specific area by fluorescent microscopy.
Figure 4. Fluorescent image of DNA ciliate gathering at the specific area by fluorescent microscopy.
Figure 5. Fluorescent image of DNA ciliate gathering at the specific area by fluorescent microscopy.
Figure 5. Fluorescent image of DNA ciliate gathering at the specific area by fluorescent microscopy.
In conclusion, we observed the DNA ciliates stayed at the spot of the substrate DNA on a glass plate.

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

In the light-irradiated gathering mode, DNA ciliates gather at a specific area responding to UV irradiation. This mode is achieved by UV-switching DNA devices and gathering of DNA ciliates.
In this page, we show the two experimental results: confirmation of UV-switching and observation of gathering DNA ciliates. It is confirmed that UV-switching system worked and that DNA ciliates gathered, so the light-irradiated gathering mode will be achieved.

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...)


Confirmation of UV-switching

Method

We did native-PAGE to check UV-switching system. (more detail...)

Results

Image of non-denaturing 20% PAGE for the confirmation of UV-switching
Image of non-denaturing 20% PAGE for the confirmation of UV-switching
  • The control bands were appeared in lane 1 to 6. Lane 4 means the bands when the loop is stable and hybridization U and B . Lane 5 means the bands when the loop is open and not spotted UVhybridization of U and D. Lane 6 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+.
  • From the picture, in lane 10, there is the band B+U and not the band U+D, so it is confirmed that when UV is not spotted, UV-switching DNA is close the loop. On the other hand, in lane 11 and 12, there is the band U+D and not the band B+U, so it is confirmed that when UV is spotted, UV-swithching DNA is open the loop.
  • In lane 4,5, and 6, there is a band which is neither monomer nor hybridized. In lane 1, 2, and 3, there is only a band, so the band is not dimer, so we thought the band is another hybridizing structure. The density of these bands is much weaker than the band of normal hybridizing structure, so we thought this band is little effect to UV-switching system.
  • Comparing lane 5 and 6, the density of the bands in lane 6 is weaker than in lane 5. We thought the density of the band U+D becomes weak by spotting UV. We thought that is a reason the band of Lane 11 and 12 are weak.
  • Comparing lane 11 and 12, the density of the band U+D is much the same, so we thought UV-switching finished in 15 minutes in this experimentation.
  • In conclusion, we confirmed UV-switching DNA changes the structure and branch migration happens when UV is spotted.

Gathering at the specific area


A fluorescent image of DNA ciliate gathering at the specific area by fluorescent microscopy.
A fluorescent image of DNA ciliate gathering at the specific area by fluorescent microscopy.

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

Results

  • 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.
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