Biomod/2011/TeamJapan/Tokyo/Project/Results: Difference between revisions

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:We use two methods to attach DNA to polystyrene beads. In both methods, we bind polystyrene beads' carboxylic acid to amino group of aminated DNA. In first method, we apply reference <sup>[1]</sup>. In this method, we used linker between polystyrene beads and aminated DNAs. The linker has amino group and carboxylic acid. The linker’s amino group combines with carboxylic acid of polystyrene beads and the linker’s carboxyric acid combines with amino group of aminated DNA, so polystyrene beads combine DNA through the linker. In second method, we use NHS and EDC to alter carboxylic acid to NHS. NHS has very high reactivity, and DNA's amino group reacts with NHS of porystyrene beads. The DNA ciliate body is developed in this process. :<br>As a motor of DNA ciliate, we used deoxyribozyme which is the enzyme comprised of DNA. Deoxyribozyme cleaves its substrate at an RNA base, if there are 2+ metal ions. Using this reaction, DNA ciliate can move.
:We use two methods to attach DNA to polystyrene beads. In both methods, we bind polystyrene beads' carboxylic acid to amino group of aminated DNA. In first method, we apply reference <sup>[1]</sup>. In this method, we used linker between polystyrene beads and aminated DNAs. The linker has amino group and carboxylic acid. The linker’s amino group combines with carboxylic acid of polystyrene beads and the linker’s carboxyric acid combines with amino group of aminated DNA, so polystyrene beads combine DNA through the linker. In second method, we use NHS and EDC to alter carboxylic acid to NHS. NHS has very high reactivity, and DNA's amino group reacts with NHS of porystyrene beads. The DNA ciliate body is developed in this process. :<br>As a motor of DNA ciliate, we used deoxyribozyme which is the enzyme comprised of DNA. Deoxyribozyme cleaves its substrate at an RNA base, if there are 2+ metal ions. Using this reaction, DNA ciliate can move.


===Principle and methods===
===Methods===
*Two experiments were needed to complete developing DNA ciliate body.<br>First experiment was creating DNA ciliate by attaching DNAs to polystyrene beads. This process is used the reaction of connecting aminated DNAs’ amino group and polystyrene beads’ carboxylic acid. We took two methods to react. Both methods are used the common reaction, but chemical materials are different. First method is used '''EDC and NHS'''. This induces transforming carboxylic acid to succinimide which is been able to react with aminated DNAs and connect.
*Two experiments were needed to complete developing DNA ciliate body.<br>First experiment was creating DNA ciliate by attaching DNAs to polystyrene beads. This process is used the reaction of connecting aminated DNAs’ amino group and polystyrene beads’ carboxylic acid. We took two methods to react. Both methods are used the common reaction, but chemical materials are different. First method is used '''EDC and NHS'''. This induces transforming carboxylic acid to succinimide which is been able to react with aminated DNAs and connect.
:[[Image:DNA beads method1.jpg|Figure1.the result of PAGE of φ200 nm polystyrene beads using NHS and EDC. |900px]]
:[[Image:DNA beads method1.jpg|Figure1.the result of PAGE of φ200 nm polystyrene beads using NHS and EDC. |900px]]
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:<br>Second experiment is confirming whether deoxyribozyme is attached to polystyrene beads and able to cleave substrate. We confirm deoxyribozyme activity by urea-PAGE. Making mixture of DNA ciliate and substrate and Zn<sup>2+</sup> ions. If DNA ciliate has deoxyribozyme activity, substrate is cleaved and the band of cleaved substrate appears as a band.
:<br>Second experiment is confirming whether deoxyribozyme is attached to polystyrene beads and able to cleave substrate. We confirm deoxyribozyme activity by urea-PAGE. Making mixture of DNA ciliate and substrate and Zn<sup>2+</sup> ions. If DNA ciliate has deoxyribozyme activity, substrate is cleaved and the band of cleaved substrate appears as a band.


===Protocol===
*Creating DNA ciliate, we use (1) and (2) protocols.
*Creating DNA ciliate, we use (1) and (2) protocols.
**(1) The method of using EDC and NHS is [[Biomod/2011/TeamJapan/Tokyo/Notebook/Protocols#How to make samples for electrophoresis|here]].
**(1) The method of using EDC and NHS is [[Biomod/2011/TeamJapan/Tokyo/Notebook/Protocols#How to make samples for electrophoresis|here]].
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**(3) The method of electrophoresis is [[Biomod/2011/TeamJapan/Tokyo/Notebook/Protocols#How to make samples for electrophoresis|here]].
**(3) The method of electrophoresis is [[Biomod/2011/TeamJapan/Tokyo/Notebook/Protocols#How to make samples for electrophoresis|here]].
**(4) The method of making sample is  
**(4) The method of making sample is  
 
<!-- 上記のプロトコルはPrinciple and methodsからうまくつながるようにつなぐ　河村-->
===Results===
===Results===
*Following is the result of electrophoresis. Result 1 is DNA ciliate using NHS and EDC. Result 2 is DNA ciliate using EDAC. Meanings of the numbers above results are here.
*Following is the result of electrophoresis. Result 1 is DNA ciliate using NHS and EDC. Result 2 is DNA ciliate using EDAC. Meanings of the numbers above results are here.
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===Discussions===
===Discussions===
*First, we explain this experimentation’s appropriateness for checking DNA ciliate body. To create DNA ciliate body, it was necessary to attach deoxyrobozyme to micrometer-sized polystyrene beads firmly. Furthermore, we had to check deoxyribozyme activity of DNA ciliate body. In this method, these two things could be checked. First, the degree of fixation can be checked. DNA ciliate is removed before loading to polyacrylamide gel, so if deoxyribozyme can’t be attached to polystyrene beads firmly, the leg band is appeared. Second, 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. Based on the above, this method is appropriate for checking DNA ciliate body.
*First, we explain this experimentation’s appropriateness for checking DNA ciliate body. To create DNA ciliate body, it was necessary to attach deoxyrobozyme to micrometer-sized polystyrene beads firmly. Furthermore, we had to check deoxyribozyme activity of DNA ciliate body. In this method, these two things could be checked. First, the degree of fixation can be checked. DNA ciliate is removed before loading to polyacrylamide gel, so if deoxyribozyme can’t be attached to polystyrene beads firmly, the leg band is appeared. Second, 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. Based on the above, this method is appropriate for checking DNA ciliate body.
*Second, we explain necessity of lanes. Lanes of 1 to 4 are needed for checking deoxyribozyme activity and the positions of each band of DNAs. Lane 1 and lane 2 are control lanes. The band in lane 1 is deoxyribozyme band, and the band of lane 2 is substrate band. Lane 3 and lane 4 are lanes to check deoxyribozyme activity. The solutions in lane 3 doesn’t contain of Zn(2+), the solution in lane 4 contains of Zn(2+). The cleaved band is appeared in lane 4, but doesn’t be appeared in lane 3. This means normal deoxyribozyme isn’t active in no metal ions solutions. Lane 4’s bottom band means position of cleaved substrate.  
<!--実験の適切性についての記述はいらない　書くにしてもResultsの中　河村-->
]*Second, we explain necessity of lanes. Lanes of 1 to 4 are needed for checking deoxyribozyme activity and the positions of each band of DNAs. Lane 1 and lane 2 are control lanes. The band in lane 1 is deoxyribozyme band, and the band of lane 2 is substrate band. Lane 3 and lane 4 are lanes to check deoxyribozyme activity. The solutions in lane 3 doesn’t contain of Zn(2+), the solution in lane 4 contains of Zn(2+). The cleaved band is appeared in lane 4, but doesn’t be appeared in lane 3. This means normal deoxyribozyme isn’t active in no metal ions solutions. Lane 4’s bottom band means position of cleaved substrate.  
: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 DNA ciliate body’s 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 DNA ciliate body’s 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.
 
<!--この内容はResultsへ。また、ここのディスカッションにはなぜビーズの直径が大きくなると凝集しやすくなるのかを推測するといいのでないでしょうか　河村-->
==1. Free moving mode==
==1. Free moving mode==
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<div id="navigation"> <div id="menu" style="position:static"> <ul> <li><a class="aMain" href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo">Home</a></li> <li><a class="aTeam" href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Team/Students">Team</a></li> <li><a class="aProject" href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project">Project</a> <!-- <ul> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project">Overview</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/introduction">Introduction</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Model">Model</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Devices">Devices</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Modes">Modes</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Results">Results</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Achievements">Achievements</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Future_works">Future works</a></li> </ul> --> <li><font color="#ffffff">Results</font> <ul> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Results">Experiments</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Simulations">Simulations</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Achievements/DNA_Devices">DNA Design</a></li> </ul></li> <!-- <li><a class="Simulation" href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Simulations">Simulations</a></li> <li><a class="DNA design" href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Achievements/DNA_Devices">DNA Designs</a></li> --> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Achievements">Achievements</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Future_works">Future works</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Notebook/Protocols">Protocols</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Notebook/Lab.notebook">Notes</a></li> <li><a class="aNotebook" href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Sponsors/">Sponsors</a></li> <li><a class="aSitemap" href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Sitemap">Sitemap</a></li>

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Results

<html><body> <table border=0 width=250 align=left> 

 <th><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Results_of_DNA_Ciliate_Body"><img src="http://openwetware.org/images/4/4a/BIOMOD_Tokyo20111031Result_figure_ciliate.png" border=0 width=200 height=200></a></th>

</tr> 

<tr align=center>
 <td><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Results_of_Free_Moving_Mode"><img src="http://openwetware.org/images/a/ac/BIOMOD_Tokyo20111031Result_figure1.png" border=0 width=200 height=200></a></td>
</tr>
<tr align=center>
 <td><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Results_of_Track_Walking_Mode"><img src="http://openwetware.org/images/0/05/BIOMOD_Tokyo20111031Result_figure2.png" border=0 width=200 height=200></a></td>
</tr>
<tr align=center>
 <td><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Results_of_Light_Irradiated_Gathering_Mode"><img src="http://openwetware.org/images/b/bf/BIOMOD_Tokyo20111031Result_figure3.png" border=0 width=200 height=200></a>
</td>
</tr>

</table> </html>


・We succeeded to attach micrometer-sized body with many DNA strands. So, we can give three functional modes to DNA ciliate.





・We confirmed that DNA ciliate can move freely and randomly alomost all area based on Brownian motion.





・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.





・We designed the DNA which react.
・When we irradiated UV, we succeeded to gather beads at the spot where we irradiate UV.


DNA ciliate body

We constructed micrometer-sized body attached DNA strands.
We use two methods to attach DNA to polystyrene beads. In both methods, we bind polystyrene beads' carboxylic acid to amino group of aminated DNA. In first method, we apply reference [1]. In this method, we used linker between polystyrene beads and aminated DNAs. The linker has amino group and carboxylic acid. The linker’s amino group combines with carboxylic acid of polystyrene beads and the linker’s carboxyric acid combines with amino group of aminated DNA, so polystyrene beads combine DNA through the linker. In second method, we use NHS and EDC to alter carboxylic acid to NHS. NHS has very high reactivity, and DNA's amino group reacts with NHS of porystyrene beads. The DNA ciliate body is developed in this process. :
As a motor of DNA ciliate, we used deoxyribozyme which is the enzyme comprised of DNA. Deoxyribozyme cleaves its substrate at an RNA base, if there are 2+ metal ions. Using this reaction, DNA ciliate can move.

Methods

  • Two experiments were needed to complete developing DNA ciliate body.
    First experiment was creating DNA ciliate by attaching DNAs to polystyrene beads. This process is used the reaction of connecting aminated DNAs’ amino group and polystyrene beads’ carboxylic acid. We took two methods to react. Both methods are used the common reaction, but chemical materials are different. First method is used EDC and NHS. This induces transforming carboxylic acid to succinimide which is been able to react with aminated DNAs and connect.
Figure1.the result of PAGE of φ200 nm polystyrene beads using NHS and EDC.
Second method is used EDAC. EDAC reacts with both aminated DNAs and polystyrene beads’ carboxylic acid.
Figure1.the result of PAGE of φ200 nm polystyrene beads using NHS and EDC.

Second experiment is confirming whether deoxyribozyme is attached to polystyrene beads and able to cleave substrate. We confirm deoxyribozyme activity by urea-PAGE. Making mixture of DNA ciliate and substrate and Zn2+ ions. If DNA ciliate has deoxyribozyme activity, substrate is cleaved and the band of cleaved substrate appears as a band.
  • Creating DNA ciliate, we use (1) and (2) protocols.
    • (1) The method of using EDC and NHS is here.
    • (2) The method of using EDAC…
  • Confirming DNA ciliate, we use (3) and (4) protocols
    • (3) The method of electrophoresis is here.
    • (4) The method of making sample is

Results

  • Following is the result of electrophoresis. Result 1 is DNA ciliate using NHS and EDC. Result 2 is DNA ciliate using EDAC. Meanings of the numbers above results are here.
  1. deoxyribozyme
  2. substrate
  3. negative control (Zn2+ 0 mM)
  4. positive control (Zn2+ 10 mM)
  5. no deoxyribozyme beads (Zn2+ 0 mM)
  6. no deoxyribozyme beads (Zn2+ 10 mM)
  7. DNA ciliate (Zn2+ 0 mM)
  8. DNA ciliate (Zn2+ 10 mM)
  • Three bands were there. From a top, A is the band of deoxyribozyme, B is the band of substrate (didn’t be cleaved), and C is the band of cleaved substrate. If there is cleaved substrate band, it means deoxyribozyme activity is appeared and deoxyribozyme is attached to polystyrene beads successfully.

[Result 1]

  • PAGE of φ200 nm polystyrene beads using NHS and EDC.
Figure1.the result of PAGE of φ200 nm polystyrene beads using NHS and EDC.


  • PAGE of φ1 um polystyrene beads using NHS and EDC.
Figure2.the result of PAGE of φ200 nm polystyrene beads using NHS and EDC.


[Result 2]

  • PAGE of φ200 nm polystyrene beads using EDAC.
Figure1.the result of PAGE of φ200 nm polystyrene beads using NHS and EDC.

Discussions

  • First, we explain this experimentation’s appropriateness for checking DNA ciliate body. To create DNA ciliate body, it was necessary to attach deoxyrobozyme to micrometer-sized polystyrene beads firmly. Furthermore, we had to check deoxyribozyme activity of DNA ciliate body. In this method, these two things could be checked. First, the degree of fixation can be checked. DNA ciliate is removed before loading to polyacrylamide gel, so if deoxyribozyme can’t be attached to polystyrene beads firmly, the leg band is appeared. Second, 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. Based on the above, this method is appropriate for checking DNA ciliate body.

]*Second, we explain necessity of lanes. Lanes of 1 to 4 are needed for checking deoxyribozyme activity and the positions of each band of DNAs. Lane 1 and lane 2 are control lanes. The band in lane 1 is deoxyribozyme band, and the band of lane 2 is substrate band. Lane 3 and lane 4 are lanes to check deoxyribozyme activity. The solutions in lane 3 doesn’t contain of Zn(2+), the solution in lane 4 contains of Zn(2+). The cleaved band is appeared in lane 4, but doesn’t be appeared in lane 3. This means normal deoxyribozyme isn’t active in no metal ions solutions. Lane 4’s bottom band means position of cleaved substrate.

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 DNA ciliate body’s 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.

1. Free moving mode
In the free moving mode, DNA ciliates moves freely and randomly in almost all area. To achieve this mode, we tried to observe DNA ciliates' Brownian motion on a glass plate. In this page, we show an experimental result of observing Brownian motion of DNA ciliates.

Method

To check DNA ciliate’s free moving mode,we used same solution when we checked deoxyribozyme activity (Link ) The solution is 3% BSA in 1x SSC and the size of DNA ciliate is 200nm (A) and 1um (B). We spotted the solution which DNA ciliates are diffused to glass plate. After that, we put cover glass on it and observed these DNA ciliates by a phase-contrast microscope and took videos.


Result

The left two videos are used DNA ciliates which are 200 nm in diameter. The right two videos are used DNA ciliates which are 1 um in diameter. The lower videos are enlarged videos of the upper videos. The lower videos are used for taking mote of single DNA ciliate.

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 <td>[Video](A)</td>
 <td>[Video](B)</td>
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 <td><iframe width="450" height="259" src="http://www.youtube.com/embed/uGRn9Z8inW4?rel=0" frameborder="0" allowfullscreen></iframe></td>
 <td><iframe width="450" height="259" src="http://www.youtube.com/embed/-zzB6UeWKoM?hl=ja&fs=1" frameborder="0" allowfullscreen></iframe></td>
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 <td><iframe width="420" height="315" src="http://www.youtube.com/embed/E1vW6eaABcQ" frameborder="0" allowfullscreen></iframe></td>
 <td><iframe width="420" height="315" src="http://www.youtube.com/embed/jKpgMfls3Kw" frameborder="0" allowfullscreen></iframe></td>
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Discussions and conclusions

First, we discuss the experimentation’s appropriate. To check free moving mode, it was necessary to check DNA ciliate’s movement in solution. By taking videos, we could see the movement of DNA ciliate in solution. Furthermore, the movement is not affected by wind because the solution is isolated from the external environment through cover glass. Because of above two things, this experimentation is appropriate for checking DNA ciliate’s movement.
Second, we discuss the movement of DNA ciliate in these videos. In both videos, many DNA ciliates moved freely and randomly in the solution. To compare two videos, the DNA ciliate which is 200 nm in diameter moves more strongly than the DNA ciliate which is 1 um in diameter. However some DNA ciliates didn't move because they were crystallization or located at the surface of slide glass or cover glass.
In conclusion, DNA ciliate’s Brownian motion is very intense and at random, so we can say we confirmed free moving mode. Especially, DNA ciliate which is 200 nm moves strongly. By using this mode, DNA ciliate can move on non-DNA glass plate and search other DNAs which can

The track walking mode
As already mentioned in the project page, we used "Deoxyribozyme-substrate reaction " and microchannel to move DNA ciliates directionally on the DNA tracks. Furthermore,we set three goals to achieve this mode:1.Conformation of deoxyribozyme activity, 2.Construction of DNA tracks and 3.Confirmation of moving directonally.(Link:) Here, we show the processes and results to achieve these goals.


1.Confirmation of Deoxyribozyme activity for substrate

We confirmed deoxyribozyme activity by using electrocataphoresis. This result has already shown in the result page written about DNA ciliate's body. (Link: ) So, we succeeded to confirm deoxyribozyme activity for substrate.

2.Construction of DNA tracks

Principles and methods of making DNA tracks

To make DNA track, four experiments were needed.
First experiment was making sample mold of microchannel. We used polyacetal resin as sample. We cut polystyrene resin by micro fine machining center and made a mold of microchannel. To make sample mold precisely, we shaved surroundings of the microchannel.
Second experiment was making PDMS mold. To begin with, we mixed PDMS and its hardener at the rate of 10:1(mass ratio). After cleaning bubble in this solution by using vacuum desiccators, next, we pour PDMS to the sample mold. After that, we heat sample mold and the solution to harden PDMS. Then, get hardened PDMS from sample mold. Microchannel we made on polyacetal-mold was transcribed to this PDMS-mold.
Figure1. A series of attaching aminated DNA to glass reaction
Figure2. Construction of DNA track
Third experiment was immobilization of DNA on glass plate. To make DNA tracks, we used microchannel and arrayed DNAs on glass plate. To attaching DNAs on glass plate, we use DSS as the linker between aminated DNA and the glass.[1] DSS linker reacts with amino groups that are exposed on the surface of the MAS-coated glass.[2] DSS linker is very highly reactive with amino groups, so DNAs can be attached on the glass plate by covalent bonding with DSS linker (Figure1). We use DSS coated MAS-coated glass, and put PDMS-mold on the glass plate. Then, we poured DNA solution into microchannel (Figure2). With this operation, DNAs are arrayed as the shape of microchannel. We can design the shapes of microchannels freely, so we can make DNA tracks with freely designed shapes.
Fourth experiment was confirming whether DNAs were arrayed as the shape of microchannel. To confirm this thing, we used fluorescent labeling complementary strands for the DNA strands of DNA track. Using hybridization of these DNAs, we were able to check whether DNAs were arrayed as the shape of microchannel by fluorescence microscopes and were able to compare with control experiment.(Figure3)
Figure3. Confirmed by DNA hybridization

Results of making DNA tracks

Figure4 is the result of making PDMS mold. We can see two right angle winding lines. They are a part of microchannels and using these microchannels, we arrayed DNAs. The result is Figure5. In Figure5, we hybridized fluorescent labeling complementary DNA strands with arrayed DNA. With the hybridization of arrayed DNAs and fluorescent labeling complementary strands, We can see two fluorescent lines whose shapes are same as the designed microchannel in PDMS mold in Figure5.
In addition, we made the microchannel which forms like human and hybridized fluorescent labeling complementary DNA strands with arrayed DNA.The result is Figure6.We can confirm that the DNA tracks arrayed as we designed. From the result of Figure5 and 6, we can say that we achieved to array DNA as complex structure and make DNA tracks.
Figure4. This figure is microchannel in PDMS-mold. This figure was observed by phase contrast.
Figure5. This figure is the result of arraying DNAs on glass plate using microchannel of Figure4 and hybridized with their complementary fluorescent labeling DNA strands. This figure was observed by fluorescent phase contrast.
Figure6: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.


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.

Mechanism

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...)

Results

1. Confirmation of UV-switching

method

result

Image of non-denaturing 20% PAGE for the confirmation of UV-switching
  • 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.
  1. U 0.225uM and Mg2+ 80mM
  2. B 0.45uM and Mg2+ 80mM
  3. D 0.225uM and Mg2+ 80mM
  4. U 0.225uM and B 0.45uM and Mg2+ 80mM
  5. U 0.225uM and D 0.225uM and Mg2+ 80mM (UV isn’t spotted)
  6. U 0.225uM and D 0.225uM and Mg2+ 80mM (UV is spotted for 60 min.)
  7. Reaction solution (UV isn’t spotted)
  8. Reaction solution (UV is spotted for 15 min.)
  9. Reaction solution (UV is spotted for 60 min.)
  10. Reaction solution and Mg2+ 80mM (UV isn’t spotted)
  11. Reaction solution and Mg2+ 80mM (UV is spotted for 15 min.)
  12. 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

A fluorescent image of DNA ciliate 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.
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