Biomod/2011/TeamJapan/Tokyo/Project/Results of Track Walking Mode: Difference between revisions

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=Track Walking Mode=


==DNA ciliate body==
[[Image:DNA ciliate body 2.jpg|center|300px]]


:As already mentioned in the project page, we attached DNA strands to polystyrene beads to make DNA ciliate.
==The track walking mode==
{|
|-
|width="300px"|[[Image:Track walking mode figure.jpg|left|250px]]
|
: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.
|}
 
 
<!--
DNA ciliate を足場上で動かすために、Deoxylibozymeとマイクロ流路を使った。
-->


: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.
<h3>1.Confirmation of Deoxyribozyme activity for substrate</h3>
: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.


===Principle and methods===
<h3>2.Construction of DNA tracks</h3>
*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]]
:Second method is used '''EDAC'''. EDAC reacts with both aminated DNAs and polystyrene beads’ carboxylic acid.
:[[Image:DNA beads method2.jpg|Figure1.the result of PAGE of φ200 nm polystyrene beads using NHS and EDC. |900px]]
:<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.


:<h4>Principles and methods of making DNA tracks</h4>


===Protocol===
:To make DNA track, four experiments were needed.
*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]].
**(2) The method of using EDAC…
*Confirming DNA ciliate, we use (3) and (4) protocols
**(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


===Results===
: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.
*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.
# deoxyribozyme
# substrate
# negative control (Zn(2+) 0 mM)
# positive control (Zn(2+) 10 mM)
# no deoxyribozyme beads (Zn(2+) 0 mM)
# no deoxyribozyme beads (Zn(2+) 10 mM)
# DNA ciliate (Zn(2+) 0 mM)
# '''DNA ciliate (Zn(2+) 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.


<h3>[Result 1]</h3>
: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.
*PAGE of φ200 nm polystyrene beads using NHS and EDC.
<table width="100%">
:[[Image:Beads gel 1.jpg|border|Figure1.the result of PAGE of φ200 nm polystyrene beads using NHS and EDC. |400px]]
<tr align="center">
  <td>
[[Image:Tokyo tech：DNAimmobilization 5.png|thumb|center|Figure1. A series of attaching aminated DNA to glass reaction|400px]]
  </td>
  <td>
[[Image:Biomod2011 Team Tokyo 111030Biomod Construction of DNA track.png|thumb|center|Figure2. Construction of DNA track|400px]]
  </td>
</tr>
</table>
:Third experiment was creating DNA tracks. 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.<sup>[1]</sup> DSS linker reacts with amino groups that are exposed on the surface of the [http://www.matsunami-glass.co.jp/english/life/clinical_g/data18.html MAS-coated glass].<sup>[2]</sup> 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.


<table width="100%">
<tr align="center">
<tr>
  <td>
: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)
  </td>
  <td align="center">
[[Image:Biomod2011 Team Tokyo 111030Biomod hybridization-Fl-DNA.png|thumb|Figure3. Confirmed by DNA hybridization|300px]]
  </td>
</tr>
</table>


*PAGE of φ1 um polystyrene beads using NHS and EDC.
:<h4>Results of making DNA tracks</h4>
:[[Image:Beads gel 2.jpg|border|Figure2.the result of PAGE of φ200 nm polystyrene beads using NHS and EDC. |300px]]


<table cellpadding=10>
  <tr>
: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. 


<h3>[Result 2]</h3>
    <td>[[Image:IMG_2051_scale_bar.jpg|thumb|center|Figure4. This figure is microchannel in PDMS-mold. This figure was observed by phase contrast.|250px]]</td>
*PAGE of φ200 nm polystyrene beads using EDAC.  
    <td>[[Image:IMG_1698_edit_R.jpg|thumb|center|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.|300px]]</td>
:[[Image:Beads gel 3.jpg|border|Figure1.the result of PAGE of φ200 nm polystyrene beads using NHS and EDC. |400px]]
    <td>[[Image:BIOMOD Tokyo 20111101 humanformmicrochannel2.png|thumb|center|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.|300px]]</td>
</tr>
</table>


===Discussions===
<h3>3.Confirmation of directional walking on DNA tracks</h3>
*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==
===Principles and methods of simulations===
{|
{|
|-
|-
|width="300px|[[Image:Free moving mode figure.jpg|center|250px]]
|440px|[[image:Tokyo-trackwalking1.png|400px]]
|
|
: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.  
: To confirm this mode, we simulated whether DNA ciliate moves intended direction on substrate DNAs track by cellular automaton framework. Cellular automaton framework is separate model. Its field is assumed that the width is infinite and cells are square-block type. Next time step state is calculated by present own value and neighbor’s value. In this simulation, probability for movement is determined by each cell’s free energy. Probability for moving to substrate is larger than moving to cleaved substrate because substrate’s energy is smaller than cleaved substrate’s energy.
:This program is the simulation of DNA ciliate’s behavior, which works base on cellular-automaton framework.
|}
|}
===Method===
*A yellow cell represents DNA ciliate.
--------------------------------
*Blue cells represent oligodeoxynucleotide substrates with a single ribose moiety.
:To check DNA ciliate’s free moving mode,we used same solution when we checked deoxyribozyme activity <!--to 理由.-->(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.
*Red cells represent oligodeoxynucleotide substrates without a ribose moiety.
*The size of each cell is one micro-meter.
*A Yellow cell makes a move depend on its located cell. The rate of moving on each cell is derived as following steps.  
 
:Firstly, we calculated the average time that DNA ciliate moves one micrometer by Brownian movement as following formula.  


::[[Image:simulation1_tokyo.png|border|80px]]
::[[Image:simulation2_tokyo.png|border|100px]]


===Result===
::R: Gas constant. 8.3145[P/mol*K]
------------------------------------
::T: Temperature. 298[K].
: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.  
::Na=Avogadro number. 6.022141×10<sup>23</sup>
<HTML><body>
::η: Viscosity 0.00089[Pa/s]
<table width="100%">
::a: Radius of DNA ciliate body. 0.5<sup>-6</sup>[m]
  <tr align="center">
::x: 10<sup>-6</sup>[m]
  <td>[Video](A)</td>
 
  <td>[Video](B)</td>
:Next, we obtained the rate based on two assumptions. One assumption is that DNA ciliate moves with cleaving a ribose moiety to another cell in 120 seconds at a rate of 0.5. The other assumption is that DNA ciliate moves on substrates without a ribose moiety in 1.2 seconds at a rate of 0.5.
</tr>
::[[Image:simulation3_tokyo.png|border|130px]]
<tr align="center">
  <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>
</tr>
<tr align="center">
  <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>
</tr>
</table>
</body></HTML>


===Discussions and conclusions===
::t: Average time to move one micro-meter. [s/μm]
--------------------------------------
::y: Steps to move at rate 0.5. [s]
: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. <!-- ここで1umの方がcrystallizationがより起こる原因とどこまでのサイズなら出来そうかを考察するべき-->
: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==
:Then, each rate is following.
{|
    On the blue cells, yellow cells do not move at a rate of 0.9941.
|-
    On the red cells, yellow cells do not move at a rate of 0.555.
|width="300px"|[[Image:Track walking mode figure.jpg|left|250px]]
    On the white cells, yellow cells do not move at a rate of 0 (means to move absolutely).
|
*Yellow cells can move to only adjacent cell at one step.
: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.  
*The direction of yellow cell' movement is stochastically dependent on the adjacent cells’ free-energy. The rates are derived as :following formula. Free-energy of blue cells is -19.02[KJ/mol]. Free-energy of red cells is 16.03[KJ/moc]. Blue cells are about 211 times more likely to move than red cells.
|}




<!--
::[[Image:simulation4_tokyo.png|border|200px]]
DNA ciliate を足場上で動かすために、Deoxylibozymeとマイクロ流路を使った。
-->


==3. Light-irradiated gathering mode==
::[[Image:simulation04.png|border|300px]]
{|
|-
|width="270px"|[[image:Tokyo-gathering1.png|250px|center]]
|
: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==
::ΔG_n: An free energy of cell n which is adjacent;
::R: Gas constant. 8.3145[P/mol*K]
::T: Temperature. 298[K].


===UV-switching system===
===Results of simulations===
:[[Image:Tokyo-gathering2.png|600px|center]]
===Simulation movie===
: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...)]]
<HTML><body><iframe width="450" height="259" src="http://www.youtube.com/embed/jbS0hgjK7q8?rel=0" frameborder="0" allowfullscreen></iframe></body></HTML>
::[http://twotree.dousetsu.com/SimulationProgram2.html Execute a Simulation]


==Results==
<table>
<tr>
  <td>[[Image:biomod-tokyo2011_simulation_figure1.png|thumb|alt=Figure1|center|Figure1:The result of the simulation. (The beads’diameter are 1um)|420x330px]]
  <td>[[Image:biomod-tokyo2011_simulation_figure2.png|thumb|alt=Figure2|center|Figure2:The result of the simulation. (The beads' diameter are 200nm)|420x330px]]</td>
</tr>
</table>


===1. Confirmation of UV-switching===
:To confirm“tracks walking mode”works correctly, we made line graphs movement of DNA ciliate based on cellular automaton. Please look at figure1 and figure2. The vertical axis shows the distance from origin. The horizontal axis shows the number of steps. Three lines are different in the DNA types of DNA track. The red line's DNA track is normal DNA track our made, so DNA ciliate moves with cleaving substrates by its legs. The blue line's DNA track is only cleaved substrate, so DNA ciliate moves without cleaving in this situation. The green line's DNA track is complementary strands for DNA ciliate’s leg, not substrate, which deoxyribozyme can't cleave, so DNA ciliate can hybridize with, but can't cleave the DNA.
--------------------------------
:From these graphs, we found that DNA ciliate moving on substrate moves faster  and more directly than the others.


===method===
[[Image:Biomod2011 Team Tokyo Simulation MeanSquareDisplacement.png|thumb|alt=Figure3|center|Figure3:The result of the simulation.|800px]]
*Doing PAGE analysis
:How to make samples for electrophoresis is [[here]].
:How to do PAGE is [[here]].


===result===
:These graphs are mean square displacement of DNA ciliate’s movement. In mean square displacement, Brownian motion is presented by linear function and forced motion is presented by quadratic function. In this simulation, the moving distance on substrate field is presented by quadratic function. We confirmed DNA ciliate is forced directly and moves intended direction on substrate in this simulation.
[[Image:Tokyo_UV-gel.jpg|thumb|920px|Image of non-denaturing 20% PAGE for the confirmation of UV-switching]]
:In conclusion, we confirmed that DNA ciliate moves by directional force, not by Brownian motion in “track walking mode”.
*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.
:[[http://openwetware.org/wiki/Image:BiomodSimulation_tokyo.zip Program Source]]
#U 0.225uM and Mg<sup>2+</sup> 80mM
#B 0.45uM and Mg<sup>2+</sup> 80mM
#D 0.225uM and Mg<sup>2+</sup> 80mM
#U 0.225uM and B 0.45uM and Mg<sup>2+</sup> 80mM
#U 0.225uM and D 0.225uM and Mg<sup>2+</sup> 80mM (UV isn’t spotted)
#U 0.225uM and D 0.225uM and Mg<sup>2+</sup> 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 Mg<sup>2+</sup> 80mM (UV isn’t spotted)
#Reaction solution and Mg<sup>2+</sup> 80mM (UV is spotted for 15 min.)
#Reaction solution and Mg<sup>2+</sup> 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 Mg<sup>2+</sup> 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 Mg<sup>2+</sup> 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 Mg<sup>2+</sup> 80mM (UV is spotted for 60 min.)) means the bands when the loop is open and spotted UV (band U-D).
===Conclusion===
*In the presence of Mg<sup>2+</sup>, the switching was caused clearly (lane 10 to 12) because of the stable effect of Mg<sup>2+</sup>.
*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===
From thease results,we can
------------------------------------------
[[Image:Biomod2011 Team TokyoIMG 1947 E2 Scale Bar.jpg|thumb|right|A fluorescent image of DNA ciliate gathering at the specific area|500px]]
===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===
===References===
*A fluorescent image of the DNA ciliates gathering at the spot of complementary DNA is here.
[1]
*Complementary DNA was attached on upper-right area in this image.
[2]
: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.

Latest revision as of 08:59, 1 November 2011


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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 creating DNA tracks. 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.Confirmation of directional walking on DNA tracks

Principles and methods of simulations

To confirm this mode, we simulated whether DNA ciliate moves intended direction on substrate DNAs track by cellular automaton framework. Cellular automaton framework is separate model. Its field is assumed that the width is infinite and cells are square-block type. Next time step state is calculated by present own value and neighbor’s value. In this simulation, probability for movement is determined by each cell’s free energy. Probability for moving to substrate is larger than moving to cleaved substrate because substrate’s energy is smaller than cleaved substrate’s energy.
This program is the simulation of DNA ciliate’s behavior, which works base on cellular-automaton framework.
  • A yellow cell represents DNA ciliate.
  • Blue cells represent oligodeoxynucleotide substrates with a single ribose moiety.
  • Red cells represent oligodeoxynucleotide substrates without a ribose moiety.
  • The size of each cell is one micro-meter.
  • A Yellow cell makes a move depend on its located cell. The rate of moving on each cell is derived as following steps.
Firstly, we calculated the average time that DNA ciliate moves one micrometer by Brownian movement as following formula.
R: Gas constant. 8.3145[P/mol*K]
T: Temperature. 298[K].
Na=Avogadro number. 6.022141×1023
η: Viscosity 0.00089[Pa/s]
a: Radius of DNA ciliate body. 0.5-6[m]
x: 10-6[m]
Next, we obtained the rate based on two assumptions. One assumption is that DNA ciliate moves with cleaving a ribose moiety to another cell in 120 seconds at a rate of 0.5. The other assumption is that DNA ciliate moves on substrates without a ribose moiety in 1.2 seconds at a rate of 0.5.
t: Average time to move one micro-meter. [s/μm]
y: Steps to move at rate 0.5. [s]
Then, each rate is following.
    On the blue cells, yellow cells do not move at a rate of 0.9941.
    On the red cells, yellow cells do not move at a rate of 0.555.
    On the white cells, yellow cells do not move at a rate of 0 (means to move absolutely).
  • Yellow cells can move to only adjacent cell at one step.
  • The direction of yellow cell' movement is stochastically dependent on the adjacent cells’ free-energy. The rates are derived as :following formula. Free-energy of blue cells is -19.02[KJ/mol]. Free-energy of red cells is 16.03[KJ/moc]. Blue cells are about 211 times more likely to move than red cells.


ΔG_n: An free energy of cell n which is adjacent;
R: Gas constant. 8.3145[P/mol*K]
T: Temperature. 298[K].

Results of simulations

Simulation movie

<html><body><iframe width="450" height="259" src="http://www.youtube.com/embed/jbS0hgjK7q8?rel=0" frameborder="0" allowfullscreen></iframe></body></html>

Execute a Simulation
Figure1
Figure1:The result of the simulation. (The beads’diameter are 1um)
Figure2
Figure2:The result of the simulation. (The beads' diameter are 200nm)
To confirm“tracks walking mode”works correctly, we made line graphs movement of DNA ciliate based on cellular automaton. Please look at figure1 and figure2. The vertical axis shows the distance from origin. The horizontal axis shows the number of steps. Three lines are different in the DNA types of DNA track. The red line's DNA track is normal DNA track our made, so DNA ciliate moves with cleaving substrates by its legs. The blue line's DNA track is only cleaved substrate, so DNA ciliate moves without cleaving in this situation. The green line's DNA track is complementary strands for DNA ciliate’s leg, not substrate, which deoxyribozyme can't cleave, so DNA ciliate can hybridize with, but can't cleave the DNA.
From these graphs, we found that DNA ciliate moving on substrate moves faster and more directly than the others.
Figure3
Figure3:The result of the simulation.
These graphs are mean square displacement of DNA ciliate’s movement. In mean square displacement, Brownian motion is presented by linear function and forced motion is presented by quadratic function. In this simulation, the moving distance on substrate field is presented by quadratic function. We confirmed DNA ciliate is forced directly and moves intended direction on substrate in this simulation.
In conclusion, we confirmed that DNA ciliate moves by directional force, not by Brownian motion in “track walking mode”.
[Program Source]

Conclusion

From thease results,we can

References

[1] [2]

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