Biomod/2011/TeamJapan/Tokyo/Project/Results of the track walking mode

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Project

The track walking mode

The purpose of the track walking mode is to move DNA ciliate directionally
on the track.
To achive this mode, we needed to come up with the mechanism of walking and the way to make DNA tracks.
We chose the "Deoxyribozyme-substrate reaction" to solve the mechanism of walking. And we also chose the microchannel to solve the problem of making of DNA tracks.

・The mechanism of "Deoxyribozyme-substrate reaction"

This reaction utilizes DNA ciliate’s deoxyribozyme legs and their substrates on the DNA tracks. A deoxyribozyme leg of the DNA ciliate cuts the substrate DNA at an inserted RNA base. Then, the leg dissociates from cut substrate and moves to the near uncut substrate. By repeating this reaction, DNA ciliate can walk along DNA track with substrates.(Figure.1)

Figure.1:The mechanism that DNA ciliate moves directionally.

・The motivation of using microchannel to make the landcape

DNA origami can be appropriately landscape for nanometer-sized moving nanomachines because it can be designed to complex structural DNA tracks. However, as the tracks for our micrometer-sized molecular robot DNA ciliate, DNA origami is not useful because it takes enormous time to make micrometer-sized track from DNA origami that DNA ciliate can move along and we may be not able to complete constructing the tracks by this summer. Therefore, we challenged to make complex structural DNA tracks by using the technology of microfluid mechanics.(Figure.2) We show you the principle of making microchannel in the result page. (Link:)
Thus,we set three goals to achieve this mode:1.Confirmation of deoxyribozyme activity, 2.Construction of DNA tracks and 3.Confirmation of moving directionally. We show the results of these in the result page. (Link:)

Figure.2:The schematic diagram of microchannel.

Result

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
Figure1. A series of attaching aminated DNA to glass reaction
Figure2. Construction of DNA track
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
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.
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.
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.
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

Execute a Simulation
Figure1:The result of the simulation. (The beads’diameter are 1um)
Figure1:The result of the simulation. (The beads’diameter are 1um)
Figure2:The result of the simulation. (The beads' diameter are 200nm)
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:The result of the simulation.
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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