Biomod/2011/TeamJapan/Sendai/Result Atomic Force Microscope/detail Observation struture+field

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Observation of the robot on the field

We used High-speed AFM(RIBM, Nano Live Vision).
silicon nitride cantilever:
  • Olympus BL-AC10EGS-A2
resonant frequency = 1.0-2.0 MHz
spring constant = 0.1-0.3 N/m
EBDTip radius < 15 nm
  • Olympus BL-AC10DS-A2
resonant frequency = 1.5 MHz
spring constant = 0.1 N/m
EBDTip radius = 24nm
Scale bar : Figure.1-26, 200 nm
Flow chart of experiments to bind the robots onto Fields
Flowchart robots + Fields
Flowchart robots + Fields

Contents

Mixing the annealing solutions.

Figure 1.overnight robot and Field(1000nm×750nm)
Figure 1.overnight robot and Field(1000nm×750nm)
Figure 2.overnight robot and Field(750nm×560nm)
Figure 2.overnight robot and Field(750nm×560nm)

We mixed the two kinds of the solution (1:1) in the tube. One solution contained annealed 3D structures and the other contained the annealed Fields. The sample (2 μL) was adsorbed on the cleaved mica surface. The sample was left at room temperature overnight. Secondly, it was washed several times with the same buffer.

  • Condition

・overnight at room temperature
・3D structure : Field = 1 : 1 (ratio of concentration)
・Mg2+ concentration 12.5 mM

  • Result

No 3D structures attached to the Fields (we were able to observe only the Fields and no 3D structures).

  • Consideration

There is a possibility of failing (not properly self-assembled) in annealing. Moreover it was considered that the temperature at mixing the 3D structures and the Fields (mix temperature), or Mg2+ concentration in the solution have effects on attachment of 3D structures and the Fields.


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Changing the Mg2+ concentration.

We changed the Mg2+ concentration in the solution to anneal the 3D structures and the Fields. After annealing, we mixed the two kinds of the solution (1:1) in the tube. One solution contained annealed 3D structures and the other contained the annealed Fields. The sample (2 μL) was adsorbed on the cleaved mica surface. The sample was left at room temperature overnight. Secondly, it was washed out several times with the same buffer.


  • Condition

・overnight at room temperature
・3D structure : Field = 1:1 (ratio of concentration)
・Mg2+ concentration 12.5 mM, 25 mM, 37.5 mM

  • Result

No 3D structures attached to the Fields (we were not able to observe the Fields and the 3D structures).

  • Consideration

It seems that there is a possibility of failing in annealing. If not so, it may be not appropriate to change the Mg2+ concentration at the annealing.
Next, we performed to mix the 3D structures and the Fields at low temperature because mix temperature may affect the attachment of those.



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Changing the temperature.

Figure 3.overnight at 4°C
Figure 3.overnight at 4°C

We mixed the two annealing solution (1:1) in the tube. One solution contained 3D structures and the other contained the Fields. The sample (2 μL) was left at 4°C overnight. The sample (2 μL) was adsorbed on the cleaved mica surface. Secondly, it was washed several times with the same buffer.

  • Condition

・overnight at 4°C
・3D structure : Field = 1 : 1 (ratio of concentration)
・Mg2+ concentration 12.5 mM

  • Result

No 3D structures attached to the Fields (we were able to observe only the Fields. There ware many aggregates).

  • Consideration

Staples of capture-leg or start-leg float in the solution in surplus. therefore, it is conceivable that the following three patterns prevent the 3D structure and the Field from attaching to each other.
⑴floating staples mutually hybridize
⑵Capture-leg of the robot and the floating start-leg hybridize
⑶Start-leg in the Field and floating capture-leg hybridize

Therefore by reducing these staples floating in surplus, we may be able to decrease undesirable hybridization.

    


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Changing the concentration of capture-leg.

Figure 4. reduce Capture-leg concentration
Figure 4. reduce Capture-leg concentration

We reduced only concentration of the staples of capture-leg in the annealing solution containing robots. We mixed the two annealing solution (1:1) in the tube. One contained 3D structures and the other contained the Fields. The sample (2 μL) was left at 4°C overnight. The sample (2 μL) was adsorbed on the cleaved mica surface. Secondly, it was washed several times with the same buffer.


  • Condition

・overnight at 4°C
・3D structure : Field = 1:1 , 1:3 (ratio of concentration)
・Mg2+ concentration 12.5 mM
・M13 : Capture-leg : Staples = 1:1:10

  • Result

No 3D structures attached to the Fields (we were able to observe only the Fields and no 3D structures).

  • Consideration

From the above reason, it is conceivable that staples of start-leg floating in the solution prevent the 3D structures and the Fields from attaching each other.



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Changing the capture-leg and start-strand concentration.

Figure 5. reduce Capture-leg and Start-leg concentration at 4°C
Figure 5. reduce Capture-leg and Start-leg concentration at 4°C

We reduced only concentration of the staples of capture-leg and the staples of start-leg in the each annealing solution. We mixed the two annealing solution (1:1) in the tube. One contained 3D structures and the other contained the Fields. The sample (2 μL) was left at 4°C overnight. The sample (2 μL) was adsorbed on the cleaved mica surface. After that it was washed several times with the same buffer.


  • Condition

・M13 : Capture-leg : Start-leg : Staples = 1:1:1:10(ratio of concentration)
・overnight at 4°C
・3D structure : Field = 1:1 , 1:3 (ratio of concentration)
・Mg2+ concentration 12.5 mM

  • Result

No 3D structures attached to the Fields.

  • Consideration

It may affect the experimental results that the length of capture-leg is short (because our robot is used the legs of same sequence as spider but don’t have spacer staples). Moreover we suppose that Robot legs may get into the inside of the 3D structure during the self-assembly; there is a possibility that there are no legs to attach to the Field.


As we have seen, we can’t confirm our robot is attachment to the Field in spite of the adjustment of Mg2+ concentration, mix temperature and leg’s concentration. We suppose the following two reasons. Moreover
⑴the length of Capture-leg is short (because our robot is used the legs of same sequence as Spider but don’t have spacer staples)
⑵Robot legs may get into the inside of the 3D structure during the self-assembly; there is a possibility that there are no legs to attach to the Field.
   



New Design

We improved our design of robot. There are Capture-leg with spacer of 20nucleotides T-linker and legs of deoxiribozime. After that, we used this robot. M13(cut) represents a M13mp18 DNA single strand extracted with restriction enzyme as a necessary part.


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Mixing the annealing solutions.

Figure 6.reduce Capture-leg and Start-leg concentration at room temperature
Figure 6.reduce Capture-leg and Start-leg concentration at room temperature

We mixed the two kinds of the solution (1:1) in the tube. One solution contained annealed 3D structures and the other contained the annealed Fields. The sample (2 μL) was adsorbed on the cleaved mica surface. The sample was left at room temperature overnight. Secondly, it was washed several times with the same buffer. We used 3D and 2D structures in this experiment


  • Condition

・overnight at room temperature
・M13 : Capture-leg : Start-leg : Staples = 1:1:1:10(ratio of concentration)
・3D or 2D structure : Field = 1:1(ratio of concentration)
・Mg2+ concentration 12.5 mM

  • Result

No 3D structures attached to the Fields. There were a lot of aggregate.


  • Consideration

The probability that capture-leg hybridized start staple is very low because the robots and the Fields move around in the solution by Brownian motion.
We decided that we first made the Fields attach to the cleaved mica surface, and dropped the robots on it later.
We thought that by this way, the Fields would be fixed and it would be easier for the robots to contact with start staples.
With this technique, the Field is fixed and a robot attaches start staple easily.
Moreover, the stability after hybridization will also increase by fixation of the Field.




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Changing the observation way

Figure 7.overnight on mica at room temperature(1)
Figure 7.overnight on mica at room temperature(1)
Figure 8.overnight on mica at room temperature(2)
Figure 8.overnight on mica at room temperature(2)


First of all, the sample solution containing the Fields (2 μL) adsorbed on the cleaved mica surface and the sample was left at room temperature for 5 min. Secondly, it was washed several times with the same buffer. Thirdly, the sample containing the triangular prism (3D structure version) was adsorbed on the same surface. Finally, it was left at room temperature for 5min.

  • Condition

・M13 : Capture-leg : Start-leg : Staples = 1:1:1:10(ratio of concentration at annealing)
・3D structure:Field = 1:1 (ratio of concentration at mixing)
・Mg2+ concentration 12.5 mM at mixing.

  • Result

No 3D structures attached to the Fields (we were able to observe only the Fields and no 3D structures).

  • Consideration

We thought the probability of robots attaching to the Fields was probably high because Brownian motion becomes small at low temperature (4 °C) and robots bind the Field's start strand easily. Therefore, we decided to leave the sample containing robots and the Fields at low temperature.



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Changing the temperature.

Figure 9.overnight on mica at 4°C
Figure 9.overnight on mica at 4°C

We mixed the two annealing solution (1:1) in the tube. One solution contained 2D structures and the other contained the Fields. The sample (2 μl) was left at 4°C overnight. The sample (2 μL) was adsorbed on the cleaved mica surface. Secondly, it was washed several times with the same buffer. We used 2D structures(M13 cut) in this experiment.

  • Condition

・M13 : Capture-leg : Start-leg : Staples = 1 : 1 : 1 : 10(ratio of concentration)
・overnight at 4°C
・2D structure:Field = 1:1 (ratio of concentration)
・Mg2+ concentration 12.5 mM

  • Result

No 2D structures attached to the Fields (there were a lot of aggregation.we were able to observe only the Fields).

  • Consideration

We were not able to observe the robots to attach the Fields at low temperature. From these figures, we supposed there were no differences between low and room temperature experiments. Therefore, we decided to change both Mg2+ concentration and temperature to attach.



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Changing the Mg2+ concentration and the temperature.

First of all, the sample solution containing the Fields (2 μL) adsorbed on the cleaved mica surface and the sample was left at room temperature for 5 min. Secondly, it was washed several times with the same buffer. Thirdly, the sample containing the triangular prism was adsorbed on the same surface. We prepared several samples which have different Mg2+ concentrations using these ways.These samples were left at multi-temperature.

  • Condition

・M13 : Capture-leg : Start-leg : Staples = 1:1:10:10(ratio of concentration)
・overnight at 4°C or room temperature
・3D structure:Field = 1:1 (ratio of concentration)
・Mg2+ concentration 12.5 mM,15 mM,20 mM


  • Result

No 3D structures attached to the Fields.

  • Consideration

We can see the DNA structures which filled the mica surface. In these structures, we can also see the DNA Fields. The robots had no choice but to hybridize the Field, because there were no space for the robots to attach the mica surface. However, no 2D structures attached to the Fields. Therefore, it seems that changing only Mg2+ concentration or temperature to wait for attaching didn't effect the robots to bind the Fields greatly. We experimented at low temperature and room temperature, but not high temperature. We thought the probability of robots attaching to the Fields may be high because Brownian motion also become big at around the melting temperature.


Figure 13. 4°C,Mg2+ 12.5mM
Figure 13. 4°C,Mg2+ 12.5mM
Figure 14.4°C,Mg2+ 15mM
Figure 14.4°C,Mg2+ 15mM
Figure 15.,room temperature,Mg2+ 12.5mM
Figure 15.,room temperature,Mg2+ 12.5mM
Figure 16.room temperature,Mg2+ 15mM
Figure 16.room temperature,Mg2+ 15mM


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Changing the temperature

First of all, the sample solution containing the Fields (2 μL) adsorbed on the cleaved mica surface and the sample was left at room temperature for 5 min. Secondly, it was washed several times with the same buffer. Thirdly, the sample containing the triangular prism was adsorbed on the same surface. We prepared several samples using these ways. These samples were left at multi-temperature.

  • Condition

・M13 : Capture-leg : Start-leg : Staples = 1:1:10:10(ratio of concentration)
・3D structure : Field = 2 : 1 (ratio of concentration)
・2D structure : Field = 2 : 1 (ratio of concentration)
・Mg2+ concentration 12.5 mM.
・Temperature 4,25°C

  • Result

No 3D and 2D structures attached to the Fields (we were not able to observe the Fields and the 3D structures).

Figure 17. 2D on the Field at room temperature
Figure 17. 2D on the Field at room temperature
Figure 18. 2D on the Field at 4°C
Figure 18. 2D on the Field at 4°C
Figure 19. 3D on the Field at room temperature
Figure 19. 3D on the Field at room temperature
Figure 20. 3D on the Field at 4°C
Figure 20. 3D on the Field at 4°C

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Changing the temperature and the time to left sample.

First of all, the sample solution which contained the Fields (2 μL) adsorbed on the cleaved mica surface and the sample was left at room temperature for 5 min. Secondly, it was washed several times with the same buffer. Thirdly, the sample containing the triangular prism was adsorbed on the same surface. We prepared several samples using these ways.These samples were left at multi-temperature for multi-time.

  • Condition

・M13 : Capture-leg : Start-leg : Staples = 1:1:10:10(ratio of concentration)
・The solution composition. 2D structures : Fields = 1 : 1 (ratio of concentration)
・Mg2+ concentration 12.5 mM.
・Left time = 1,4,7 hour
・Temperature 40,50°C

  • Result

No 2D structures attached to the Field (we were not able to observe the Fields and the 2D structures).

  • Consideration

We can see the DNA structures which filled the mica surface. In the structures, we can also see the DNA Fields, but no 2D structures attached to the Field. We thought that electrostatic repulsion interrupted 2D structures to attach the Fields. We also thought the force was so large that 2D or 3D structures wasn't able to attach the Fields.

Figure 21. 40°C, 1 hour on mica
Figure 21. 40°C, 1 hour on mica
Figure 22. 50°C, 1 hour on mica
Figure 22. 50°C, 1 hour on mica
Figure 23. 40°C, 1 hour in tube
Figure 23. 40°C, 1 hour in tube
Figure 24. 40°C, 4 hour in tube
Figure 24. 40°C, 4 hour in tube
Figure 25. 50°C, 1 hour in tube
Figure 25. 50°C, 1 hour in tube
Figure 26. 50°C, 4 hour in tube
Figure 26. 50°C, 4 hour in tube

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Conclusion

As we have seen, we can’t confirm our robot is attachment to the Field in spite of the adjustment of Mg2+ concentration, mix temperature and leg’s concentration. We suppose the following reasons.
⑴the length of Capture-leg is short (because our robot is used the legs of same sequence as Spider but don’t have spacer staples)
⑵Robot legs may get into the inside of the 3D structure during the self-assembly; there is a possibility that there are no legs to attach to the Field.
⑶electrostatic repulsion interrupted 2D structures to attach the Fields.

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Solution

In order to solve the problem we assume as shown above, we indicate below the possible solutions.

  • Structures:extending the capture-leg.

Detaching a robot from the Field to reduce the influence of minus charge.

  • Mg2+ concentration : mixing Field and 2D or 3D structures with other Mg2+ concentration

Eraseing the power of the minus which work between a robot and the Field.

  • Using PNA for 2D or 3D structures.

Using a robot made from a material without minus charge.

  • Not using DNA for the Fields.

Making the Field which does not receive the influence of minus charge.

  • Changing the concentration of the robot on a large scale.

Increasing the probability that the robots attach to the Fields.


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