Biomod/2012/Titech/Nano-Jugglers/Results: Difference between revisions
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We introduce the photo-switching DNA system into Biomolecular Rocket in order to control its moving direction. | We introduce the photo-switching DNA system into Biomolecular Rocket in order to control its moving direction. | ||
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: | : To investigate the relationship between the strength of UV light and the irradiation time for dissociation of the photoresponsive DNA and determine the valid time for UV light irradiation, we examined two types of strength of UV light. | ||
Figure 3-2.1 represents spctrum of Abs in condition of UV-light(30 mW/cm<sup><small>2</small></sup>) irradiation. Abs of A+B around 260 nm was increasing gradually from 0 minutes to 5 minutes. This result means dsDNA was completely dissociated after irradiating UV-light for 5 minutes. Moreover, Abs of A+B around 330 nm was decreasing from 0 minutes to 5 minutes. This means trans-formed azobenzene changed its form to cis-formation. Therefore, we achieved photoresponsive DNA which was designed by us would be dissociated by irradiating UV-light for 5 minutes. | |||
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Revision as of 07:12, 27 October 2012
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} </style> </head> <BODY> <div id="biomodlink"> <<a href="http://openwetware.org/wiki/Biomod">BIOMOD</a>|<a href="http://openwetware.org/wiki/Biomod/2012">2012</a>|Titech Nano-Jugglers </div> <div id="header"> <div id="navigation"> <div id="menu"> <ul> <li><a href="http://openwetware.org/wiki/Biomod/2012/Titech/Nano-Jugglers"><br>Home<br><br></a></li> <li><a href="http://openwetware.org/wiki/Biomod/2012/Titech/Nano-Jugglers/Team/Students"><br>Team<br><br></a></li> <li><a href="http://openwetware.org/wiki/Biomod/2012/Titech/Nano-Jugglers/Project"><br>Project<br><br></a></li> <li><a href="http://openwetware.org/wiki/Biomod/2012/Titech/Nano-Jugglers/Results">Results<br>&<br>Methods</a></font></li> <li class="ach"><a href="http://openwetware.org/wiki/Biomod/2012/Titech/Nano-Jugglers/Achievements"><br>Achievements<br><br></a> <li class="sup"><a href="http://openwetware.org/wiki/Biomod/2012/Titech/Nano-Jugglers/Protocols"><br>Suppl. Info.<br><br></a></li> <li class="none"><a href="http://openwetware.org/wiki/Biomod/2012/Titech/Nano-Jugglers/Acknowledgement"><br>Acknowledgements<br><br></a></li> </ul> </div> </div> </div> </BODY> </html>
Results
<html><body><td align="center" width="300px"><A href=#0._Construction_of_Biomolecular_Rocket title="Body"><img src="http://openwetware.org/images/b/b5/BM.jpg" border=0 width=310 height=240></a></td></body></html> |
Construction of Biomolecular Rocket
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<html><body><A href=#1._Power_supply_for_the_rail-free_movement_of_Biomolecular_Rocket title="Rail-free"><img src="http://openwetware.org/images/7/7f/Rail-free%E3%80%80movement_kinesin.jpg" border=0 width=310 height=220></a></body></html> Power supply for the rail-free movement of the Biomolecular Rocket |
<html><body><A href=#2._Realization_of_high-speed_movement_of_Biomolecular_Rocket title="High-speed"><img src="http://openwetware.org/images/8/89/High-speed_movement.jpg" border=0 width=310 height=220></a></body></html> Realization of high-speed movement of the Biomolecular Rocket |
<html><body><A href=#3._Introduction_of_a_photo-switchable_DNA_system_for_the_directional_control title="Control"><img src="http://openwetware.org/images/d/dd/Control_image.jpg" border=0 width=310 height=220></a></body></html> Introduction of a photo-switchable DNA system for the directional control |
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0. Construction of Biomolecular Rocket
<html><body><font size="5">We constructed Biomolecular Rocket with a microbead, catalysts, and designed DNAs.</font></body></html> Biomolecular Rocket is composed of a micrometer-sized body and many catalytic engines. The body consists of a microbead with a diameter of 10 μm, and catalytic engines consist of platinum nanoparticles or catalase molecules. The catalytic engines are conjugated to the body using a DNA-based linker in a spatially selective manner.
>>see more methods
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<html><body><td align="center"><img src="http://openwetware.org/images/4/4d/Charts.jpg" border=0 width=200 height=440></a></td></body></html> |
0.1. Selective coating of the body
<html><body><font size="5">We succeeded in selective coating of a micrometer-sized bead by vapor deposition of Au and Cr.</font></body></html>
- >>see more methods
<html><body><td align="center"><img src="http://openwetware.org/images/a/a5/Vapor_deposition.jpg" border=0 width=330 height=110></a></td></body></html> - >>see more methods
- Figure 0.1a, b and c are microscope images of 40 μm microbeads. Figure 0.1a shows microbeads before vapor deposition of metals. Figure 0.1b shows microbeads after vapor deposition of Au (gold) on the microbeads. Figure 0.1c shows microbeads after additional vapor deposition of Cr on the Au-deposited microbeads. The microbeads had three types of surface areas because the angular alignment of the beads was changed when Cr (chromium) was deposited on the Au-deposited microbeads.
- Similarly, Figure 0.1e, f and g are microscope images of 10 μm microbeads. Figure 0.1e shows microbeads before vapor deposition of metals. Figure 0.1f shows microbeads after vapor deposition of Au on the microbeads. Figure 0.1g shows microbeads after additional vapor deposition of Cr on the Au-deposited microbeads. The 10 μm microbeads probably had three types of surface areas. From these result, we conclude that selective coating of microbeads for Biomolecular Rocket was achieved.
0.2. Design of linker DNA strands
- <html><body><font size="5">We designed DNA sequences for the linkers to attach catalytic engines to the microbead body.</font></body></html>
- >>see more methods
We designed two kinds of DNA sequences with different stability. One is DNA sequence L. This sequence forms a very stable duplex with its complementary sequence L* at a room temperature. Another is DNA sequence S. The duplex of DNA sequence S and S* has less stability than DNA sequence L and L* but is stable at a room temperature. |
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- Figure 0.2a and 0.2b show calculated melting profiles of duplexes L-L* and S-S*, respectively. Calculated melting temperatures of these duplexes were higher than a room temperature.
0.3. Selective DNA conjugation to the microbead body of Biomolecular Rocket
0.3.1. Selective conjugation of DNA to polystyrene surface area
<html><body><td align="center"><img src="http://openwetware.org/images/f/f5/Polystyrene_EDAC.jpg" border=0 width=300 height=240></a></td></body></html> <html><body><font size="5">We succeeded in selective DNA conjugation to the polystyrene surface area of the microbead body.</font></body></html>
- >>see more methods
We conjugated amino-modified DNA sequence L* to the calboxylated polystyrene surface area of the microbead body with amide binding. To investigate the selective conjugation of L*, we hybridized a fluorophore-modified DNA sequence L with L* on the body and observed the DNA-conjugated microbead bodies.
- >>see more methods
- Figure 0.3.1a, b, and c are bright-field microscope images of selectively-coated microbead bodies (10 μm in diameter). Figure 0.3.1a', b', and c' are fluorescence microscope images of the selectively-coated microbead bodies. Figure 0.3.1a and a' shows selectively DNA-L*-conjugated microbeads with fluorophore-modified DNA L. Fluorescence of L was observed only where the polystyrene surface was exposed after the vapor deposition of metal. Figure 0.3.1b and b' shows selectively DNA-L*-conjugated microbeads without fluorophore-modified DNA L, and no fluorescence was observed. Figure 0.3.1c and c' shows non-DNA-conjugated microbeads with fluorophore-modified DNA L, and no fluorescence was observed. From these results, we conclude that selective conjugation of DNA L* to polystyrene surface area of the microbead bodies was achieved.
0.3.2. Selective conjugation of DNA to metal surface area
<html><body><td align="center" width="150px"><img src="http://openwetware.org/images/8/87/Metal_SAM.jpg" border=0 width=300 height=240></a></td></body></html> <html><body><font size="5">We succeeded in selective DNA conjugation to the Au surface area of the microbead body.</font></body></html>
- >>see more methods
We conjugated thiol-modified DNA sequence S* to the Au surface area of the microbead body with Au-thiol bonding. To confirm the selective conjugation of S* to an Au surface, we observed an Au-deposited cover glass after DNA conjugation instead of selectively Au-coated microbeads after DNA conjugation. We investigated the wetting properties of the Au surface before and after DNA conjugation because the wetting investigation showed the selective conjugation of DNA more clearly than the fluorescence observation like Fig. 0.3.1.
- >>see more methods
- Figure 0.3.2a shows the Au glass plate with a non-DNA spot (1), a non-thiol-modified DNA spot (2), and a thiol-modified DNA spot (3). Only at the spot (3), residual water was observed. The water remained by the wetting property of the spot (3); the higher wetting property of spot (3) than those of spots (1) and (2) was expected to be caused by the Au surface with thiol-modified DNA conjugation. Therefore, we conclude that selective conjugation of thiol-modified DNA to the Au surface area of the microbead body was also achieved.
0.4. Catalyst attachment with DNA hybridization
<html><body><td align="center"><img src="http://openwetware.org/images/1/19/Conjugation_catalyst.jpg" border=0 width=300 height=230></a></td></body></html> <html><body><font size="5">Catalyst engines are attached to the microbead body of Biomolecular Rocket with DNA hybridization reactions between L and L<sup>*</sup>, and S and S<sup>*</sup>.</font></body></html>
- >>see more methods
- >>see more methods
- From the results of sections 0.1 and 0.3, a selectively DNA-conjugated microbead body of Biomolecular Rocket is constructed. In addition, we prepared catalytic engines with DNA-tag sequences (L and S) that are complementary to the DNA sequences (L* and S*) on the body. Therefore, by mixing the DNA-tagged catalytic engines and selectively DNA-conjugated body, Biomolecular Rocket would be constructed in a self-assembled manner. Experiments Are currently in progress.
<html><body><font color="red">1. Power supply for the rail-free movement of the<br> Biomolecular Rocket</font></body></html>
- <html><body><font size="5">We achieved the power supply for the rail-free movement of the Biomolecular Rocket.</font></body></html>
We utilized H2O2 for fuel and catalysts of H2O2 for catalytic engines to realize the rail-free movement of the Biomolecular Rocket. Catalysts such as platinum and catalase catalyze the decomposition of H2O2 into H2O and O2; emitted bubbles of O2 launch Biomolecular Rocket.
Here, we investigated the following points to realize the power supply for the rail-free movement of the Biomolecular Rocket.
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<html><body><td align="center"><img src="http://openwetware.org/images/b/b3/WikiRFBM.jpg" border=0 width=240 height=240></a></td></body></html> |
1.1. Power supply for rail-free movement by using catalase catalytic engine
- <html><body><font size="5">We succeeded in the rail-free movement by O<sub>2</sub> bubble emission with catalase.</font></body></html>
- We utilized the catalase for catalytic engines, which catalyzes the decomposition of H2O2 into H2O and O2. Here, we conjugated the catalase to the polystyrene area of a microbead with a hemispherical Au surface and a hemispherical polystyrene surface (Fig. 1.1).
- >>see more methods
- Movie 1.1 shows the rail-free movement of a 10 μm microbead with a hemispherical Au surface and a hemispherical polystyrene surface by O2 bubble emission with catalase. The 10 μm beads without catalase did not move at all (Movie 1.2). Thus, we conclude that we succeeded in the rail-free movement by O2 bubble emission with catalase.
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1.2. Power supply for rail-free movement by using platinum catalytic engine
- <html><body><font size="5">We achieved bubble emissions with platinum micro-particles in solution of H<sub>2</sub>O<sub>2</sub>.</font></body></html>
- We provided 1 μm platinum particles, and Cr coating to create platinum hemispherical area. Then added 3% H2O2 solution and observed their movement.
- >>see more methods
- Movie 1.2 reveals the catalytic reaction of 3% H2O2 and 1 μm beads that have platinum hemispherical area. We can recognize that bubbles are emitted in H2O2 solution. These bubbles are emitted from 1 μm beads that have platinum hemispherical area by decomposing H2O2. From this result, we conclude that power supply for rail-free movement by using platinum catalytic engine was achieved.
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1.3. DNA hybridization in solution of H2O2
- <html><body><font size="5">We revealed that diluted H<sub>2</sub>O<sub>2</sub> solutions did not affect DNA hybridization and a H<sub>2</sub>O<sub>2</sub> solution was suitable for a fuel for the Biomolecular Rocket. </font></body></html>
- >>see more methods
- We confirmed that DNA duplexes were stably formed in diluted H2O2 solutions by the native polyacrylamide gel electrophoresis (PAGE). Figure 1.3 shows the gel image of the PAGE. As shown in the lanes 1-3, a double-stranded DNA was formed in 1% and 5% H2O2 solutions.
2. Realization of the high-speed movement of the
Biomolecular Rocket
- <html><body><font size="5">We confirmed the high-speed movement of the Biomorecular Rocket by bubble emission.</font></body></html>
Catalytic engines emit large amount of bubbles, and thus, supplies powerful driving force to the Biomolecular Rocket. By the analyses of the movement of platinum particles and numerical simulations, we found that the speed of the Biomolcular Rocket was faster than that of kinesin. We carried out the experiments and simulations as follows.
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<html><body><img src="http://openwetware.org/images/2/26/Hst.jpg" width=300 height=230></body></html> |
2.1. Power supply for the high-speed movement with platinum catalytic engines
- <html><body><font size="5">We succeeded in high speed movement with platinum catalytic engines.</font></body></html>
- >>see more methods
- Movie 2.1.1a shows the movement of platinum particles in 3% H2O2 solution. Movie 2.1.1b is the movement of platinum particles observed by a high-speed camera (the top left of the movie) and its analyzed results (the right of the movie). The analyzed results show the values of acceleration (top), velocity (the second from the top), coordinate x (third), and coordinate y (bottom) of platinum movement. From the results, we found the relationships between the bubble radius growth and the speed of platinum. Using the relationships and the kinetic parameters, we did numerical simulations of the movement of the Biomolecular Rocket (see Section 2.2).
- Figure. 2.1.1 shows the graph of the mean square displacement of the platinum particle. From this data, we calculated that the value of diffusion constant was 80.3 mm2/s2, and the mean velocity was 9.0 mm/s. Platinum particles moved at about 9,000 time’s faster speed than kinesin.
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<html><iframe width="440" height="330" src="http://www.youtube.com/embed/7pBw4FWEt3I" frameborder="0" allowfullscreen></iframe></html>
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<html><iframe width="440" height="330" src="http://www.youtube.com/embed/E1rtI0mS5Zs" frameborder="0" allowfullscreen></iframe></html>
Movie. 2.1.1 Analyses of the speed of platinum in solution of H2O2.
(a) Platinum movement in solution of H2O2 (b) Analises of the speed of plutinum by High-speed camera
Fig. 2.1.2. The mean square of displacement of a platinum particle. |
2.2. Numerical estimation of the speed of Biomolecular Rocket
- <html><body><font size="5">The numerical simulation revealed that Biomolecular Rocket can move at ten times faster than kinesin moves.</font></body></html>
- The simulation also demonstrated that Biomolecular Rocket with many platinum catalysts on the body can move twice faster than a microbead with a hemispherical platinum surface moves because of the difference in the catalytic areas between them (Fig. 2.2).
- >>see more simulation models
- Figure 2.2 shows mean velocity of kinesin motor, 10 μm sized bead that have platinum hemispherical area (catalytic motor without DNA), and 10 μm of Biomolecular Rocket. The velocity of Biomolecular Rocket is calculated as 11.2 μm/s, the velocity of catalytic motor without DNA is 6.8 μm/s. We assumed that Kinesin will move straightforward at 1.0 μm/s, therefore, we can speculate that biomolecular rocket will move faster than kinesin, and surface enlargement of catalyst will surely increase the speed of Biomolecular Rocket.
3. Introduction of a photo-switchable DNA system for the directional control
- <html><body><font size="5">We developed a photo-switchable DNA system for the directional control with a photoresponsive DNA.</font></body></html>
The photo-switchable DNA system consists of an azobenzene-based photoresponsive DNA. The structure of the photoresponsive DNA is changed by UV light irradiation, then the photoresponsive DNA duplex is dissociated into DNA single strands. By the dissociation of DNA, catalytic engines are dissociated from the body of Biomolecular Rocket. As a result, the directional change of the force generated by O2 bubble is caused, and the direction of the Biomolecular Rocket is changed. Here, we developed and investigated the photo-switchable DNA system by the following experiments and simulations.
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<html><body><td align="center" width="150px"><img src="http://openwetware.org/images/8/8a/DC.jpg" border=0 width=300 height=230></a></td></body></html> |
3.1. Design of photoresponsive DNA
<html><body><td align="center" width="150px"><img src="http://openwetware.org/images/4/45/Photo_design.jpg" border=0 width=210 height=210></a></td></body></html> <html><body><font size="5">We designed photoresponsive DNA strands for a photo-switchable DNA system to achieve the directional control of Biomolecular Rocket.</font></body></html>
>>see more methods
We designed a photoresponsive DNA strand based on DNA sequence S*; four azobenzene molecules were introduced into S*. Figure 3.1.1 shows that absorbance spectra of DNA strands (A) photoresponsive DNA sequence S*, (B) DNA sequence S, and (A+B) the duplex of them at a room temperature. The absorbance of A+B around 260nm was less than those of A and B. This indicated that the hybridization of A with B was stable at a room temperature.
Fig.3.1 Absorbance spectra of DNA strands (A) photoresponsive DNA sequence S*, (B) DNA sequence S, and (A+B) the duplex of them at a room temperature. |
3.2. Achievement of the photo-switchable DNA system
<html><body><td align="center" width="150px"><img src="http://openwetware.org/images/f/f3/Azobenzene_image_dissociation.jpg" border=0 width=240 height=180></a></td></body></html> <html><body><font size="5">We have succeeded in development of a photo-switchable DNA system.</font></body></html>
- >>see more methods
We introduce the photo-switching DNA system into Biomolecular Rocket in order to control its moving direction.
- >>see more methods
- To investigate the relationship between the strength of UV light and the irradiation time for dissociation of the photoresponsive DNA and determine the valid time for UV light irradiation, we examined two types of strength of UV light.
Figure 3-2.1 represents spctrum of Abs in condition of UV-light(30 mW/cm2) irradiation. Abs of A+B around 260 nm was increasing gradually from 0 minutes to 5 minutes. This result means dsDNA was completely dissociated after irradiating UV-light for 5 minutes. Moreover, Abs of A+B around 330 nm was decreasing from 0 minutes to 5 minutes. This means trans-formed azobenzene changed its form to cis-formation. Therefore, we achieved photoresponsive DNA which was designed by us would be dissociated by irradiating UV-light for 5 minutes.
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Fig. 3.2.1 Spectrum analysis of photoresponsive DNA duplex(A+B) in condition of UV-light(30 mW/cm2) irradiation |
- Then, we tested the dissociation of photoresponsive DNA under the condition of different strength of UV-light(180 mW/cm2). First, from Figure 3.2.2b, we could confirm that dsDNA was dissociated gradually from 0 seconds to 50 seconds because maximum Abs around 260 nm was increasing. Second, Figure 3.2.2c shows that trans-formed azobenzene decreased because Abs around 330 nm was decreasing from irradiation for 0 seconds to 50 seconds. Finally, Figure 3.2.2 shows cis-formed azobenzene increased. As we did experiences for many times, we noticed that there was maximum wave length around 480 nm. By researching, we reached the fact that Abs around 480 nm shows the existence of cis-formed azobenzene. So, we can say that cis-formed azobenzene increased because Abs around 480 nm was increasing from irradiation for 0 seconds to 50 seconds. To summarize, we concluded that photo-seichable DNA system works in 50 seconds' irradiation of UV-light(180 mW/cm2). In brief, we can conclude that photoresponsive dsDNA was dissociated completely in the irradiation of UV-light (180 mW/cm2) for 50 seconds.
- From these results, we conducted that Dissociation of photoresponsive DNA by UV-light irradiation was achived.
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Fig. 3.2.2 Spectrum analysis of photoresponsive DNA duplex(A+B) in condition of UV-light(180 mW/cm2) irradiation |
3.3. Control direction of Biomolecular Rocket movement in simulation
- <html><body><font size="5">Biomolecular Rocket surely changes its direction by a photo-switchable DNA system , nevertheless they are affected by viscous resistance force and Brownian dynamics.</font></body></html>
- >>see more simulation models
- Figure 3.3 is a Pathways of 10um sized Biomolecular Rockets images. Each pathways show the movement of the Biomolecular Rocket which detaches catalytic engines after the Biomolecular Rocket is irradiated UV light at 10 seconds, 15 seconds, 20 seconds, 25 seconds passed, and a pathway with no irradiation of UV Light.
- From Figure 3.3, we concluded that the Biomolecular Rocket can change its direction immediately after whole dissociation of engines have occurred, nevertheless viscous resistance and Brownian movement prevented their controlled movement. Considering about the results of “Directional control with photo-switchable DNA system”, the Biomolecular Rocket needs a little more time to change its direction after irradiation of the UV light. From these results, we found that Biomolecular Rocket surely changes its direction in simulation.
- If you want to see all of our methods, click here