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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

Construction of Biomolecular Rocket

We constructed Biomolecular Rocket composed of a micrometer-sized body and many catalytic engines. The catalytic engines were conjugated to the body using DNA-baed linkers in a spatially selective manner.

Shown in detail below

<html><body><A href=#1._Power_supply_for_the_rail-free_movement 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

<html><body><A href=#2._Increasing_driving_force_for_the_high-speed_movement title="High-speed"><img src="http://openwetware.org/images/8/89/High-speed_movement.jpg" border=0 width=310 height=220></a></body></html>
Increasing driving force for the high-speed movement

<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

We realized rail-free movement by power generation with catalytic reactions of platinum and catalase.

Shown in detail below

We realized the high-speed movement by power generation with catalytic reactions of platinum and catalase, andanalyzed the speed.
We carried out numerical simulations of the high-speed movement of Biomolecular Rockets.

Shown in detail below

We developed photo-switchable DNA for the control of Bimolecular Rocket using UV light irradiation.
We investigated directional control of Biomolecular Rocket in the simulation.

Shown in detail below

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.
We constructed the Biomolecular Rocket through the following four steps.

1. The microbead body was selectively coated by vapor deposition of metals (Au and Cr).
2. We designed DNA sequences for spatially-selective hybridization of catalytic engines.
3. The DNA molecules were conjugated to a designated metal surface of the microbead body.
4. Catalyst engines were attached to the microbead body with selective hybridization of DNAs
we designed.

>>see more methods of construction of Biomolecular Rocket

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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

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 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 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.

40 μm silica beads	a	b	c
10 μm polystyrene beads	d	e	f
Image of micro-beads	<html><body><img src="http://openwetware.org/images/d/d5/Polystyrene.jpg" border=0 width=240 height=180></body></html>	<html><body><img src="http://openwetware.org/images/4/41/After_first_deposition.jpg" border=0 width=240 height=180></body></html>	<html><body><img src="http://openwetware.org/images/3/30/After_second_deposition.jpg" border=0 width=240 height=180></body></html>
Fig. 0.1 Selective coating of micro-beads by vapor deposition.

0.2. DNA design

<html><body><font size="5">We designed DNA strands in order to hybridize stably at constant temperature.</font></body></html>

>>see more methods

We designed 2 types of DNA duplex that must hybridize stably at constant temperature by NUPACK. We called the longer one as DNA sequence L, and the shorter one as DNA sequence S. We called their complementary strands as DNA sequence L^* and DNA sequence S^*.

DNA sequence L 5’-CGTCTATTGCTTGTCACTTCCCC-3'	DNA sequence S 5’-AATACCCAGCC-3’
DNA sequence L^* 5'-GGGGAAGTGACAAGCAATAGACG-3'	DNA sequence S^* 5’-GGCTGGGTATT-3’

In both Figure 0.2a and 0.2b, T_m is far from R_t. So, we achieved DNA design for hybridizing stably at room temperature.

a	b
Fig. 0.2 Analysis of melting temperature of hybridization with each DNA strands. a Melting profile of DNA sequence L and L^* duplex b Melting profile of DNA sequence S and S^* duplex Red line shows T_m (melting temperature), and Blue line shows R_t (Room temperature, 24 °C).

0.3. DNA conjugation

0.3.1. Selective conjugation of DNA to polystyrene surface area

<html><body><font size="5">We succeeded in DNA conjugation to polystyrene surface area by using EDAC.</font></body></html>

For visualizing the results of DNA conjugation to polystyrene surface area, we hybridized FAM complementary DNA and observed them under blue light by microscope.

>>see more methods

Figure 0.3.1a, b, and c are images of 10 μm selective coated beads under visible light. Figure 0.3.1a', b', and c' are images of 10 μm selective coated beads under blue light. Figure 0.3.1a anda' shows selective coated beads after conjugated DNA to polystyrene area and hybridize fluorescent cDNA. Figure 0.3.1b and b' shows selective coated beads after conjugated DNA to polystyrene area. Figure 0.3.1c and c' shows selective coated beads after mixing fluorescent DNA. Selective coated beads exhibit fluorescence because fluorescent cDNA was excite by blue light. From these results, we conclude that selective conjugation of DNA to polystyrene surface area.

Under visible light	a	b	c
Under blue light	a'	b'	c'
conditions	<html><body><img src="http://openwetware.org/images/d/d3/EDACBEADS1.JPG" border=0 width=280 height=150></a></body></html>	<html><body><img src="http://openwetware.org/images/3/38/EDACBEADS2.JPG" border=0 width=280 height=150></a></body></html>	<html><body><img src="http://openwetware.org/images/2/23/EDACBEADS3.JPG" border=0 width=280 height=150></a></body></html>
Fig. 0.3.1 Confirmation of DNA conjugation to polystyrene surface area by fluorescence complementary DNA strand.

0.3.2. Selective conjugation of DNA to metal surface area

<html><body><font size="5">We succeeded in DNA conjugation onto Au surface area by the reaction of SAM.</font></body></html>

For visualizing the results of DNA conjugation to metal surface area, we hybridized fluorescent complementary DNA to Au plate surface.

>>see more methods

Figure 0.3.2a is a images of Au plate that DNA conjugated partially. Figure 0.3.2b is a images after soaking up DNA buffer of Au plate. Figure 0.3.2a is a images after washing away of partially DNA conjugated Au plate. Final concentration of phosphate and NaCl buffer are the same in these spot. But 1, 2 and 3 are added NaCl concentration immediately after 24 hours of incubation, the other hand 4, 5, and 6 are added NaCl as salt aging process. Au plate reveals partially hydrophilic, this is because physical property of Au surface was assimilated to physical property of DNA. From these result, we conclude that selective conjugation of DNA to metal surface area.

a	b	c
d<html><body><img src="http://openwetware.org/images/1/1a/Auplate1.jpg" border=0 width=300 height=160></a></body></html>	e<html><body><img src="http://openwetware.org/images/1/12/Auplate2.jpg" border=0 width=300 height=160></a></body></html>	f<html><body><img src="http://openwetware.org/images/f/fd/Auplate3.jpg" border=0 width=300 height=160></a></body></html>
g	Fig. 0.3.2 Confirmation of DNA conjugation to metal area a Incubated for 48 hours at a gold plate b Soaked up the water after incubation c Washed away gold plate surface by 3 × SSC after soaking up the water d Image of spot 1, 4 e Image of spot 2, 5 f Image of spot 3, 6 g Condition of gold plate

0.4. Catalyst attachment with DNA hybridization

<html><body><font size="5">We tried to attach catalyst to the body of Biomolecular Rocket with DNA hybridization.</font></body></html>

DNA hybridization enable us to attach each materials in that DNA strand transits to take thermodynamically stable forms.

>>see more methods

To verificate this experiments, we tried to attach Pt particles to Au plate (instead of Au deposited beads) , but there is a few difference between DNA conjugated materials and natural materials. So, We must prove the technology of attaching catalyst. DNA strands are nano-scale, and to conjugate micro-scale beads and platinum engines was difficult.

We also design DNA strands of DNA sequence L and DNA sequence S that have the first 15 bases from 5' end as a linker (TTTTTTTTTTTTTTT). This DNA was also designed not to make unexpected structures. Probably, this linker part will allow much leeway for attaching the body, in that decrease the effect of steric hindrance.

Poly T linked DNA sequence L	5’-CGTCTATTGCTTGTCACTTCCCC-3'
Poly T linked DNA sequence S	5’-AATACCCAGCC-3’

1. Power supply for the rail-free movement

    <html><body><font size="5">In order to realize rail-free movement, we looked at bubble emission of catalyst reaction.</font></body></html>
    Platinum or catalase catalysts decompose H₂O₂ and emit H₂O and O₂ bubbles. Since the driving force created by divergence of bubbles, and rocket proceeds by dissociation of oxygen, rail does not require.
    Power supply for the rail-free movement, we had to check following parts.

1. DNA hybridization is not affected damage and denaturate that comes from H₂O₂
2. Platinum catalyst can supply efficient energy for rail-free movement
3. Catalase catalyst can supply efficient energy for rail-free movement

↑Page Top

1.1. DNA hybridization in solution of H₂O₂

<html><body><font size="5">We succeeded in ascertaining the stability of DNA duplex in 1%-5% H<sub>2</sub>O<sub>2</sub> solution.</font></body></html>

To visualize the stability of DNA duplex in 1%-5% H₂O₂ solution, we use PAGE electrophoresis. It shows the difference of molecular weight of nucleic acid that comes from denaturetion or hybridization in the form of bands.

>>see more Method

Figure 1.1 is a image of DNA hybridization after immersing in solution of H₂O₂. Comparing between 4, 5, 6 & 7, these 4 lines appear lower than the white line, and line 4-5 and 6-7 are few differences. If H₂O₂ affects ssDNA destroy(tear up, denature), line 5 and 7 will appear in the lower position or becomes unclear. Judging from appearances, differences between positive and negative control ware few. Comparing 1, 2, 3 and 8, these lines are completely appear in the same position. Differences between these lines are only concentration of H₂O₂. So, we conclude that there is no influence of H₂O₂ for DNA hybridizations however DNA is immersed to H₂O₂ within 90 minutes. So we proved that there is no effect of H₂O₂ for dsDNA as well as ssDNA.

Fig. 1.1 Result of DNA hybridization after immersing in solution of H₂O₂

dsDNA bands show in lane 1, 2, 3 & 8. In lane 4, 5, 6 & 7 show ssDNA bands. Lane 1 & 8 are the same, lane 2 & 3 are immersed in H₂O₂ solution (1% and 5%) for 90 minutes. In lane 5 & 7 show the results of ssDNA in solution of H₂O₂. In lane 4 & 6 are the control bands of ssDNA.

1.2. Verification of platinum hemisphere behavior in solution of H₂O₂

We tried to verify whether platinum hemisphere moves forward in solution of H₂O₂.
We provide 1 μm platinum particles, and Cr coating to create platinum hemisphere. Then added 3% H₂O₂ solution and observed.

>>see Method

1.3. Verification of catalase hemisphere behavior in solution of H₂O₂

We also tried to verify whether catalase hemisphere moves in solution of H₂O₂.

Catalase has Catalytic ability of decomposing H₂O₂, like platinum.
>>see Method

2. Increasing driving force for the high-speed movement

Rocket gets much stronger driving force by increasing catalytic surface area.
In order to realize top speed of molecular robot, we check the following parts.

1. Analyses of the speed of platinum to get data of the relationship between
bubble conditions and platinum behavior
2. Compare the speed of kinesin, platinum hemisphere and Biomolecular Rocket to
dictate which is the fastest one.

Catalytic engine produced sufficient energy to move quickly, but further accelerate the Biomolecular Rocket, we conjugated numerous platinum catalytic engines to a micro-sized rocket body. Emission of bubbles depends on the surface area of catalyst, so if the catalytic surface area is expanded, it is obvious that our rocket will be able to emit more bubbles and speeding up.

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2.1. Analyses of the speed of platinum in solution of H₂O₂ by High-speed camera

We succeeded in analyses of the speed of plutinum in solution of H₂O₂ by High-speed camera.

>>see Method

a <html><iframe width="440" height="330" src="http://www.youtube.com/embed/7pBw4FWEt3I" frameborder="0" allowfullscreen></iframe></html>	b <html><iframe width="440" height="330" src="http://www.youtube.com/embed/E1rtI0mS5Zs" frameborder="0" allowfullscreen></iframe></html>
Fig.2.1 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

Figure 2.1b disclosed the values of Acceleration, Velocity, Coordinate x, and Coordinate y of platinum movement. Not only that, by observation of these values, we could determine relationships between bubble radius growth and the speed of platinum, so we were able to simulate of movement of our rocket.

2-2.Simulation for speeding-up of Biomolecular Rocket movement

From our numerical simurations of high-speed.We carried out numerical simulations of the high-speed movement of Biomolecular Rockets.

3. Introduction of a photo-switchable DNA system for the directional control

Direction of the rail-free movement of our rocket can be controlled, since we introduced a photo-switchable DNA system.
In order to realize Directional control of our rocket, we conducted the following parts.

1. Introduction of a photo-switchable DNA system for the directional control
2. Research the relationship between the strength of UV light and the time
of dissociation to determine the valid time
3. Verification of the relationship between the movement of Biomolcular
Rocket and dissociation of catalyst engines

Photoresponsive DNA structure is changed by UV light irradiation, then dissociation of double strand DNA will happen. Take advantage of this reaction in region-specific manner, we can control the direction of our rocket.

↑page Top

3.1. Design of photoresponsive DNA

We designed photoresponsive DNA strands in order to control the direction of Biomolecular Rocket.

    In this project, we ensured that photoresponsive DNA could hybridize their cDNA at room temperature by Abs. Numerical values of Abs depends on the concentration of material corresponding to the absorption wavelength.
    >>see Method

    We called ssDNA as A:Photoresponsive DNA, and B:Complementary DNA of A.

Figure 3.1 reveals that Abs of A+B around 260nm was below those of A and B, and also Abs of A+B around 330 nm was less than that of A. This is because the concentration of azobenzene decreased by hybridization of A with B.

Calculation an average absorbance of A and B Abs around 260 nm, theorical value of A+B was 0.168. Compared to this, measured value of absorbance is 22.6% lower. We thought this difference comes from that DNA formed duplex and interactions between base pairs, so decrease the UV absorbance relative to single strands.

In this point, we believe that the complementary photo-responsive DNAs can form duplex. So, we concluded that these results mean A and B hybridized successfully.

a	b
Fig.3.1 Abs of photoresponsive DNA results, wave length around 260 nm reveals the concentration of DNA. Around 330 nm reveals the concentration of trans-formed azobenzene.

3.2. Dissociation of photoresponsive DNA by UV-light irradiation

We have scceeded in dissociation of photo-switchable DNA by UV-light irradiation.

Photo-switchable DNA duplex can easily dissociate its duplex by irradiating UV-light. We put this switching system in the rocket in order to control our rocket. To concirm the dissociation of photo-switchable DNA duplex, we checked Abs.

>>see Method

To investigate the relationship between the strength of UV light and the time of dissociation to determine the valid time, we examine 2 type of UV light.

Fig.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.

a	b
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).

nbsp; Fig.3-2.2 shows photoresponsive dsDNA was dissociated completely after the irradiation of UV-light for 50 seconds. There are three evidences.

First, from Fig.b), we could find that dsDNA was dissociated gradually from 0 seconds to 50 seconds because maximum Abs around 260 nm was increasing. Second, Fig.c) shows that trans-formed azobenzene decreased because Abs around 330 nm was decreasing from irradiation for 0 seconds to 50 seconds. Finally, Fig.d) 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.

So, we concluded that photo-seichable DNA system achieved after 50 seconds irradiation of UV-light(180 mW/cm²).

a	b
c	d
Fig.3.2.2 Spectrum analysis of photoresponsive DNA duplex(A+B) in condition of UV-light(180 mW/cm²) irradiation

3-3.Simulation for directional control of Biomolecular Rocket

sss

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If you want to see all of our methods, click here

Biomod/2012/Titech/Nano-Jugglers/Results

Results

0. Construction of Biomolecular Rocket

0.1. Selective coating of the body

0.2. DNA design

0.3. DNA conjugation

0.3.1. Selective conjugation of DNA to polystyrene surface area

0.3.2. Selective conjugation of DNA to metal surface area

0.4. Catalyst attachment with DNA hybridization

1. Power supply for the rail-free movement

1.1. DNA hybridization in solution of H₂O₂

1.2. Verification of platinum hemisphere behavior in solution of H₂O₂

1.3. Verification of catalase hemisphere behavior in solution of H₂O₂

2. Increasing driving force for the high-speed movement

2.1. Analyses of the speed of platinum in solution of H₂O₂ by High-speed camera

2-2.Simulation for speeding-up of Biomolecular Rocket movement

3. Introduction of a photo-switchable DNA system for the directional control

3.1. Design of photoresponsive DNA

3.2. Dissociation of photoresponsive DNA by UV-light irradiation

3-3.Simulation for directional control of Biomolecular Rocket

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Biomod/2012/Titech/Nano-Jugglers/Results

Results

0. Construction of Biomolecular Rocket

0.1. Selective coating of the body

0.2. DNA design

0.3. DNA conjugation

0.3.1. Selective conjugation of DNA to polystyrene surface area

0.3.2. Selective conjugation of DNA to metal surface area

0.4. Catalyst attachment with DNA hybridization

1. Power supply for the rail-free movement

1.1. DNA hybridization in solution of H2O2

1.2. Verification of platinum hemisphere behavior in solution of H2O2

1.3. Verification of catalase hemisphere behavior in solution of H2O2

2. Increasing driving force for the high-speed movement

2.1. Analyses of the speed of platinum in solution of H2O2 by High-speed camera

2-2.Simulation for speeding-up of Biomolecular Rocket movement

3. Introduction of a photo-switchable DNA system for the directional control

3.1. Design of photoresponsive DNA

3.2. Dissociation of photoresponsive DNA by UV-light irradiation

3-3.Simulation for directional control of Biomolecular Rocket

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1.1. DNA hybridization in solution of H₂O₂

1.2. Verification of platinum hemisphere behavior in solution of H₂O₂

1.3. Verification of catalase hemisphere behavior in solution of H₂O₂

2.1. Analyses of the speed of platinum in solution of H₂O₂ by High-speed camera