Biomod/2012/Titech/Nano-Jugglers/Results

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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-bead linkers in a spatially selective manner.

        Shown in detail below


Power supply for the rail-free movement of the Biomolecular Rocket

Realization of high-speed movement of the Biomolecular Rocket

Introduction of a photo-switchable DNA system for the directional control
  • We realized the 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, and analyzed the speed.
  • We carried out numerical simulations of the high-speed movement of the Biomolecular Rockets.

      Shown in detail below

  • We developed a photo-switchable DNA system for controlling of the Bimolecular Rocket using UV light irradiation.
  • We investigated directional control of the Biomolecular Rocket in the simulations.

      Shown in detail below




0. Construction of Biomolecular Rocket

We constructed Biomolecular Rocket with a microbead, catalysts, and designed DNAs.

    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

0.1. Selective coating of the body

We succeeded in selective coating of a micrometer-sized bead by vapor deposition of Au and Cr.

>>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 (chromium) 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 results, we conclude that selective coating of microbeads for Biomolecular Rocket was achieved.
  40 μm silica beads     a
40 μm silica beads
    b
After first deposition
    c
After second deposition
  10 μm polystyrene beads       d
10 μm polystyrene beads
    e
After first deposition
    f
After second deposition
  Image of micro-beads
    Fig. 0.1 Selective coating of micro-beads by vapor deposition.

0.2. Design of linker DNA strands

We designed DNA sequences for the linkers to attach catalytic engines to the microbead body.
>>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.
  DNA sequence L

  5’-CGTCTATTGCTTGTCACTTCCCC-3'  

  DNA sequence S

  5’-AATACCCAGCC-3’  

  DNA sequence L*

  5'-GGGGAAGTGACAAGCAATAGACG-3'  

  DNA sequence S*

  5’-GGCTGGGTATT-3’  

    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.
  a
DNA Tm
  b
Photoresponsive DNA Tm
Fig. 0.2. (a) The melting profile of duplex L-L*. (b) The melting profile of duplex S-S*. RT and Tm indicate a room temperature (24°C) and a melting temperature, respectively. We calculated the melting profiles using NUPACK software.

0.3. Selective DNA conjugation to the microbead body of Biomolecular Rocket

0.3.1. Selective conjugation of DNA to polystyrene surface area


We succeeded in selective DNA conjugation to the polystyrene surface area of the microbead body.

>>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.

    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.
  Bright-field microscope images       a    
DNA conjugated beads and FAM
    b    
DNA conjugated beads
    c    
Beads and FAM
  Fluorescence microscope images       a'    
DNA conjugated beads and FAM
    b'    
DNA conjugated beads
    c'    
Beads and FAM
  Experimental conditions  
Fig. 0.3.1 Microscope images of the microbead bodies of Biomolecular Rocket. (a) and (a') Selectively DNA-conjugated microbeads with complementary fluorophore-modified DNA. (b) and (b') Selectively DNA-conjugated microbeads without complementary fluorophore-modified DNA. (c) and (c') non-DNA-conjugated microbeads with complementary fluorophore-modified DNA.

0.3.2. Selective conjugation of DNA to metal surface area


We succeeded in selective DNA conjugation to the Au surface area of the microbead body.

>>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.

    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.
    a    
Au plate
    b    
condition
    c
Fig. 0.3.2. (a) An Au glass plate with a non-DNA spot (1), a non-thiol-modified DNA spot (2), and a thiol-modified DNA spot (3).
(b) A layout drawing of the spots (1)-(3).
(c) Experimental condition for the spots (1)-(3).

0.4. Catalyst attachment with DNA hybridization

Catalyst engines are attached to the microbead body of Biomolecular Rocket with DNA hybridization reactions between L and L*, and S and S*.

>>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.



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1. Power supply for the rail-free movement of the Biomolecular Rocket

We achieved the power supply for the rail-free movement of the Biomolecular Rocket.
    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.

  1. We found that catalase molecules as catalytic engines could emit sufficient amount of bubbles for the rail-free movement.
  2. We found that platinum particles as a catalytic engines could emit sufficient amount of bubbles for rail-free movement.
  3. We found that the H2O2 did not affect the stability of DNA linkers for attaching catalytic engines to the body in the H2O2 solution.

1.1. Power supply for rail-free movement by using catalase catalytic engine

We succeeded in the rail-free movement by O2 bubble emission with catalase.
    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.
Fig. 1.1  Image of a microbead with a hemispherical Au surface and a hemispherical polystyrene surface in 3% H2O2 solution.
Movie. 1.1   The real-time video of the moving 10 μm bead with a catalase-conjugated hemisphere area in 3% H2O2 solution.

1.2. Power supply for rail-free movement by using platinum catalytic engine

We achieved bubble emissions with platinum micro-particles in solution of H2O2.
    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.
Fig. 1.2 Image of a microbead with a hemispherical Cr surface and a hemispherical platinum surface in 3% H2O2 solution.

Movie. 1.2     Image of the catalytic reaction of 3% H2O2 and 1 μm beads that have platinum hemispherical area.

1.3. DNA hybridization in solution of H2O2

We revealed that diluted H2O2 solutions did not affect DNA hybridization and a H2O2 solution was suitable for a fuel for the Biomolecular Rocket.
>>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.
Electrophoresis
Fig. 1.3    A gel image of a native PAGE of DNA in H2O2 solutions. Lanes 1-3, 8: a double stranded DNA (D+R) in diluted H2O2 solutions eith concentrations 0% (lanes 1 and 8), 1% (lane 2), and 5% (lane 3). In lanes 4-7, single stranded DNAs in 0% and 5% H2O2 solutions were shown. DNA sequences R and D are shown at the upper right of the image.


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2. Realization of the high-speed movement of the Biomolecular Rocket

We confirmed the high-speed movement of the Biomorecular Rocket by bubble emission.

    Catalytic engines emit large amount of bubbles, and thus, supply 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.
  1. Observations of the movement of platinum particles using a high-speed camera and analyses of the speed.
  2. Numerical simulations of the movement of the Biomolecular Rocket

2.1. Power supply for the high-speed movement with platinum catalytic engines

We succeeded in high speed movement with platinum catalytic engines.
>>see more methods and full length movie

    Movie 2.1.1a shows the movement of 0.15-0.40 μm platinum particles in 3% H2O2 solution. Movie 2.1.1b is the movement of platinum particles observed by a high-speed camera (the upper 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. 2.1.2 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.
a

b

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 displacement of a platinum particle.

2.2. Numerical estimation of the speed of Biomolecular Rocket

>>see more simulation models
The numerical simulation revealed that Biomolecular Rocket can move at ten times faster than kinesin moves.
    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).
    Figure 2.2 shows mean speed of kinesin motor, 10 μm sized bead that have platinum hemispherical area (catalytic motor without DNA), and 10 μm of Biomolecular Rocket. The speed of Biomolecular Rocket is calculated as 11.2 μm/s, the speed 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.
Simulation of high-speed
Fig. 2.2    Image of each molecular motor’s speed with time



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

We developed a photo-switchable DNA system for the directional control with a photoresponsive DNA.

    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.

  1. Design of a photo-switchable DNA system for the directional control
  2. Investigation of the dissociation rate of the photo-switchahble DNA duplex by UV light irradiation experiments
  3. Investigation of the directional control of Biomolecular Rocket with the photo-switchable DNA system by numerical simulations

3.1. Design of photoresponsive DNA

We designed photoresponsive DNA strands for a photo-switchable DNA system to achieve the directional control of Biomolecular Rocket.

>>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.

Spectrum analysis

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

We have succeeded in development of a photo-switchable DNA system.
>>see more methods

    We introduce the photo-switching DNA system into Biomolecular Rocket in order to control its moving direction.

   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 3.2.1a represents the absorbance spectra in the condition of UV-light irradiation with the strength of 30 mW/cm2. The absorbance of the DNA duplex A+B at the wavelength of 260 nm gradually increased from 0 minutes to 5 minutes (Fig. 3.2.1b). This result means that the DNA duplex A+B was dissociated after 5 minutes irradiation of UV light. Moreover, the absorbance of the DNA duplex A+B at 330 nm decreased from 0 minutes to 5 minutes (Fig. 3.2.1b). This means that the trans-formed azobenzene changed its form to cis-formation. Therefore, we conclude that the dissociation of the photoresponsive DNA can be achieved by 5 minutes irradiation of UV-light (30 mW/cm2).
    a
Spectrum analysis
    b
Wave length around 260 nm and 330 nm
Fig. 3.2.1. (a)    Absorbance spectra of photoresponsive DNA duplex (A+B) in condition of UV light irradiation (30 mW/cm2). (b) Time course of the absorbance change during UV light irradiation.


    Similarly, we studied the dissociation rate of photoresponsive DNA under the condition of UV light irradiation with the strength of 180 mW/cm2 (Fig. 3.2.2a). Figure 3.2.2b represents that the DNA duplex A+B was gradually dissociated from 0 seconds to 50 seconds because the maximum absorbance at 260 nm increased. Figure 3.2.2c shows that the trans-formed azobenzene changed to the cis-fomed one by the UV irradiation because the absorbance at 330 nm decreased from 0 seconds to 50 seconds. Figure 3.2.2d shows that the cis-formed azobenzene increased because the absorbance at 480 nm increased from 0 seconds to 50 seconds. In summary, the photo-switchable DNA system worked by 50-second irradiation of UV-light (180 mW/cm2). From these results, we conclude that the dissociation of photoresponsive DNA by UV-light irradiation was achived.
    a
Spectrum analysis
    b
Wave lengte around 260 nm
    c
Wave lengte around 330 nm
    d
Wave lengte around 480 nm
Fig. 3.2.2    Spectrum analysis of photoresponsive DNA duplex(A+B) in condition of UV-light(180 mW/cm2) irradiation

3.3. Directional control of Biomolecular Rocket by the photo-switchable DNA system

    By numerical simulations, we found that Biomolecular Rocket changes its direction by the UV light irradiation even though Biomolecular Rocket is affected by the viscous resistance force and Brownian dynamics of water.

>>see more Simulation Models

    Figure 3.3 shows the trajectories of Biomolecular Rockets of 10 μm in diameter by the numerical simulation. Each trajectory shows the movement of Biomolecular Rocket that detaches catalytic engines after the irradiation of UV light when 10 seconds, 15 seconds, 20 seconds, and 25 seconds passed. This simulation results suggested that Biomolecular Rocket can change its direction immediately after all catalytic engines are dissociated even though the viscous resistance and Brownian movement prevented the controlled movement of Biomolecular Rocket. From these results, we conclude that Biomolecular Rocket changes its direction by UV light irradiation.
Control simulation
Fig. 3.3 Trajectories of Biomolecular Rockets of 10 μm in diameter by the numerical simulation. Each trajectory shows the movement of Biomolecular Rocket that detaches catalytic engines after the irradiation of UV light when 10 seconds, 15 seconds, 20 seconds, and 25 seconds passed.
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