Biomod/2011/TeamJapan/Tokyo/Project: Difference between revisions

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=Project=
=Project=
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|width="320px"|[[Image:DNA ciliate概念図.png|light|450px]]
|width="320px"|[[Image:Biomod2011 Team Tokyo DNA ciliate three mode.png|450px]]
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<p>The aim of our project is the construction of an autonomous micrometer-sized molecular robot. We designed and constructed a micrometer-sized molecular robot, “DNA ciliate”, named after water microorganisms, ciliate. The DNA ciliate has three independent functional modes (see the left figure): free moving mode, track walking mode, and light-irradiated gathering mode.</p>
<p>The aim of our project is the construction of an autonomous micrometer-sized molecular robot. We designed and constructed a micrometer-sized molecular robot, “DNA ciliate”, named after water microorganisms, ciliate. The DNA ciliate has three independent functional modes (see the left figure): free moving mode, track walking mode, and light-irradiated gathering mode.</p>
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==Three independent modes of the DNA ciliate==
==Three independent modes of the DNA ciliate==
==　　1. Free moving mode==
==1. Free moving mode==
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:The movement by Brownian motion is described by the right equation (1). The left side of the equation is the mean square displacement of a DNA ciliate. r is the displacement from a default position during the time Δt. The diffusion constant D is described by the right equation (2), where T is a temperature, NA is the Avogadro number, η is a viscosity coefficient, and a is the radius of the DNA ciliate body. The diffusion constant is inversely proportional to the radius of the body. Thus, a DNA ciliate with a smaller body can move more broadly.
:The movement by Brownian motion is described by the right equation (1). The left side of the equation is the mean square displacement of a DNA ciliate. <i>R</i> is the displacement from a default position during the time <i>Δt</i>. The diffusion constant <i>D</i> is described by the right equation (2), where <i>T</i> is a temperature, <i>N<sub>A</sub></i> is the Avogadro number, <i>η</i> is a viscosity coefficient, and a is the radius of the DNA ciliate body. The diffusion constant is inversely proportional to the radius of the body. Thus, a DNA ciliate with a smaller body can move more broadly.
|width="300px"|[[image:Tokyo-freemoving1.png|center|280px|The equation of Brownian motion.]]
|width="300px"|[[image:Tokyo-freemoving1.png|center|280px|The equation of Brownian motion.]]
[[image:Tokyo-freemoving2.png|center|240px|The equation of Brownian motion.]]
[[image:Tokyo-freemoving2.png|center|240px|The equation of Brownian motion.]]
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|width="270px"|[[Image:Biomod2011 Team Tokyo111028TrackWalkingMode.png|right|250px]]
|width="270px"|[[Image:Biomod2011 Team Tokyo111028TrackWalkingMode.png|right|250px]]
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:The mechanizm of track walking is to move DNA ciliate directionally on the track.
:In the track walking mode, the DNA ciliate walks directionally along a track. The track is constructed from arrayed single-stranded DNA molecules that are substrates for the deoxyribozymes attached on the DNA ciliate body. DNA tracks with arbitrary shapes can be constructed using DNA nanotechnology, microfluidic technology, etc.
: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.
: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.
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===The mechanism of track walking===
===The mechanism of track walking===
:This reaction utilizes 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.
:The cleaving activity of the deoxyribozymes on the DNA ciliate body is used when the DNA ciliates work at the track walking mode. The reaction scheme of the track walking is presented in the right figure. First, a deoxyribozyme hybridizes with a substrate DNA composing a track. The deoxyribozyme has an RNA cleaving activity and cleaves a ribose site (yellow site in the right figure) included in the substrate DNA. After the cleavage, the cleaved and shorten substrate and the deoxyribozyme dissociate because the thermodynamically stability between the deoxyribozyme and the shorten substrate DNAs decreases. The dissociated deoxyribozyme can hybridize with another uncleaved substrate DNA near the cleaved substrate DNA. The deoxyribozyme more easily hybridizes with an uncleaved substrate than a cleaved one. Thus, the DNA ciliate can walk along the DNA track by repeating this reaction.
|width="450px"|[[Image:Tokyo-trackwalking2.png|Figure.1:The mechanism that DNA ciliate moves directionally.|420px|center]]
|width="450px"|[[Image:Tokyo-trackwalking2.png|Figure.1:The mechanism that DNA ciliate moves directionally.|420px|center]]
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===Construction of DNA tracks===
===Construction of DNA tracks===
: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.(The light figure) We show you the principle of making microchannel in the result page. (Link:[[Biomod/2011/TeamJapan/Tokyo/Project/Results#2.The track walking mode|here]]) 
:Although nanometer-sized DNA machines such as a DNA spider walks along a nanometer-sized DNA track constructed from DNA origami and DNA nanostructure, the DNA ciliate walks along a micrometer-sized DNA track (shown in the right figure) because the DNA ciliate has a micrometer-sized body. The micrometer-sized DNA tracks can be constructed using DNA nanotechnology, microfluidic technology, etc. Here, the micrometer-sized DNA tracks were constructed using a microchannel. Substrate DNAs were immobilized on a glass plate as a self-assembled monolayer through a silane coupling reaction.  
|width="330px"|[[Image:Tokyo-trackwalking3.png|280px|center|Figure.2:The schematic diagram of microchannel.]]
|width="330px"|[[Image:Tokyo-trackwalking3.png|280px|center|Figure.2:The schematic diagram of microchannel.]]
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==　　3. Light-irradiated gathering mode==
==3. Light-irradiated gathering mode==
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|width="270px"|[[Image:Biomod2011 Team Tokyo111028Light-irradiatedGatheringMode02.png|250px|right]]
|width="270px"|[[Image:Biomod2011 Team Tokyo111028Light-irradiatedGatheringMode02.png|250px|right]]
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:In the light-irradiated gathering mode, DNA ciliates gather at a specific area responding to UV irradiation. This mode is achieved by UV-switching DNA devices.
:In the light-irradiated gathering mode, DNA ciliates gather at a specific UV-irradiated UV area; i.e., the DNA ciliate can be controlled by UV irradiation. This mode is achieved by a UV-switching DNA device with UV-responsive bases, azobenzenes. Before UV-irradiattion, this DNA device does not hybridize with deoxyribozyme of the DNA ciliate. After UV irradiation, azobenzenes are isomerized and the DNA device can hybridize with the deoxyribozyme of DNA ciliate. By this reaction, the hybridization of DNA ciliates with arrayed DNAs on a glass plate is controlled.
:The UV-switching DNA device has the stem-loop structure which has UV-responsive bases, azobenzenes. Before UV-irradiattion, this DNA device doesn’t trap deoxyribozyme legs of DNA ciliate. After UV irradiation, azobenzenes are isomerized and the DNA device traps deoxyribozyme legs of DNA ciliate. By this reaction, DNA ciliates gather at UV-irradiated area. By using this system, we think movement of DNA ciliates can be controlled.
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===UV-switching system===
===UV-switching DNA device and the mechanism of light-irradiated gathering===
:Deoxyribozyme, blocking DNA, and UV-switching DNA are used in this system. The UV-switching DNA hybridizes with blocking DNA and deoxyribozyme legs of DNA ciliates don’t hybridize with blocking DNA. This structure is closed state. By UV irradiation, the stem-loop of UV-switching DNA becomes open and branch migration starts, so deoxyribozyme legs of DNA ciliates become trapped by UV-switching-DNA. This structure is open state.
:The UV-switching DNA device is composed of a UV-switching DNA and a blocking DNA. The UV-switching DNA includes a complementary sequence of the deoxyribozyme on the DNA ciliate. However, the deoxyribozyme cannot hybridize with the UV-switching DNA before UV irradiation because the UV-switching DNA has a stem-loop structure and the blocking DNA at the complementary sequence of the deoxyribozyme (the “closed” state in the right figure). After UV irradiation, the stem-loop structure of the UV-switching DNA open, caused by the UV-responsive structural transition of azobenzenes in the UV-switching DNA. When the UV-switching DNA is open state, the deoxyribozyme can partly hybridizes with the UV-switching DNA and finally wholly hybridizes with the UV-switching DNA as a result of the branch migration of them.
:UV-switching DNA has a stem-loop structure which contains two azobenzenes. By spotting UV, azobenzenes are isomerized and this loop becomes open. This opened loop has a complementary part for deoxyribozyme. This UV-switching DNA is complimentary for blocking DNA.
|width="450px"|[[Image:Tokyo-gathering2.png|450px|center]]
|width="450px"|[[Image:Tokyo-gathering2.png|450px|center]]
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Project

The aim of our project is the construction of an autonomous micrometer-sized molecular robot. We designed and constructed a micrometer-sized molecular robot, “DNA ciliate”, named after water microorganisms, ciliate. The DNA ciliate has three independent functional modes (see the left figure): free moving mode, track walking mode, and light-irradiated gathering mode.

The DNA ciliate consists of a micrometer-sized body and cilia. The micrometer-sized body is made of a microsphere such as a polystyrene microbead, a glass microbead, etc. The cilia are made of deoxyribozymes (single-stranded DNAs with cleaving activity). In the free moving mode, the DNA ciliate move around in a broad space based on the Brownian motion. In addition, the DNA ciliate can walk along a single-stranded DNA track using the deoxyribozyme activity. The DNA ciliate can be gathered up at a UV-irradiated area. The construction of the DNA ciliate is shown in detail below.

The body of the DNA ciliate

DNA ciliates'body
DNA ciliates'body
The DNA ciliate consists of a micrometer-sized body and cilia. The body of the DNA ciliate is made of a polystyrene microbead with a diameter of about 1 μm. The cilia made of deoxyribozymes are attached on the surface of the body with the amide bond (a chemical covalent bond).
Figure.1:The pattern diagram of creating DNA ciliate
Figure.1:The pattern diagram of creating DNA ciliate

Three independent modes of the DNA ciliate

1. Free moving mode
The DNA ciliate is an autonomously moving molecular robot. The first moving mode is the free moving based on the Brownian motion in an aqueous media. By the free moving mode, the DNA ciliate can move around and explore in a broad range of space. This motion is similar to a random motion of microorganisms when the microorganisms search food.

The principle of free moving

The movement by Brownian motion is described by the right equation (1). The left side of the equation is the mean square displacement of a DNA ciliate. R is the displacement from a default position during the time Δt. The diffusion constant D is described by the right equation (2), where T is a temperature, NA is the Avogadro number, η is a viscosity coefficient, and a is the radius of the DNA ciliate body. The diffusion constant is inversely proportional to the radius of the body. Thus, a DNA ciliate with a smaller body can move more broadly.
The equation of Brownian motion.
The equation of Brownian motion.
The equation of Brownian motion.
The equation of Brownian motion.

2. Track walking mode
In the track walking mode, the DNA ciliate walks directionally along a track. The track is constructed from arrayed single-stranded DNA molecules that are substrates for the deoxyribozymes attached on the DNA ciliate body. DNA tracks with arbitrary shapes can be constructed using DNA nanotechnology, microfluidic technology, etc.

The mechanism of track walking

The cleaving activity of the deoxyribozymes on the DNA ciliate body is used when the DNA ciliates work at the track walking mode. The reaction scheme of the track walking is presented in the right figure. First, a deoxyribozyme hybridizes with a substrate DNA composing a track. The deoxyribozyme has an RNA cleaving activity and cleaves a ribose site (yellow site in the right figure) included in the substrate DNA. After the cleavage, the cleaved and shorten substrate and the deoxyribozyme dissociate because the thermodynamically stability between the deoxyribozyme and the shorten substrate DNAs decreases. The dissociated deoxyribozyme can hybridize with another uncleaved substrate DNA near the cleaved substrate DNA. The deoxyribozyme more easily hybridizes with an uncleaved substrate than a cleaved one. Thus, the DNA ciliate can walk along the DNA track by repeating this reaction.
Figure.1:The mechanism that DNA ciliate moves directionally.
Figure.1:The mechanism that DNA ciliate moves directionally.

Construction of DNA tracks

Although nanometer-sized DNA machines such as a DNA spider walks along a nanometer-sized DNA track constructed from DNA origami and DNA nanostructure, the DNA ciliate walks along a micrometer-sized DNA track (shown in the right figure) because the DNA ciliate has a micrometer-sized body. The micrometer-sized DNA tracks can be constructed using DNA nanotechnology, microfluidic technology, etc. Here, the micrometer-sized DNA tracks were constructed using a microchannel. Substrate DNAs were immobilized on a glass plate as a self-assembled monolayer through a silane coupling reaction.
Figure.2:The schematic diagram of microchannel.
Figure.2:The schematic diagram of microchannel.

3. Light-irradiated gathering mode
In the light-irradiated gathering mode, DNA ciliates gather at a specific UV-irradiated UV area; i.e., the DNA ciliate can be controlled by UV irradiation. This mode is achieved by a UV-switching DNA device with UV-responsive bases, azobenzenes. Before UV-irradiattion, this DNA device does not hybridize with deoxyribozyme of the DNA ciliate. After UV irradiation, azobenzenes are isomerized and the DNA device can hybridize with the deoxyribozyme of DNA ciliate. By this reaction, the hybridization of DNA ciliates with arrayed DNAs on a glass plate is controlled.

UV-switching DNA device and the mechanism of light-irradiated gathering

The UV-switching DNA device is composed of a UV-switching DNA and a blocking DNA. The UV-switching DNA includes a complementary sequence of the deoxyribozyme on the DNA ciliate. However, the deoxyribozyme cannot hybridize with the UV-switching DNA before UV irradiation because the UV-switching DNA has a stem-loop structure and the blocking DNA at the complementary sequence of the deoxyribozyme (the “closed” state in the right figure). After UV irradiation, the stem-loop structure of the UV-switching DNA open, caused by the UV-responsive structural transition of azobenzenes in the UV-switching DNA. When the UV-switching DNA is open state, the deoxyribozyme can partly hybridizes with the UV-switching DNA and finally wholly hybridizes with the UV-switching DNA as a result of the branch migration of them.
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