Biomod/2011/TeamJapan/Tokyo/Project/Model of the track walking mode

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<div id="navigation"> <div id="menu" style="position:static"> <ul> <li><a class="aMain" href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo">Home</a></li> <li><a class="aTeam" href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Team/Students">Team</a></li> <li><a class="aProject" href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project">Project</a> <!-- <ul> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project">Overview</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/introduction">Introduction</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Model">Model</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Devices">Devices</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Modes">Modes</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Results">Results</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Achievements">Achievements</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Future_works">Future works</a></li> </ul> --> <li><font color="#ffffff">Results</font> <ul> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Results">Experiments</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Simulations">Simulations</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Achievements/DNA_Devices">DNA Design</a></li> </ul></li> <!-- <li><a class="Simulation" href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Simulations">Simulations</a></li> <li><a class="DNA design" href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Achievements/DNA_Devices">DNA Designs</a></li> --> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Achievements">Achievements</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Future_works">Future works</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Notebook/Protocols">Protocols</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Notebook/Lab.notebook">Notes</a></li> <li><a class="aNotebook" href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Sponsors/">Sponsors</a></li> <li><a class="aSitemap" href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Sitemap">Sitemap</a></li>

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Model of the track walking mode

Introduction

Track walking modeでは、DNA trackに沿って、 DNA ciliateを一方向的に歩かせることを目的としている。 その達成のためには、当然ながら、 「歩行に用いる機構(mechanism,reactionとか)」と、 「DNA trackの作りかた」を考える必要がある。

我々は、 「歩行に用いる機構」として「Deoxyribozyme-substrate」反応を選び、 「DNA tracksの作り方」としてマイクロ流路を用いることにした。

このページでは、それぞれの方法について詳しく説明する。

This mode utilizes DNA ciliate’s 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.


Deoxyribozyme-substrate interaction

↑どのような反応機構を用いて歩かせるか、という意味の見出しにする。

Deoxyribozyme-substrate reaction.png
Substrate and deoxyribozyme are used in this system. First, deoxyribozyme hybridizes with substrate. Second, deoxyribozyme cleaves substrate at ribose adenine and make two products. Third, the shouter product separates and the longer product:cleaved substrate hybridizes with deoxyribozyme. Forth, the hybridization becomes fragile and deoxyribozyme hybridizes with neighbor substrate. DNA ciliate repeats this reaction and move directly.

次に、反応に必要なDNAのデザインについて語る。

Sequence design

[Substrate]

Simplified image of substrate
  • 5' -(NH2)-TTTTTTTTTT TTTTCACTAT[rA]GGAAGAG-3'
  • Size: 28bases
    • Substrate is used for the scaffold of the track walking mode. It contains an RNA base at 21st from its 5' end. When substrate hybridizes with deoxyribozyme, substrate works as deoxyribozyme’s substrete by be cut at the ribose. The last 18bases from 3'end of substrate are equal to cleaved substrate in other pages.
    • Those 18bases are same to the substrate of the DNA spider's leg (TTTTCACTAT[rA]GGAAGAG).(ref) In molecular spider, substrate is used as a part of DNA origami, but we can’t use DNA origami as a scaffold (link), so we designed substrate as a independent DNA strand.
    • We designed the first 10 bases from 5' end as a linker (TTTTTTTTTT). This is also designed not to make unexpected structures. As a result, we decide using this linker.
    • The 5' end is aminated to be fixed firmly on a glass plate.


DNA tracks

DNA trackの敷き方としてどのような方法を考えたか (マイクロ流路を用い、ガラス表面にDSSを用いて固定しようと考えた、ということと、その理由まで述べられればベスト)。

DNA origami can be appropriately landscape for nanometer-sized moving nanomachines because it can be designed to complex structural DNA tracks. In fact, DNA origami is used to make landscape of DNA origami. 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.
For this reason, we decided to make the tracks of our DNA ciliate by arraying DNA molecules directly to glass plate. To array DNA at any shapes of track, we used microfluidic channel.
To make DNA track, four small goals were needed.
First small goal was making sample mold of microchannel. We used polyacetal resin as sample. We cut polystyrene resin by micro fine machining center and made a mold of microchannel. To make sample mold precisely, we shaved surroundings of the microchannel.
Second small goal was making PDMS mold. To begin with, we mixed PDMS and its hardener at the rate of 10:1(mass ratio). After cleaning bubble in this solution by using vacuum desiccators, next, we pour PDMS to the sample mold. After that, we heat sample mold and the solution to harden PDMS. Then, get hardened PDMS from sample mold. Microchannel we made on polyacetal-mold was transcribed to this PDMS-mold.
Figure1. A series of attaching aminated DNA to glass reaction
Figure2. Construction of DNA track
Third small goal was creating DNA tracks. To make DNA tracks, we used microchannel and arrayed DNAs on glass plate. To attaching DNAs on glass plate, we use DSS as the linker between aminated DNA and the glass.[1] DSS linker reacts with amino groups that are exposed on the surface of the MAS-coated glass.[2] DSS linker is very highly reactive with amino groups, so DNAs can be attached on the glass plate by covalent bonding with DSS linker (Figure1). We use DSS coated MAS-coated glass, and put PDMS-mold on the glass plate. Then, we poured DNA solution into microchannel (Figure2). With this operation, DNAs are arrayed as the shape of microchannel. We can design the shapes of microchannels freely, so we can make DNA tracks with freely designed shapes.
Fourth little goal was confirming whether DNAs were arrayed as the shape of microchannel. To confirm this thing, we used fluorescent labeling complementary strands for the DNA strands of DNA track. Using hybridization of these DNAs, we were able to check whether DNAs were arrayed as the shape of microchannel by fluorescence microscopes and were able to compare with control experiment.(Figure3)
Figure3. Confirmed by DNA hybridization

Protocol

  • Creating sample mold, we use (1) protocols.
    • (1) The method of using micro fine machining center is here.
  • Creating PDMS mold, we use (2) protocols.
    • (2) The method of making PDMS mold is here.
  • Creating DNA tracks, we use (3) or (4) protocols.
    • (3) The method of using APS agent and normal glass plate is here.In this method, attaching DNAs on glass beads is inefficient, so we don't use results of this protocol.
    • (4) The method of using MAS coated glass plate is

References

[1]DSS and BS3 Crosslinkers

http://www.piercenet.com/instructions/2160418.pdf

[2]MAS coated glass slide - MATSUNAMI GLASS IND.,LTD.

http://www.matsunami-glass.co.jp/english/life/clinical_g/data18.html