Biomod/2011/TeamJapan/Tokyo/Project/DNA tracks

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DNA tracks

Introduction

In already-developed moving molecular nanomachines, DNAs are often used as their track or landscape. For example, DNA spider is guided by prescriptive DNA origami landscapes. 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.
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.

Principle and methods

To make DNA track, four experiments were needed.
First experiment 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 experiment 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
Figure1. A series of attaching aminated DNA to glass reaction
Figure2. Construction of DNA track
Figure2. Construction of DNA track
Third experiment 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 experiment 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
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
  • Confirming DNA tracks, we use (5) protocol.
    • (5) The method of DNA-DNA hybridization…

Results

Figure4 is the result of making PDMS mold. We can see two right angle winding lines. They are a part of microchannels and using these microchannels, we arrayed DNAs. The result is Figure4. In Figure4, we hybridized fluorescent labeling complementary DNA strands with arrayed DNAs. With the hybridization of arrayed DNAs and fluorescent labeling complementary strands, We can see two fluorescent lines whose shapes are same as the designed microchannel in PDMS mold in Figure5. From the results of Figure4 and 5, we can say that we achieved to array DNAs as any shapes and make DNA tracks.
Figure4. This figure is microchannel in PDMS-mold. This figure was observed by phase contrast.
Figure4. This figure is microchannel in PDMS-mold. This figure was observed by phase contrast.
Figure5. This figure is the result of arraying DNAs on glass plate using microchannel of Figure4 and hybridized with their complementary fluorescent labeling DNA strands. This figure was observed by fluorescent phase contrast.
Figure5. This figure is the result of arraying DNAs on glass plate using microchannel of Figure4 and hybridized with their complementary fluorescent labeling DNA strands. This figure was observed by fluorescent phase contrast.


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

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