Biomod/2011/TeamJapan/Tokyo/Project/Results of the light irradiated gathering mode

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Light-irradiated gathering mode

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 and gathering of DNA ciliates.
In this page, we show the two experimental results: confirmation of UV-switching and observation of gathering DNA ciliates. It is confirmed that UV-switching system worked and that DNA ciliates gathered, so the light-irradiated gathering mode will be achieved.

Mechanism

UV-switching system

The UV-switching DNA has a stem-loop structure and short blocking DNA, which blocks hybridization of deoxyribozyme. After UV irradiation, this loop becomes open, and hybridize with the deoxyribozyme. (more detail...)

Results & Discussions

1. Confirmation of UV-switching

Image of non-denaturing 20% PAGE for the confirmation of UV-switching
Image of non-denaturing 20% PAGE for the confirmation of UV-switching

method

  • Doing PAGE analysis
How to make samples for electrophoresis is here.
How to do PAGE is here.

result

  • Non-denaturing 20% PAGE result is here.
U…UV-switching-trap-DNA
B…Blocking DNA
D…Deoxyribozyme DNA
Reaction solution…A 0.225uM and B 0.45uM and D 0.225uM
All solutions are in 5x SSC (sodium citrate 75mM)
  • From left, these bands mean followings.
  1. U 0.225uM and Mg(2+) 80mM
  2. B 0.45uM and Mg(2+) 80mM
  3. D 0.225uM and Mg(2+) 80mM
  4. U 0.225uM and B 0.45uM and Mg(2+) 80mM
  5. U 0.225uM and D 0.225uM and Mg(2+) 80mM (UV isn’t spotted)
  6. U 0.225uM and D 0.225uM and Mg(2+) 80mM (UV is spotted for 60 min.)
  7. Reaction solution (UV isn’t spotted)
  8. Reaction solution (UV is spotted for 15 min.)
  9. Reaction solution (UV is spotted for 60 min.)
  10. Reaction solution and Mg(2+) 80mM (UV isn’t spotted)
  11. Reaction solution and Mg(2+) 80mM (UV is spotted for 15 min.)
  12. Reaction solution and Mg(2+) 80mM (UV is spotted for 60 min.)
  • The control bands were appeared in lane 1 to 6. Lane 4 (U 0.225uM and B 0.45uM and Mg(2+) 80mM) means the bands when the loop is stable and hybridization U and B (band U-B). Lane 5 (U 0.225uM and D 0.225uM and Mg(2+) 80mM (UV isn’t spotted)) means the bands when the loop is open and hybridization of U and D. Lane 6 (U 0.225uM and D 0.225uM and Mg(2+) 80mM (UV is spotted for 60 min.)) means the bands when the loop is open and spotted UV (band U-D).

discussion

  • In the presence of Mg(2+), the switching was caused clearly (lane 10 to 12) because of the stable effect of Mg(2+).
  • Before UV irradiation (lane 10), the UV-switching DNA was closed state (band U-B). After UV irradiation (lane 11, 12), the band shifted to the position of hybridized state (band U-D). Thus, the UV-switching device we designed worked successfully as we intended.

2. Gathering at the specific area

A fluorescent image of DNA ciliate gathering at the specific area
A fluorescent image of DNA ciliate gathering at the specific area

method

  • Attaching complementary DNA of deoxyriboazyme on a glass plate
  • Making the situation which deoxyribozymes hybridize with complementary DNA on the glass plate
How to make the situation for hybridization is here
  • Putting DNA ciliates on the glass plate
  • Waiting for 2 hours
  • Observing the DNA ciliates under an fluorescent microscope

result

  • A fluorescent image of the DNA ciliates gathering at the spot of complementary DNA is here.
  • Complementary DNA was attached on upper-right area in this image.
There was no DNA in lower-left area in this image.

discussion

  • DNA ciliates gathered at the spot of complementary DNA, and didn't gather at another area. Following this result, it was confirmed that DNA ciliates can gather at the specific area after UV irradiation.
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