Biomod/2013/Todai/Experiment

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       <a name="Assembling_of_DNA structure (scaffold+staple+cholesterol)">1.Assembly of DNA structure (scaffold+staple)<sup>[2]</sup></a>
       <a name="Assembling_of_DNA structure (scaffold+staple+cholesterol)">1.Assembly of DNA structure (scaffold+staple)<sup>[2]</sup></a>
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Revision as of 02:43, 19 September 2013


Experiment-Todai nanORFEVRE-

 Experiment


 Contents

 Pilot Study


2-2. Floatation assay of liposome and DNA origami
Result of agarose gel electrophoresis of the sample of flotation assay
The result of 1% agarose gel electrophoresis(100V,30min). In this measurement, the fluorescence of Cy5, which is integrated into DNA origami(Rect tile[1]) ,is observed. Fraction1 is the liquid in the top layer, and fraction 5 is in the bottom layer. Fraction 6 is the sample retrieved from precipitation. DNA origami solely was also loaded on the extreme right lane.
Fluorescence intensity of the samples of flotation assay(DNA Rect tile +liposome)
Although the size of liposome might change during the flotation assay(data not shown), the intensity of the fluorescence of NIL(Nile Red, ex 500nm, em 550~700nm ) suggests the amount of lipid membrane,liposome. The fluorescence spectrum of water was subtracted as background

[Discussion]

To confirm the flotation assay, mixed tiles(DNA origami) and liposomes were assayed. Five samples (fraction 1,2,...,5, from the top) were retrieved from supermetant liquid and a sample(fraction 6) from precipitation by the addition of buffer used in assay. When the sample, tile mixed with liposomes, were assayed, tiles were observed in the top layer. The distribution of liposomes is observed by the fluorescence of NIL(Nile Red).


3. Comparision of dimerization method

Result of urea gel electrophoresis of the sample of click reaction
[Discussion]

Copper(Ⅰ) catalyzed click reaction was used to dimerize of oligo DNA(length of 20bp and 14bp). The time cause of the reaction indicate that the click reaction is so quick(<5min).


results of the electrophoresis of DNA-tube
[Discussion]

We examined how DNA-tube was synthesized efficiently by using the method which is introduced in “Rapid Folding of DNA into Nanoscale Shapes at Constant Temperature” (Jean-Philippe J. Sobczak et al, Science, 2012, 338, 1458) [2]. In the figure above, two bands derived from scaffold or DNA-tube were showed with cursors. At 56.4℃, the scaffold band was diminished. In contrast, the DNA-tube band was concentrated. In fact, the ratio of DNA- tube band to scaffold band was the greatest at this temperature, which means we succeeded in synthesizing DNA- tube efficiently and improving the yield of DNA-tube.


Result of agarose gel electrophoresis of T-motif (wheel)
The result of 1.5% agarose gel electrophoresis(100V) for 33min.
[Discussion]

In this measurement, it is difficult to distinguish between final structure band and monomer band. Next time, Native PAGE will be used instead of agarose gel.


 Protocols

1.Assembly of DNA structure (scaffold+staple)[2]

[Reagent]

M13mp18ss (0.1μM) 5μL
staple mix (0.68μM) 4μL
10x buffer 1μL

[Procedure][2]
  • 5μL of M13mp18ss, 4μL of staple mix and 1μL of 10×buffer were mixed, pipetting.
  • The solution was kept at 45 degree C for 4h.

2.Flotation assay

[Reagent]

150mM aqueous KCl solution 3mL
POPC 3mg
Chloroform (99.0%) 3mL
40μM Nile Red solution 0.1mL

[Procedure]
  • POPC were dissolved in 3mL of Chloroform.
  • A lipid film was formed by evaporating 3mL of POPC solution in a 50mL eggplant flask, using a rotational evaporator for 5 minutes.
  • The flask was kept under vacuum overnight to evaporate remaining chloroform.
  • The lipid film was resuspended in 3mL of a 150mM aqueous KCl solution.
  • The solution was filtered through 200nm polar filter with extruder to even the size of liposome.
  • The size of liposome was measured with DLS (Viscotek 802 DLS).
  • The solution was kept at 3 degree C until usage.

[Reagent]
・Sucrosebuffer
a) 0.375M Sucrose buffer
HEPES-KOH(pH7.6) 50mM
KCl 100mM
MgCl2 10mM
Sucrose 0.375M
b) 1.25M Sucrose buffer
HEPES-KOH(pH7.6) 50mM
KCl 100mM
MgCl2 10mM
Sucrose 1.25M
c) 1.6M Sucrose buffer
HEPES-KOH(pH7.6) 50mM
KCl 100mM
MgCl2 10mM
Sucrose 1.6M

・liposome
・DNA origami(Rect tile[1])
~30nM

[Procedure]
  • sample + sucrose buffer were layered in this order on centrifuge tubes.
  • The tubes were centrifugalized.
  • Samples were fructionized.
  • Precipitation was resuspended with sucrose buffer.
  • Samples were measured by the fluorescence of NIL(ex 500nm, em 550nm~700nm) and agarose gel electrophoresis.

3. Comparision of dimerization method

[Reagent]

azide solution (10μM) 3μL
alkyne solution (10μM) 3μL
CuSO4 solution (50mM) 1μL
THTA solution (100mM) 1μL
sodium ascorbate solution (100mM) 1μL

[Procedure][4]
  • The above all solutions were mixed, using a vortex.
  • The solution was kept at room temperature.



 Reference

Rothemund, P. W.
Nature 440, 297–302 (2006)
Jean-Philippe J. Sobczak, Thomas G. Martin, Thomas Gerling, Hendrik Dietz
Science, 2012, 338, 1458
Masayuki Endo, Ryoji Miyazaki, Tomoko Emura, Kumi Hidaka, and Hiroshi Sugiyama
Journal of the American Chemical Society, 2012, 134, 2852-2855
Shogo Hamada, Satoshi Murata Prof.
Angewandte Chemie International Edition,2009,48(37),6820–6823








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