Material & Method
Nano Face-Off Synthesis
Optimizing the Condition of Folding DNA Origami – MgCl2 Concentration
1. Mix the following components
2. Run the following procedure by PCR machine
(i) heat up to 80°C; incubate for 10 mins
(ii) 80°C to 61°C at 4 min / °C
(iii) 60°C to 25°C at 20 min / °C
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3. Run gel electrophoresis to find out the optimal concentration of MgCl2
Optimizing the Condition of Folding DNA Origami – Molar Ratio between Scaffold and Staple
1. Mix the following component
2. Run the following procedure by PCR machine
(i) heat up to 80°C; incubate for 10 mins
(ii) 80°C to 61°C at 4 min / °C
(iii) 60°C to 25°C at 20 min / °C
​
3. Run gel electrophoresis to find out the optimal concentration of MgCl2
The Folding of DNA Origami - Base
-1. Mix the following component
2. Run the following procedure by PCR machine
(i) heat up to 80°C; incubate for 10 mins
(ii) 80°C to 61°C at 4 min / °C
(iii) 60°C to 25°C at 20 min / °C
The Folding of DNA Origami - Left lid and Right lid
-1. Mix the following component
- 1. Mix the following component
2. Run the following procedure by PCR machine
(i) heat up to 80°C; incubate for 10 mins
(ii) 80°C to 61°C at 4 min / °C
(iii) 60°C to 25°C at 20 min / °C
Steps for running gel electrophoresis
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Mix 1xTAE buffer 150mL and agarose gel powder 1.5g, and heat up the mixture with microwave till the powder is completely dissolved and the solution becomes transparent.
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Wait till the solution cools down a bit. Pour the mixture into the glue plate and put on the suitable tooth comb; leave it for 50 minutes for solidification.
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Put the solid agar gel with the plastic case into the electrophoresis groove.
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Mix 20μL of sample solution and 2.2μL of 10X loading dye, and load each sample to wells. (the first well and the last well is loaded with DNA markers.)
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Add 3μL of (10^-5M) EtBr solution to the electrophoresis groove evenly.
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Put on the lid and open the switch to run the electrophoresis with 110V half-voltage for an hour. (cover with tin foil to prevent EtBr from decomposing)
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After the electrophoresis, put the agar gel in the UV box for observation.
DNA Origami Purification
Amicon Ultra-0.5 mL Centrifugal Filters are used to remove the excess staples.
The following protocol refers to the handbook of the product.
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Assemble the filter and the centrifuge tube, load 200μl 1x TE buffer with 10mM MgCl2 into the filter.
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Spin down at 14000 g for 3 mins.
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Remove the liquid in the collection tube.
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Upside down the filter and put back into the collect tube.
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Spin down at 1000 g for 2 mins.
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Remove the liquid in the centrifuge tube.
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Turn the filter back to the original direction, load 200μl of sample in filter.
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Spin down at 7000 g for 10 mins.
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Discard the filtrate.
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Upside down the filter and put it into a new collection tube.
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Spin down at 500 g for 4 mins.
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Add buffer into the collection tube to the initial volume(200μl)
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Collect the sample.
Transmission Electron Microscope (TEM)
Sample Preparation
​TEM enables us to see the structures of our products and to measure the exact scales, for more information see instruments.
Sample preparation
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Drop 7µL DNA origami solution onto the copper grids.
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Let the origami settle onto the grid at room temperature for 5 mins.
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Excess sample is removed by filter paper.
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The grin then is negatively stained by one drop of 2% uranyl acetate solution
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Leave it for 2 mins.
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Remove the excess dye by filter paper.
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Air dry for 20 mins.
Observation
The grids are ready for observation. This is performed using an Hitachi H-7650 TEM, at 75kV.
Assembly
Adding Blockers
1. Add blocking staples solution to base solution. The mole of blocking staples is twenty times as the scaffold.
2. Put the mixture of base and blocker in Mini-Shaking-Hybridisation Oven, and shake at 25 ℃ for one hour.
Adding Hinge
1. Add hinge staples solution to lid solution. The mole of hinge staples is five times as the scaffold.
2. Put the mixture of lid and hinge in Mini-Shaking-Hybridisation Oven, and shake at 25 ℃ for one hour.
3. Purify the solution of lid with hinge to remove excess staples.
Combinig the Components
1. Mix the blocked base and purified left lid with hinge and purified right lid with hinge
2. Put the mixture in Mini-Shaking-Hybridisation Oven, and shake at 30 ℃ overnight.
3. Purify the solution of assemblies to remove excess staples.
Starting to flip
*Signal L is ssDNA which let the left lid to flip, while signal R is another ssDNA which let the right lid to flip.
*The mole of signal we add is ten times as the blocker
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1. To flip left lid: Add signal L to the mixture of Base and Both Lid
To flip right lid: Add signal R to the mixture of Base and Both Lid
To flip Both lids: Add signal L and signal R to the mixture of Base and Both Lid
2. Put the mixture in Mini-Shaking-Hybridisation Oven, and shake at 30 ℃ overnight.
​Reference
1. Martin, T G and Dietz, H. s.l. "Magnesium-free self-assembly of multi-layer DNA objects . " Nature Communications, 2012.3:1103.
2. Douglas, Shawn M., Ido Bachelet, and George M. Church. "A logic-gated nanorobot for targeted transport of molecular payloads." Science 335.6070 (2012): 831-834.
3. Han, D., Pal, S., Nangreave, J., Deng, Z., Liu, Y., & Yan, H. (2011). DNA origami with complex curvatures in three-dimensional space. Science, 332(6027), 342-346.