Result
Electropherogram
--The Optimization of Folding Condition
MgCl2 concentration
Magnesium ion creates a positive charge environment around the negative charge of DNA, preventing them from destabilization by electrostatic repulsion. According to this property, the appropriate concentration of magnesium ion is crucial for the folding process of DNA origami
In the beginning, we tested several concentration of magnesium chloride to find out the optimal folding condition for 3 components.(Figure 1, 2, 3)
Figure 1. Determining the optimal concentration of MgCl2 for Base folding
In the part of base, according to the electrophoresis result shown in Figure 1 , the structures folded under 20mM,25mM and 30mM of MgCl2 were stuck in the wells, whereas 10mM and 15mM groups showed larger and more visible bands. We further compared the intensity of band in 10mM and 15mM groups, and the latter was slightly stronger than the former. As a result, we chose 15mM as the optimal concentration of MgCl2 in folding process.
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Figure 2. Determining the optimal concentration of MgCl2 for Left Lid folding
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Figure 3. Determining the optimal concentration of MgCl2 for Right Lid folding
In the part of two lids, according to the electrophoresis result shown in Figure 2 and Figure 3, there are two different bands in each well. We assumed that the band between 5kb and 4kb is the band of our lids, since there’s a slightly band shift compared to the unfolded scaffold. The bands of lids appear to be the most obvious in 15mM group, so all future synthesis will be performed at this concentration.(Note: For Lane 4 in Figure 3, no bands were observed since the sample isn't loaded into the well properly.)
Staple-Scaffold Ratio
In order to find out the optimal proportion between scaffold and staple, we tested different ratios of scaffold and staple in the folding process.
Figure 4. Staple : Scaffold ratio test in base folding
According to the electrophoresis result shown in Figure 4, we chose staple:scaffold=10:1 to fold our base since some misfolding structures trapped in the wells in 5X staples and 7X staples groups .
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Figure 5. Staple : Scaffold ratio test in Left Lid folding
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Figure 6. Staple : Scaffold ratio test in Right Lid folding
According to the electrophoresis result shown in Figure 5 and Figure 6, the intensity of band in 5X staples group was slightly stronger than the intensity of band in 7X staples and 10X staples groups. Therefore, we chose staple:scaffold=5:1 to fold lids.
Sample Purification
Utilizing a centrifugal filter (Amicon® Ultra-0.5), we separate our product from the mixture of unfolded structures and excess staples. Since we assume our product is uniformly bigger than the small fragments, it should remain right up in the filter after the centrifuging process, whereas the leftovers will fall out of the filter. We then perform electrophoresis to confirm the efficiency of the filter, labeling “U” as the remaining mixture and “D” as the filtered fluids of the centrifugal purifying process. For ideal purificationing results, there shall be a band indicating enough folded product in the “U”, leaving most of the excess DNA fragments in the “D”. Noted that “Ref.” means the reference group, which is the unpurified sample.
Figure 7 Base purification test
Base on Figure 7, we assume the band being near 5kb to be our Base. At the lane of U3, though the band of the Base had slightly faded, the vague band of excess staples almost disappeared completely. Thus, we set the times of Base purification to be 4 times.
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Figure 8 Left Lid purification test
As Figure 8 shows, given that the band between 4kb and 5kb is our Left Lid, while the blurring band below indicates the unbounded staples, Repeating the purification process for 4 times should be the most preferable. The brightness of the band of our Left Lid stays almost the same from U1 through U5, but the band of excess staples fades significantly at U4. Therefore, we decided to purify the Left Lid for 4 times.
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Figure 9 Right Lid purification test
As Figure 9 shows, since we assumed the band between 4kb and 5kb is our Right Lid, while the blurry band indicates the unbounded staples, “U3” has a reasonable brightness of the band of our Right Lid. Moreover, the band of unbounded staples fades significantly at U3. Thus, we concluded that repeating the purification process for 3 times should be the most applicable.
All in all, we determined the optimal MgCl2 concentration, staple-scaffold ratio, and times of repeating the purifying process for each components with the result of electrophoresis. The conclusion is organized in the chart below.
Sample Assembly and Purification
Figure 10 Sample Assembly and Purification test
* : Purified once after assembled.
** : Purified twice after assembled.
(“Assembled” was meant to involve all the components, including the base, lids, blocker, and hinge.)
After confirming the folding of our components, we tested the assembly of them. To get rid of excess blockers and hinges DNA, we also performed the purifying process after the assembly.
Corresponding to the previous results, both Right and Left Lids exist between 4kb and 5kb, and the base appeared near 5kb. Notably, the components weren’t purified in these three lanes. Since blockers and hinges are all short DNA fragments, we expect that no obvious band shifts will happen upon adding them, which matches the experiment results. In Figure 10 we could also observe that for Left and Right Lids, the bands of excess DNA slightly faded more than the band of Left/Right Lids + Hinges after the purifying process, so we decided to purify both Right/Left Lid + Hinge before assembling them with the base. Finally, the products are barely seen in the lane of Assembled**, so we decided to purify the Assembled components once only.
TEM Imaging
To assure the 3D conformation of the structure and examine the so-called “face-off” action, we took the Transmission Electron Microscope (TEM) Imaging (Hitachi H-7650) as our tool. Before loading our sample onto the sample grid, we processed it through a short period of Ultrasonication. Though ultrasonication may irreversibly shock a few of the samples into scraps, it significantly prevents our sample from tangling together.
​We first individually observed whether three components successfully formed the expected structure by TEM.
Components
Figure 11 Base
Figure 12 Left Lid
Figure 13 Right Lid
As shown in Figure 11, we could see obvious E-shape objects in the image of base group, the size of which is about 50-60 nm. In TEM photo of lids (Figure 12, Figure 13), we could clearly recognize uniform L-shape objects which met the expected size (30 nm) we designed.
Basic Assembly
Figure 14 Base with both lids
After confirming the structure of individual components, we combined the blocked base and both lids with hinges and other DNA oligos to form the whole nano-device. Figure 14. illustrated the expected side view of the initial assembly of the Base and the Lids, which looks like a lying “8”, or the Chinese character “æ—¥” (/rì/). In TEM image, as our expectation, a object with the form of “æ—¥” was clearly recognized.
“Face-off”/ Flipping action
Figure 15 flipped left lid
Figure 16 flipped right lid
To further identify if Nano Face-off is functional as our mechanism design, we first triggered the movement by adding corresponding signals to the left and right side separately. As we can see in Figure 15 and Figure 16, after the signal stimulation, one of the lid flipped up and the other lid remained shut. (Although the structure form shown in Figure 16 was not as obvious as Figure 15.)
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In the assembled Nano Face-Off, adding the corresponding signal (ssDNA) will activate the flipping of one side. As shown in Figure 15 and 16, the results indeed proved that we can control the flip individually using different signals. Although in theory, we can acquire result that indicates the flip of both lids, we haven't been able to find out the desired forms under TEM. Nevertheless, we will keep on searching and thrive to prove that in the near future.
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