The objective of this section is to decide the perfect condition for monomer annealing. There are different aspects to annealing the perfect monomer: MgCl2 concentration, temperature, and annealing time.
Annealing of monomer - Deciding MgCl2 concentration
For this part, it did not take a lot of trials to find the perfect annealing condition as the design is 2D and has been well documented. The temperature gradient used in annealing is as seen in fig 1.1 while the gel electrophoresis result of the different MgCl2 concentrations can be seen on fig 1.2.
Alhough fig 1.2. did not show the differences between concentrations really well, intensity analysis using imageJ showed the most concentrated bands by density are the ones at 10 and 12 mM MgCl2 concentrations. The structure is an equivalent of about 5700 BP dsDNA, however it can be seen that the upper less darker band is at around 4 kb. This might have been influenced by the structure shape, which causes the flow through the gel to be different than predicted. Furthermore, the 2 bands in the lanes might also be caused by the monomers taking different conformations.
Annealing of monomer - AFM observation
AFM observation showed that the annealing produced proper monomers as predicted as seen in fig 1.3a and 1.3b.
Annealing of monomer - Conclusion
Even though gel electrophoresis result showed 2 bands that might mean that the monomers are taking different conformations that can influence the rate of flow through the agarose gel, AFM observation showed that the monomers are folded properly and that the conformations might be negligible.
Connection design overview
After successful of monomer annealing, we moved to the next target:
establishing the connection between the monomers.
Firstly, the first connection design is based on toe-hold method:
replacing monomer’s existing staple strands to the ones that are more
thermodynamically stable.
Second, we tried complementary strands methods: protruding staple
strands that have weak secondary structures and can make stable
connection with the counter partners.
Third, we tried the bridge method: protruding strands that are not
complementary, but can be connected by the bridge strands.
First connection - Incubation temperature
For the first connection design, the bridge strands were added at a ratio of monomer:bridge 2:1. These strands were added, and the monomers were incubated at a certain constant temperature for 4 hours. Fig 2.1. and fig 2.2. show the results of annealing at MgCl2 concentration of 10 and 12 MgCl2 concentration respectively.
Unfortunately, after 4 hours of incubation, the bridge did not form connection between the monomers as there was not any band showing the formation of dimers referring to fig 2.1 and 2.2.
First connection - Bridge concentration adjustment
As the previous experiment failed to show the formation of dimers, concentration of the replacement strands or bridges were adjusted. Previously, the ratio of monomer:bridge was 2:1, now the ratio was changed to 2:1 and 1:5 and the gel electrophoresis results are shown in fig 3.1. and 3.2. respectively.
The gel electrophoresis result showed that the dimers were not formed.
First connection - Monomer annealing condition adjustment
After changing the incubation condition and failing to form dimers. We tried changing the condition of annealing of the monomers in case that the monomers might not have annealed properly and leading to the failure of formation of dimers. Therefore, we changed the annealing temperature of the monomer as described in fig 4.1. The resulting gel electrophoresis is available on fig 4.2.
The result did not change compared to annealing using the previous condition. Therefore, it can be concluded that the problem is not the annealing of the monomers.
First connection - Final incubation temperature adjustment
As it was assumed that the monomers annealed properly, a final incubation incubation temperature change under different bridge concentrations was attempted. Results are as shown in fig 5.1.
First connection - AFM observation
AFM observation showed some monomers possibly forming dimers at a very low efficiency. Therefore, it can be concluded that the connection is unsufficient for this project.
First connection - Conclusion
After the different treatments, the connection did not appear to form. Therefore, it can be concluded that this connection design is not strong enough or that this connection design is not feasible to assist the swarming effect of the monomers.
Second connection - 3T spacer
In this section, the 2 different monomers with complementary protruding strands will be named monomer An and Bn where n indicates the number of spacers. The annealing condition for this section uses the annealing condition previously decided. The first experiment utilizes the protruding strands with 3T spacers and the result of gel electrophoresis available on fig 6.2.
By comparing lanes 2 and 3 with lanes 4 to 8, the dimers might have formed as there are darker bands above the monomer bands shown by 2 and 3, however there are ambiguous multiple bands and AFM was needed to confirm the existence of dimers. 29.37°C incubation temperature seemed to be the most appropriate temperature for the joining of monomers.
Second connection - 8T spacer
After the experiment for 3T spacer, 8T spacer experiment was also performed. The annealing and incubation conditions are unchanged. Results are shown in fig 6.4a and b.
Second connection - AFM observation
After taking pictures of around 140 samples of the second connection (3T spacers), around 25% of the monomers appeared to form dimers. The representative image is represented in fig 6.5a and the bar graph in fig 6.5b.
Unfortunately, due to technical difficulties, the AFM observation for 8T spacer was not taken well enough to be considered in the conclusion.
Second connection - Conclusion
The result of gel electrophoresis did not differ between 3T and 8T spacers. Furthermore, the connection yielded a relatively low yield of connection by just looking at the gel electrophoresis results. However, the formed dimers showed clean dimerization between the monomers, therefore it will be brought on to the next experiments.
Third connection - Bridge mediated connection
We also tested the other connection method by using bridge that connects the protruding strands. The monomers will be labeled as A and C. The ratio of the different monomers are 1:1 and the bridge will be 5-fold of the monomers. Fig 7.1b shows the results of the analysis done of the gel by measuring the area of the bands. The result shows an efficiency of around 37-48% in forming dimers and is significantly higher than the ones that are expected to only monomers.