This shows how the DNA strands come together. Three double stranded strings of DNA are denatured and then when slowly cooled will come together to form the Y shape. However after the denaturing each strand of DNA has an equal chance of bonding to the original piece of DNA as it does to the correct origami side. Therefore the more complex the structure the less likely it is that that structure will fully form.
This is a sequence we came up with for a Y shaped origami structure.
The highlighted colours correspond to half of one arm which is complementary to half of another arm of the same colour. At the ends of each coloured string is the binding site for a zinc finger.
This structure will self assemble into a shape with arms of length 150 base pairs with the 9 base pair long binding site on the ends.
This sequence had to have various boundary conditions, such as reasonable CG content, so that the melting temperature isn't massively out of the required range. The strings also couldn't be allowed to form secondary structures.
As you can see the probability of a structure fully forming decreases exponentially as the complexity increases. However, even though for larger number of arms there is a very high chance of a structure forming it is unlikely for all the arms to form. Therefore, for our experiments it would be better to focus on using structures with fewer number of arms to save time and money.
The previous design, which used DNA Origami required lengths of DNA to be synthesised. This is very expensive and time consuming. In order to minimise costs we need to be able to make the structures using already available DNA.
As you can see from the images above to accomplish this we will cut correct lengths of DNA out of already available plasmid inserts then denature them, binding two single stranded pieces of DNA together to form a string of twice the length. Doing this allows origami to be used to form a structure as the sides of the single strands will have a complementary pair. This also allows for quick PCR.
We decided to use plasmids from the last two years iGEM part registry.
