Biomod/2014/NTU/Method

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Experiment

Method

start

Before we synthesize the final product, we have to perform many pre-tests to ensure all the components work and define the best condition of temperature and concentration in our experiment. Here, we will introduce all the components, their function and the related pre-tests.

Guide staples

One important function of the jellyfish dimer is drug delivery. However, the origami monomer may not form a dimer solely with the aptamer lock and its complementary sequence. For example, monomer A has one of its aptamer lock bound to the complementary sequence on monomer B, but the other complementary sequence on monomer B is bound to another aptamer lock on monomer C, instead of the other aptamer lock on monomer A.

Therefore, we design four short sequences, which are called "guide staples." The guide staples are like clamps, connecting the outermost ring of two monomers. In order to maintain the feature of drug release, we need to remove the guide staples after the dimers form. We do so by adding 10 times the concentration of sequences complementary to the guide staples.

We divide our design into several parts. First, we build the bowl-like part without the lock and the inner tentacle, to prevent the long ssDNA from interacting with the scaffold and staple in the folding process. Once the bowl-like part is completed, we will add the locks and tentacles. On the inner side of the bowl, we leave 10 places to attach our tentacles. Our method is to design staples with hairpins at the end, and the tentacles with the hairpin's complementary sequence at its 5' end. The tentacles near the bottom of our jellyfish (at ring 4) are composed of those sequences and the H5 aptamer. The tentacles near the edge of jellyfish are composed of hairpin complementary sequence, aptamer complementary sequence, and poly C/G.

Protect strand

If we simply mix two kinds of tentacles, they will bind together even when not attached to the scaffold.

To avoid above problem, we put on aptamer first and add another ssDNA called" protect strand," which is designed to have higher affinity to the aptamer than " aptamer complementary sequence" do. Due to lower mismatch number of protect strand, we can add the outer tentacle partly consisted of poly C/G onto the bowl-like part.
We can test the efficacy of the protect strand by compare the ability of aggregation to the monomer that only attached with poly C/G sequence.We expect the design will the same aggregation effect as monomer with poly C&G.

While all tentacles bind to the hairpin, the next step is to remove the "protect strand" so that our tentacles will anneal with each other when there's no H5 protein in the solution. According to Nupack simulation, excess level of protect strand complementary sequence will achieve this.

To sum up, the affinity order of the four strands is "protection strand complementary sequence"-"protection strand" > "protection strand"-"inner aptamer"> "inner aptamer"-"Poly C/G" > "Poly C/G"- "protection strand complementary sequence"

Lock

We use the H5 aptamer and its complementary sequence as lock. An appropriate lock should stay closed when the H5N2 virus is absent, and should be opened when the H5 protein binds to the aptamer. We have to strike a balance with the strength of the affinity, and finally design a poly T mismatch sequence that has 36 thymine residues. We will test whether the lock is tight enough to remain close on the dimer and whether it is loose enough to be opened by the H5 protein.

reference :http://www.nupack.org/partition/show/507908?token=Ny03HpkgSo&time_refresh=1.0
Douglas, S. M., Bachelet, I., & Church, G. M. (2012). A logic-gated nanorobot for targeted transport of molecular payloads. Science, 335(6070), 831-834. doi: 10.1126/science.1214081

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