The Y-DNA was synthesized from three ssDNAs, each of which has partial complementary sequences to the other two ssDNAs. It occurred to us that we can utilize this logic gate to control the formation of Y-DNA by making the logic gate one of the ssDNAs.
From the PAGE result (Figure 24.), we can see that the first step cleavage (self-cleavage) of the logic gates are equally effective under the EcoRI, original and the mixed buffers. What is better is that the EcoRI buffer alone is enough for the formation and digestion. Furthermore, our optimized new logic gate still carried out the same function as we predicted.
Next, we use the determined reaction condition to produce Y-DNA and its polymerization. We put the logic gate, two ssDNAs and Cu2+ into the system, denatured and annealed. We can see the band of self-cleaved logic gate, the two-ssDNA. Later, EcoRI and T4 ligase were added to create the sticky ends and link them together. In Figure 25, lane 1, 2 and 3 are the marker. The bright spots near the top of gel in lane 4 & 5 imply that polymers have been produced. Lane 4 should be the Y-DNA polymer, while lane 5 is the polymer of self-cleaved logic gate molecules after EcoRI digestion. The polymer in lane 4 should be 2D, while lane 5 is one dimensional.
In Figure 26 are the AFM results of the polymers from lane 4 & 5. We can see that there are many white dots in the left image, indicating the existence of Y-DNA polymer. The right image has no white dots in it, showing no Y-DNA polymer produced in Lane 5.