Team Tianjin this year focuses on two DNAzymes, 8-17 and Cu2+ DNAzyme, which are capable of catalytic cleavage of substrate. A typical DNAzyme contains three regions: a conservative sequence that serves as the catalytic core, and two binding arms that are on either side of the core. For 8-17, it has the conventional structure. It first binds to the substrate, then works with the cofactor - Pb2+- to cut the substrate. For Cu2+ DNAzyme, it needs Cu2+ as cofactor. The two DNAzyme are incredibly sensitive to its corresponding ion, demonstrating an amazing selectivity.
Based on the selectivity, we designed this new logic gate molecules by combining 8-17 with Cu2+ DNAzyme. The molecules can only cleave substrate with the presence of both Pb2+ and Cu2+. This new logic gate design is rapid and accurate in response due to the unique property of ion activated DNAzyme.
The role of logic gate is to control, so it is applied to control two systems. One is the Y-DNA, the other is the origami delivery system.
For Y-DNA, NuPack simulation was used to optimize our design. The improved gate molecules control the formation and decomposition of Y-DNA, while the EcoRI digestion and T4 ligation control the polymerization. First, the self-cleavage induced by Cu2+ guarantees the forming Y-DNA with other two ssDNAs. Second, sticky ends are created by EcoRI and Y-DNA are ligated to form a large polymer. Finally, Pb2+ is added to activate 8-17 to cleave one branch of Y-DNA, making the whole polymer disintegrate. Our design showed its astounding advantage in Y-DNA controlling.
Furthermore, we intend to use the origami as a container for miRNA, and use the multiple turnover of 8-17 in the logic gate to achieve constant release of miRNA. This mechanism can be applied to construct an origami amplifier.
The approach of combining two DNAzymes to form a logic system is truly amazing. We can foresee much more new applications with our design in the future.