Orthogonal activation of a DNA molecular reaction network for proteinase-free and highly specific mRNA imaging in live cells

Construction of artificial DNA molecular reaction networks (DMRNs) represents an effective approach in biosensing. Here we developed a reaction network consisting of a dual catalytic hairpin assembly-mediated tandem molecular circuit combined with strand displacement reaction (DCHA-SDR) for proteinase-free and highly specific mRNA imaging in living cells. The strand displacement reaction (SDR) was designed to be orthogonally activated in exact order by two fuel strands that separately split into inactive species and grafted into the dual catalytic hairpin assembly-mediated molecular tandem circuit (DCHA). The fuel strands can be activated only when the specific mRNA is introduced, and DCHA is initiated by using two different pieces of mRNA sequences as target. In this way, the target mRNA can be automatically matched twice to obtain an accurate and amplified recognition. Comparative analysis indicates that this orthogonal strategy for target identification is superior to the strategy consisting of single CHA-mediated molecular circuit and strand displacement reaction (SCHA-SDR). Moreover, we demonstrate that the DCHA-SDR system comes with the benefit of proteinase-free progress, which makes the approach suitable for operation in living cells. Final application in various cancer cells and normal cells reveals its enormous potential for highly specific mRNA imaging in biological systems.