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Enzyme-free amplification techniques based on dynamic DNA self-assembly (DDSA) have recently been developed for the in situ detection of mRNA in living cells. However, signal generation in traditional DDSA amplifiers is mainly dependent on the random diffusion of dissociative probes in a bulk solution, which is generally accompanied by poor kinetics and interference from complex biological systems. In this work, a new amplifier based on the design of an intramolecular catalytic hairpin assembly (intra-CHA) is proposed for the FRET imaging of mRNA in living cells. Compared with that in the free catalytic hairpin assembly (free-CHA), probes H1 and H2 in intra-CHA were simultaneously fixed on a DNA tetrahedron. The distance between them was closer, the local concentration of H1 and H2 in intra-CHA was theoretically approximately 808-times higher than that in free-CHA, and the initial reaction rate was enhanced 15.6 fold. Due to the spatial confinement effect, the reaction kinetics for target-catalyzed signal generation were significantly improved. By virtue of the three-dimensional nanostructure, H1 and H2 in the intra-CHA amplifier entered cells without any transfection or nanocarrier, and the probes and their products were free from biological interference, providing much higher signal stability for the reliable imaging of mRNA in living cells.

An intramolecular catalytic hairpin assembly on a DNA tetrahedron for mRNA imaging in living cells: improving reaction kinetics and signal stability