Dual-enzyme-propelled unbounded DNA walking nanomachine for intracellular imaging of lowly expressed microRNA

Despite the progress on the analysis of miRNA either in vitro or in vivo , intracellular imaging of lowly expressed microRNA remains a challenge. Here we develop a novel dual–enzyme–propelled DNA walking nanomachine, which is tailored to accomplish this mission. The nanomachine is constructed with nanoparticles–loaded DNA tracks, on which the targeted miRNA working as a single–foot DNA walker can move autonomously under the catalysis of two cooperative enzymes. Cleavage of the DNA tracks like a “burnt–bridge” mechanism is thereafter triggered, resulting in an amplified fluorescent signal. After the comprehensive study and optimization of the DNA nanomachine, miR–892b, a significantly down–regulated miRNA in breast cancer cells, is selected as a model target. Sensitivity detection in vitro is achieved with a superior detection limit of 4 pM. While being delivered into cells, the DNA nanomachine is available for the imaging of the lowly expressed microRNA, which is totally missing using the conventional fluorescence in situ hybridization (FISH) method. Up–regulation or down–regulation of the miRNA by exogenous regulatory factors can be also well evaluated. This DNA nanomachine provides a competitive approach for the analysis of miRNA, and has the potential to be extended to some other biomolecules.