Leveraging Demethylase Activation in DNA Circuits to Overcome Signal Leakage for Reliable MicroRNA Bioimaging

Abstract DNA circuits show great potential in monitoring intracellular biomarkers based on their high programmability, predictability, and unique signal amplification capabilities, yet face challenges from uncontrollable signal leakage caused by the complex intracellular environment. Herein, a demethylase‐activated DNA‐assembly (DAD) circuit is designed for the reliable and robust imaging of cellular microRNA, by incorporating the sequential activation of a hybridization chain reaction (HCR) amplifier system. The DAD circuit consists of a demethylase‐activated DNAzyme module and a microRNA‐recognizing HCR signal‐amplifying module. The m 6 A‐modified DNAzyme sequence of DNAzyme module, initially possessing a temporally caged substrate‐cleavage activity, is integrated into the HCR probe for effectively blocking its miRNA‐sensing capacity. In the presence of ALKBH5 demethylase, the methyl‐modifying unit of DNAzyme is removed, thus restoring its catalytic substrate‐cleaving activity. This process exposed the previously caged toehold region of HCR probe, thereby activating the signal‐amplifying module for sensing miRNA. By leveraging sequential activation, this DNA circuit can substantially enhance the signal‐to‐background ratio, enabling highly sensitive miRNA detection for the efficient differentiation of cancerous and normal cells. Furthermore, the DAD circuit established a relationship between demethylase enzyme and miRNA, thus paving a reliable way for investigating more complicate biological processes and intricate signaling pathways within cells.