Harnessing\nDemethylase-Regulated Catalytic DNA Circuit\nfor In-Situ Investigation of the Regulatory Connection with MicroRNA

Insight into the epigenetic modulation-correlated molecule\ninteractions\nhas significant implications for the in-depth understanding of intracellular\nintricate biological networks. However, there is currently a lack\nof reliable biological tools for elucidating the potential correlation\nbetween epigenetic regulators and relevant genes, e.g., microRNAs\n(miRNAs). Herein, an alkB homologue 5 (ALKBH5, a key epigenetic regulator)-modulated\ncatalytic DNA circuit (ACD) was constructed by grafting a N6-methyladenosine\n(m<sup>6</sup>A)-caged I-R3 DNAzyme into the circuitry components\nfor achieving the on-site miRNA imaging in living cells. Specifically,\nthe catalytic activity of I-R3 DNAzyme could be effectively suppressed\nby the m<sup>6</sup>A modification situated at its highly sequence-conserved\ncore region and then be selectively restored through the ALKBH5-mediated\ndemethylation pathway. And the ALKBH5-activated I-R3 DNAzyme allowed\nthe highly efficient DNA cleaving reaction in the presence of DNAzyme\ncofactors, resulting in the liberation of catalytic hairpin assembly\n(CHA) reactants. Subsequently, target miRNA triggered the CHA circuit\nto produce a duplex DNA product while releasing the miRNA analyte.\nThe liberated miRNA could autonomously trigger the next round of the\nCHA assembly cycle for generating the amplified fluorescence readout.\nBy virtue of the stimuli-responsive activation and the CHA amplification\ncircuit, the ACD system achieved highly specific and sensitive imaging\nof miRNA in tumor cells. Moreover, this efficiently and reliably ALKBH5-activated\nDNA circuit is demonstrated to reveal the underlying relationship\nbetween activator ALKBH5 and miRNA. Overall, the developed ACD system\nprovides a promising tool for the robust on-site profiling of epigenetic-involved\nsignal pathways, thus displaying great potential in bioanalytical\napplications.