Spatial Confinement–Accelerated DNAzyme Nanoassembly for Demethylase Imaging and Chemoresistance Evaluation in Living Cells

Nucleobase-editing processes, exemplified by reversible DNA methylation, constitute a central regulatory layer in gene expression and cellular signaling yet remain difficult to interrogate with high sensitivity and selectivity in complex biological environments. DNAzymes offer a programmable catalytic platform with intrinsic substrate recognition and signal amplification capability; however, their application to nucleobase-editing enzyme systems is fundamentally limited by low signal gain and insufficient selectivity. Herein, we report a spatial confinement-accelerated demethylase-sensing platform based on a compact DNAzyme nanoassembly (RCA-m6Dz-ZnO NPs) that converts the site-specific nucleobase demethylation into a methylation-gated catalytic amplification. An m6A-caged DNAzyme is embedded within a rolling circle amplification scaffold that enforces the nanoscale colocalization of DNAzyme catalyst and substrate. Demethylation by ALKBH5 restores DNAzyme's catalytic activity, triggering DNA cleavage, programmed nanostructure disassembly, and amplified fluorescence output. The spatial confinement enforces intramolecular turnover rate, thus accelerating reaction kinetics while enhancing nuclease resistance. This base-modification-dependent catalytic gating further ensures high selectivity against closely related demethylases, enabling precise intracellular discrimination. Leveraging this capability, the nanoassembly further identifies the coupling between ALKBH5 downregulation and CK2α-driven glycolytic reprogramming, highlighting a mechanistic link between epigenetic regulation and metabolic adaptation. By translating the enzyme activity into discrete DNA reaction outputs, this work establishes a generalizable framework for mapping resistance-associated signaling pathways and advancing activity-based precision diagnostics in living systems.