A demethylation-activated fluorescent DNA aptamer strategy for visualising DNA alkylation repair in living cells

Profiling the spatiotemporal dynamics of DNA demethylases is critical for deciphering the mechanisms of epigenetic regulation and genomic maintenance. However, existing fluorescent strategies often suffer from false-positive signals in living cells, primarily arising from non-specific nuclease degradation or instability of the complex amplification components. Herein, we present a generalisable "demethylation-activated" fluorescent light-up DNA aptamer (FLAP) strategy for high-contrast imaging of DNA alkylation repair in living cells. Our design relies on a precise "caging" strategy: site-specific methyl lesions (e.g., O⁶-meG) are engineered into the ligand-binding domain of the Bibb Lettuce aptamer, which disrupts its folding and suppresses fluorescence. Upon specific enzymatic repair, the aptamer structure is restored, triggering a robust fluorescence "turn-on" signal. This mechanism effectively minimises false-positive signals. The optimised probe detects MGMT activity with high sensitivity (LOD: 1.64 nM) and enables direct visualisation of active demethylation processes in MCF-7 cells, revealing distinct responses to inhibitors. Highlighting the modularity of the platform, we extended the design to detect AlkBH2 (LOD: 0.81 nM) simply by substituting the lesion with 1-methyladenine (1-meA). This work establishes a versatile and programmable framework for converting transient DNA repair events into quantifiable optical signals, providing a powerful tool for exploring epigenetic dynamics and cancer pharmacology.