Aberrant demethylase activity is intimately associated with cancer initiation and progression, necessitating sensitive detection methods for biological applications.We developed epigenetically modified DNAzyme-powered walkers (EMOWAs) immobilized on gold nanoparticles for simultaneous detection of O6-methylguanine-DNA methyltransferase (MGMT) and fat mass and obesity-associated protein (FTO). The 8-17 DNAzymes were inactivated by incorporating N6-methyladenine or O6-methylguanine modifications at catalytic cores, which are specifically removed by target demethylases to restore enzymatic activity. Signal amplification was achieved through autonomous walker movement along nanoparticle surfaces. The dual-EMOWA@AuNP system achieved detection limits of 8.8 ± 0.3 pM for MGMT and 5.7 ± 0.2 pM, representing approximately 100-fold improvement over existing methods. The orthogonal metal ion dependencies (Mg2+/Zn2+) ensured cross-reactivity-free multiplexed detection. Cellular imaging successfully distinguished T98G, MCF-7, and U87 cancer cell lines based on their demethylase expression profiles, with fluorescence intensities showing excellent correlation with Western blot analysis (R2 > 0.98). In vivo studies in tumor-bearing mice demonstrated specific tumor accumulation, precise signal localization, and minimal systemic toxicity. This work establishes a sensitive, specific, and biocompatible platform for multiplexed visualization of demethylase activities in living cells and animals, providing a valuable tool for understanding cancer epigenetics and guiding targeted therapeutic development.
Li et al. (Mon,) studied this question.