Integration of Demethylation-Switchable Deoxyribozyme with Controllable Self-Assembly of Branched DNA Nanostructures Enables In Situ Monitoring of FTO Demethylation in Living Cells

Cellular profiling of fat mass and obesity-associated protein (FTO) and in situ monitoring of their demethylation programs still face major challenges due to limited sensitivity and high complexity of the context, limiting comprehensive knowledge of their biological functions. Here, an integrated sensing system (cD-CHA) is developed using N6-methyladenine (m⁶A) caged deoxyribozyme (cD) coupled with three-branched catalytic hairpin assembly circuits (3b-CHA) for monitoring demethylation processes of FTO in living cells. Initially, the FTO removes methyl groups of the cD structure, allowing for site-specific cleavage of the substrates and liberation of the initiator sequences, which trigger the cascaded assembly. Unlike conventional catalytic hairpin assembly, dimers were used as assembly units instead of a mixture of monomers, resulting in the formation of a multibranched DNA assembly product. In addition, the products exhibit a significantly limited movement within the enclosed intracellular milieu, which makes this system particularly suitable for truly in situ visualization of FTO and the observation of its demethylation in living cells. Moreover, our strategy can accurately evaluate the repair activity of FTO after various drug treatments and differentiate cancer cells from normal ones according to the expression level of FTO, providing a simple but reliable tool for clinical diagnosis and drug discovery.