Fat mass and obesity-associated protein (FTO) is the first reported N⁶-methyladenosine (m⁶A) RNA demethylase. The dysregulation of FTO demethylation is strongly associated with various human cancers in a m⁶A-dependent manner. Herein, a homogeneous electrochemiluminescence (ECL) method for the determination of FTO was proposed based on the target-regulated DNAzyme cleavage. Moreover, the ECL signal was highly enhanced by host-guest interaction between β-cyclodextrin (β-CD) and tri-n-propylamine (TPrA). The m⁶A caged DNAzyme 17E-Me acted as a padlock, while the FTO served as the corresponding key. As the key, FTO could specifically remove m⁶A modification, restoring the cleavage activity of DNAzyme 17E. With the assistance of the Zn²⁺ cofactor, the substrate strand was cleaved at a specific site, and the ECL indicator of Ru(phen)₃²⁺ was discharged to produce an ECL signal. On the contrary, 17E-Me was blocked and no cleavage reaction occurred without the key. For the ECL detection, the electrode modification of β-CD@AuNPs concentrated Ru(phen)₃²⁺ species through electrostatic adsorption and gathered TPrA molecules through host-guest interaction with β-CD, which resulted in an intense ECL response. The results demonstrated the ECL intensity linearly correlated with the logarithm of the FTO concentration (from 0.0001 to 100 nM) with a low detection limit (30 fM). The IC₅₀ value for FTO inhibitors rhein and meclofenamic acid were 35.6 μM and 20.3 μM, respectively. The strategy was further validated for FTO detection in MCF-7 cell lysates and Hela cell lysates. This work reveals that this strategy is promising for developing homogeneous ECL method for detection of FTO and screening of the demethylase inhibitors.

Shuai Qiao

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