A Demethylation-Switchable Aptamer Design Enables Lag-Free Monitoring of m⁶A Demethylase FTO with Energy Self-Sufficient and Structurally Integrated Features

Cellular context profiling of modification effector proteins is critical for an in-depth understanding of their biological roles in RNA <i>N</i>⁶-methyladenosine (m⁶A) modification regulation and function. However, challenges still remain due to the high context complexities, which call for a versatile toolbox for accurate live-cell monitoring of effectors. Here, we propose a demethylation-switchable aptamer sensor engineered with a site-specific m⁶A (DSA-m⁶A) for lag-free monitoring of the m⁶A demethylase FTO activity in living cells. As a proof of concept, a DNA aptamer against adenosine triphosphate (ATP) is selected to construct the DSA-m⁶A model, as the "universal energy currency" role of ATP could guarantee the equally fast and spontaneous conformation change of DSA-m⁶A sensor upon demethylation and ATP binding in living organisms, thus enabling sensitive monitoring of FTO activity with neither time delay nor recourse to extra supply of substances. This ATP-driven DSA-m⁶A design facilitates biomedical research, including live-cell imaging, inhibitor screening, single-cell tracking of dynamic FTO nuclear translocation upon starvation stimuli, FTO characterization in a biomimetic heterotypic three-dimensional (3D) multicellular spheroid model, as well as the first report on the in vivo imaging of FTO activity. This strategy provides a simple yet versatile toolbox for clinical diagnosis, drug discovery, therapeutic evaluation, and biological study of RNA demethylation.