A Demethylation-Switchable\nAptamer Design Enables\nLag-Free Monitoring of m<sup>6</sup>A Demethylase FTO with Energy\nSelf-Sufficient and Structurally Integrated Features

Cellular\ncontext profiling of modification effector proteins is\ncritical for an in-depth understanding of their biological roles in\nRNA <i>N</i><sup>6</sup>-methyladenosine (m<sup>6</sup>A)\nmodification regulation and function. However, challenges still remain\ndue to the high context complexities, which call for a versatile toolbox\nfor accurate live-cell monitoring of effectors. Here, we propose a\ndemethylation-switchable aptamer sensor engineered with a site-specific\nm<sup>6</sup>A (DSA-m<sup>6</sup>A) for lag-free monitoring of the\nm<sup>6</sup>A demethylase FTO activity in living cells. As a proof\nof concept, a DNA aptamer against adenosine triphosphate (ATP) is\nselected to construct the DSA-m<sup>6</sup>A model, as the “universal\nenergy currency” role of ATP could guarantee the equally fast\nand spontaneous conformation change of DSA-m<sup>6</sup>A sensor upon\ndemethylation and ATP binding in living organisms, thus enabling sensitive\nmonitoring of FTO activity with neither time delay nor recourse to\nextra supply of substances. This ATP-driven DSA-m<sup>6</sup>A design\nfacilitates biomedical research, including live-cell imaging, inhibitor\nscreening, single-cell tracking of dynamic FTO nuclear translocation\nupon starvation stimuli, FTO characterization in a biomimetic heterotypic\nthree-dimensional (3D) multicellular spheroid model, as well as the\nfirst report on the in vivo imaging of FTO activity. This strategy\nprovides a simple yet versatile toolbox for clinical diagnosis, drug\ndiscovery, therapeutic evaluation, and biological study of RNA demethylation.