A Demethylation-Switchable Aptamer Design Enables Lag-Free Monitoring of m<sup>6</sup>A Demethylase FTO with Energy Self-Sufficient and Structurally Integrated Features
Yakun Shi, Yutian Lei, Meng Chen, Hansu Ma, Taorong Shen, Yanfei Zhang, Xing Huang, Wanxuan Ling, Si‐Yang Liu, Yihang Pan, Zong Dai, Yuzhi Xu
Abstract
Cellular context profiling of modification effector proteins is critical for an in-depth understanding of their biological roles in RNA N 6 -methyladenosine (m 6 A) 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 m 6 A (DSA-m 6 A) for lag-free monitoring of the m 6 A demethylase FTO activity in living cells. As a proof of concept, a DNA aptamer against adenosine triphosphate (ATP) is selected to construct the DSA-m 6 A model, as the “universal energy currency” role of ATP could guarantee the equally fast and spontaneous conformation change of DSA-m 6 A 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-m 6 A 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.