Structure-Guided Design of an m1A Demethylation-Activated Aptamer for Live-Cell Monitoring of ALKBH3 Demethylase Activity

Abstract AlkB homolog 3 (ALKBH3) is a demethylase that regulates nucleic acid modification dynamics, and dysregulated ALKBH3 activity is associated with various diseases, including cancers and neurodegenerative disorders. However, accurate monitoring of ALKBH3 demethylase activity in living cells remains challenging. Here, we report a structure-guided strategy for engineering N1-methyladenine (m1A) demethylation-activated DNA aptamers for monitoring ALKBH3 demethylase activity in living cells. Guided by the high-resolution structure of a recently reported adenosine triphosphate DNA aptamer, we design a site-specific m1A modification that leads to an inactive conformation, which can switch to an active conformation upon m1A demethylation by ALKBH3 (m1Aapt). We further integrate this m1Aapt with a tetrahedral DNA nanostructure (TDN) to construct an m1Aapt-TDN sensor for comparative monitoring of ALKBH3 demethylation activity in living cells. Finally, we show that the m1Aapt-TDN sensor detects reduced ALKBH3 activity in cells expressing pathogenic CAG-repeat RNA and monitors glutamine metabolism-associated fluctuations in ALKBH3 activity. Our work presents a structure-guided strategy for converting aptamers into demethylation-activated sensors, offering molecular tools for monitoring demethylase activity in living cells.