Developments of the mDZ-RhoBAST Probe for Super-Resolution Imaging of FTO Demethylation in Live Cells

Single-molecule localization microscopy (SMLM) provides precise, high-resolution visualization of target biomolecules in cellular imaging. Fluorescent light-up aptamers (FLAPs) offer a versatile approach for labeling RNA of interest in live cells by binding to fluorogens through adaptive recognition. Several FLAPs can generate blinking fluorescent signals in conjunction with fluorogens, enabling single-molecule imaging of RNA in living cells. An example of this is RhoBAST, which has been successfully used for single-molecule imaging of RNA due to its fast exchange kinetics with fluorogens. However, single-molecule imaging of RNA activities regulated by proteins in living cells presents a greater challenge. Here, we introduce mDZ-RhoBAST, a nucleic acid probe, to visualize the demethylation process by fat mass and obesity-associated protein (FTO) in living cells. This probe combines the 6mA-modified DNAzyme with the RhoBAST FLAP, activated by FTO, restoring the DNAzyme's cleavage activity and releasing RhoBAST to bind the target fluorogen. Our approach achieved super-resolution imaging of the demethylation process of endogenous FTO, providing real-time insights into its demethylation processes in vivo.