A fluorescent methylation-switchable probe for highly sensitive analysis of FTO N⁶-methyladenosine demethylase activity in cells.

N⁶-Methyladenosine (m⁶A) is one of the most abundant epigenetic modifications on mRNA. It is dynamically regulated by the m⁶A demethylases FTO and ALKBH5, which are currently attracting intense medical interest because of their strong association with several human diseases. Despite their clinical significance, the molecular mechanisms of m⁶A demethylases remain unclear, hence there is tremendous interest in developing analytical tools to facilitate their functional studies, with a longer term view of validating their therapeutic potentials. To date, no method exists which permits the analysis of m⁶A-demethylase activity in cells. To overcome this challenge, herein, we describe the first example of a fluorescent m⁶A-switchable oligonucleotide probe, which enables the direct detection of FTO demethylase activity both in vitro and in living cells. The m⁶A probe provides a simple, yet powerful visual tool for highly sensitive detection of demethylase activity. Through the use of m⁶A-probe, we were able to achieve real-time imaging and single-cell flow cytometry analyses of FTO activity in HepG2 cells. We also successfully applied the probe to monitor dynamic changes in FTO activity and m⁶A methylation levels during 3T3-L1 pre-adipocyte differentiation. The strategy outlined here is highly versatile and may, in principle, be adapted to the study of a range of RNA demethylases and, more widely, other RNA modifying enzymes. To the best of our knowledge, the present study represents not only the first assay for monitoring FTO activity in living cells, but also a new strategy for sensing m⁶A methylation dynamics.