A general strategy exploiting m5C duplex-remodelling effect for selective detection of RNA and DNA m5C methyltransferase activity in cells.

RNA:5-methylcytosine (m5C) methyltransferases are currently the focus of intense research following a series of high-profile reports documenting their physiological links to several diseases. However, no methods exist which permit the specific analysis of RNA:m5C methyltransferases in cells. Herein, we described how a combination of biophysical studies led us to identify distinct duplex-remodelling effects of m5C on RNA and DNA duplexes. Specifically, m5C induces a C3'-endo to C2'-endo sugar-pucker switch in CpG RNA duplex but triggers a B-to-Z transformation in CpG DNA duplex. Inspired by these different 'structural signatures', we developed a m5C-sensitive probe which fluoresces spontaneously in response to m5C-induced sugar-pucker switch, hence useful for sensing RNA:m5C methyltransferase activity. Through the use of this probe, we achieved real-time imaging and flow cytometry analysis of NOP2/Sun RNA methyltransferase 2 (NSUN2) activity in HeLa cells. We further applied the probe to the cell-based screening of NSUN2 inhibitors. The developed strategy could also be adapted for the detection of DNA:m5C methyltransferases. This was demonstrated by the development of DNA m5C-probe which permits the screening of DNA methyltransferase 3A inhibitors. To our knowledge, this study represents not only the first examples of m5C-responsive probes, but also a new strategy for discriminating RNA and DNA m5C methyltransferase activity in cells.

A fluorescent methylation-switchable probe for highly sensitive analysis of FTO N 6-methyladenosine demethylase activity in cells.

N 6-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.

A strategy based on nucleotide specificity leads to a subfamily-selective and cell-active inhibitor of N6-methyladenosine demethylase FTO.

The AlkB family of nucleic acid demethylases are of intense biological and medical interest because of their roles in nucleic acid repair and epigenetic modification. However their functional and molecular mechanisms are unclear, hence, there is strong interest in developing selective inhibitors for them. Here we report the identification of key residues within the nucleotide-binding sites of the AlkB subfamilies that likely determine their substrate specificity. We further provide proof of principle that a strategy exploiting these inherent structural differences can enable selective and potent inhibition of the AlkB subfamilies. This is demonstrated by the first report of a subfamily-selective and cell-active FTO inhibitor 12. The distinct selectivity of 12 for FTO against other AlkB subfamilies and 2OG oxygenases shall be of considerable interest with regards to its potential use as a functional probe. The strategy outlined here is likely applicable to other AlkB subfamilies, and, more widely, to other 2OG oxygenases.