N6-methyladenosine (m6A) was recently discovered to be a widespread base modification present in thousands of mammalian mRNAs. In addition to its prevalence throughout the transcriptome, researchers have also revealed that m6A is a reversible modification, suggesting that dynamic regulation of mRNA methylation is a novel and widespread RNA regulatory mechanism in cells. Much of our understanding of the physiological importance of m6A comes from studies of m6A demethylases. Indeed, disruption of the m6A demethylase FTO leads to dysfunction of dopaminergic pathways in the brain and an abnormal response to cocaine in mice. Additionally, FTO genetic mutations have been strongly associated with diseases including melanoma and breast cancer in humans. Despite the importance of FTO for human health and disease, tools which directly measure FTO activity have not been developed. In this proposal, we use a simple yet innovative RNA aptamer-based strategy to develop assays which measure the activity of FTO and other m6A demethylating enzymes. The results of this Phase I SBIR will be the development of an optimized fluorescence assay for measuring m6A demethylase activity and demonstration of the compatibility of this tool for high-throughput screening of small-molecule FTO inhibitors. This application will enable us to develop the first commercially-viable assay for measuring FTO activity, which will fulfill an important need in the RNA methylation and FTO research communities. Furthermore, these experiments will enable the development of HTS assays for the identification of novel inhibitors of FTO and other RNA demethylase enzymes.
Disruption of the FTO gene has been strongly associated with a variety of human diseases, and FTO was recently shown to influence dopaminergic pathways and the response to drugs of abuse. Researchers recently discovered that FTO functions to demethylate the highly prevalent base modification N6-methyladenosine (m6A) in mRNAs; however, tools for measuring FTO activity have not yet been developed. The goal of this project is to develop an assay for measuring m6A demethylase activity which can eventually be used in high-throughput screens to identify small molecule inhibitors of FTO and other m6A demethylases. This tool will enable the discovery of drugs which target m6A demethylating enzymes and will facilitate the development of novel therapies for human disease.
