biostudies-literatureMa MMedical Research CouncilEuropean Commission FP7Wellcome Trust183-7https://www.ebi.ac.uk/biostudies/studies/S-EPMC7617487biostudies-literatureUnknown444(2)Genomewide-association studies have revealed that SNPs (single nucleotide polymorphisms) in FTO (fat mass and obesity-associated) are robustly associated with BMI (body mass index) and obesity. FTO is an Fe(II) 2-OG (2-oxoglutarate)-dependent dioxygenase that can demethylate 3-meT (3-methylthymine) in single-stranded DNA, as well as 3-meU (3-methyluracil) and N6-methyl adenosine in RNA. In the present paper we describe the development of an RNase-cleavage assay measuring the demethylation activity of FTO on 3-meU. RNase A cleaves at the 3'-end of pyrimidines, including uracil, and a methyl group at position three of uracil inhibits cleavage. An oligonucleotide probe was designed consisting of a DNA stem, an RNA loop containing a single 3-meU as the only RNase A-cleavage site, a fluorescentThe Biochemical journalKinetic analysis of FTO (fat mass and obesity-associated) reveals that it is unlikely to function as a sensor for 2-oxoglutarate.PMC7617487FP7_266408G0600717BG0900554241592084812FP7_241592G0600717Ron DHarding HPMa MYeo GSO'Rahilly SfalseKinetic analysis of FTO (fat mass and obesity-associated) reveals that it is unlikely to function as a sensor for 2-oxoglutarate.Genomewide-association studies have revealed that SNPs (single nucleotide polymorphisms) in FTO (fat mass and obesity-associated) are robustly associated with BMI (body mass index) and obesity. FTO is an Fe(II) 2-OG (2-oxoglutarate)-dependent dioxygenase that can demethylate 3-meT (3-methylthymine) in single-stranded DNA, as well as 3-meU (3-methyluracil) and N6-methyl adenosine in RNA. In the present paper we describe the development of an RNase-cleavage assay measuring the demethylation activity of FTO on 3-meU. RNase A cleaves at the 3'-end of pyrimidines, including uracil, and a methyl group at position three of uracil inhibits cleavage. An oligonucleotide probe was designed consisting of a DNA stem, an RNA loop containing a single 3-meU as the only RNase A-cleavage site, a fluorescent2012-01-01T00:00:00Z2012 Jun2025-04-18T14:00:54.49Z2025-04-04T14:46:59.587ZS-EPMC76174872243570710.1042/bj2012006510.1042/BJ20120065