Antibody-free fluorine assisted metabolic sequencing of RNA N4-acetylcytidine

More researches have revealed that N4-acetylcytidine (ac4C) affected a variety of cellular and biological processes. In order to better understand the ac4C roles in biology and disease, we present an antibody-free, fluorine assisted metabolic sequencing method to detect RNA N4-acetylcytidine, called ‘FAM-seq’. We have successfully applied FAM-seq to profile ac4C landscapes in humans. By comparing with the classic ac4C antibody sequencing method, we demonstrated that FAM-seq is a convenient and specific method for transcriptome-wide detection of ac4C. This method holds promise to detect nascent RNA ac4C modifications. Cells were fed with the pro-metabolite sodium fluoroacetate, which can be converted to its CoA metabolite fluoroacetyl-CoA in vivo by enzymes. Cellular RNA could therefore be acetylated with the fluoroacetyl group at supposed N4-acetylcytidine sites. The resulting fluoroacetamides could be converted to biotin or fluorophore tags by fluorine-thiol displacement reaction with high yield.