Selective oxidation and quantification of m5C in ribonucleosides and RNA in an epigenetic context

Abstract

Selective modification of methylated bases in RNA and DNA is central to the field of epigenetics and furthermore fundamental for developing next-generation sequencing and liquid biopsy applications. While in DNA the detection and modification of 5-methylcytidine (5mdC) is fairly advanced, current strategies for detecting this modification (m5C) in RNA face significant technical challenges. Chemical conversion methods, such as bisulfite sequencing, remain widely used but induce substantial degradation of RNA and DNA, leading to incomplete conversion and reduced sequence complexity. Here we demonstrate the first example of a TET-biomimetic, selectively oxidizing the C–H bond of the methyl group in m5C in RNA. We also developed an isotope-labelling mass spectrometry-based strategy for quantification of the hydroxymethyl- (hm5C), formyl- (f5C) and carboxy-derivatives (ca5C), respectively. By changing substrate and iron complex ratios and the reaction time, the product spectrum can be tuned to obtain either more f5C or ca5C as a product. The iron complex’ ligand system can be further modified to fine-tune product yields and distribution.