Global and single-nucleotide resolution detection of 7-methylguanosine in RNA.

RNA modifications, including N -7-methylguanosine (m ⁷ G), are pivotal in governing RNA stability and gene expression regulation. The accurate detection of internal m ⁷ G modifications is of paramount significance, given recent associations between altered m ⁷ G deposition and elevated expression of the methyltransferase METTL1 in various human cancers. The development of robust m ⁷ G detection techniques has posed a significant challenge in the field of epitranscriptomics. In this study, we introduce two methodologies for the global and accurate identification of m ⁷ G modifications in human RNA. We introduce borohydride reduction sequencing (Bo-Seq), which provides base resolution mapping of m ⁷ G modifications. Bo-Seq achieves exceptional performance through the optimization of RNA depurination and scission, involving the strategic use of high concentrations of NaBH ₄ , neutral pH and the addition of 7-methylguanosine monophosphate (m ⁷ GMP) during the reducing reaction. Notably, compared to NaBH ₄ -based methods, Bo-Seq enhances the m ⁷ G detection performance, and simplifies the detection process, eliminating the necessity for intricate chemical steps and reducing the protocol duration. In addition, we present an antibody-based approach, which enables the assessment of m ⁷ G relative levels across RNA molecules and biological samples, however it should be used with caution due to limitations associated with variations in antibody quality between batches. In summary, our novel approaches address the pressing need for reliable and accessible methods to detect RNA m ⁷ G methylation in human cells. These advancements hold the potential to catalyse future investigations in the critical field of epitranscriptomics, shedding light on the complex regulatory roles of m ⁷ G in gene expression and its implications in cancer biology.