SCARPET: site-specific quantification of methylated and nonmethylated adenosines reveals mA stoichiometry.

mA has different stoichiometry at different positions in different mRNAs. However, the exact stoichiometry of mA is difficult to measure. Here, we describe SCARPET (site-specific cleavage and radioactive-labeling followed by purification, exonuclease digestion, and thin-layer chromatography), a simple and streamlined biochemical assay for quantifying mA at any specific site in any mRNA. SCARPET involves a site-specific cleavage of mRNA immediately 5' of an adenosine site in an mRNA. This site is radiolabeled with P, and after a series of steps to purify the RNA and to remove nonspecific signals, the nucleotide is resolved by TLC to visualize A and mA at this site. Quantification of these spots reveals the mA stoichiometry at the site of interest. SCARPET can be applied to poly(A)-enriched RNA, or preferably purified mRNA, which produces more accurate mA stoichiometry measurements. We show that sample processing steps of SCARPET can be performed in a single day, and results in a specific and accurate measurement of mA stoichiometry at specific sites in mRNA. Using SCARPET, we measure exact mA stoichiometries in specific mRNAs and show that Zika genomic RNA lacks mA at previously mapped sites. SCARPET will be useful for testing specific sites for their mA stoichiometry and to assess how mA stoichiometry changes in different conditions and cellular contexts.