SCARPET: site-specific quantification of methylated and nonmethylated adenosines reveals m⁶A stoichiometry

SCARPET accurately predicts m⁶A stoichiometry using methylated RNA standards. (A,B) SCARPET accurately predicts the stoichiometry of a methylation site in a mixture of two 42-nt long synthetic RNA oligonucleotides—one unmethylated and the other containing a defined m⁶A modification in a common GGACU consensus site for m⁶A. The two synthetic RNAs were mixed such that the m⁶A stoichiometry ranged from 0% to 100%. The mixture was added to 1 µg 2×-poly(A) HepG2 mRNA to simulate the conditions for biological m⁶A site determination in an mRNA. SCARPET predicted the m⁶A stoichiometry with high accuracy and showed excellent linearity (R² = 0.973) to the input m⁶A fraction (B). ³²P-labeled m⁶A and A nucleosides were run as TLC standards in A.