m⁶A has different stoichiometry at different positions in different mRNAs. However, the exact stoichiometry of m⁶A 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 m⁶A 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 ³²P, 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 m⁶A at this site. Quantification of these spots reveals the m⁶A stoichiometry at the site of interest. SCARPET can be applied to poly(A)-enriched RNA, or preferably purified mRNA, which produces more accurate m⁶A 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 m⁶A stoichiometry at specific sites in mRNA. Using SCARPET, we measure exact m⁶A stoichiometries in specific mRNAs and show that Zika genomic RNA lacks m⁶A at previously mapped sites. SCARPET will be useful for testing specific sites for their m⁶A stoichiometry and to assess how m⁶A stoichiometry changes in different conditions and cellular contexts.

中文翻译：

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SCARPET：甲基化和非甲基化腺苷的位点特异性定量揭示了 m6A 化学计量


m6A 在不同 mRNA 的不同位置具有不同的化学计量。然而，m6A 的精确化学计量很难测量。在这里，我们描述了 SCARPET（位点特异性切割和放射性标记，然后进行纯化、核酸外切酶消化和薄层色谱），这是一种简单且简化的生化测定，用于量化任何 mRNA 中任何特定位点的 m6A。 SCARPET 涉及紧邻 mRNA 中腺苷位点 5’ 的 mRNA 的位点特异性切割。该位点用 32P 进行放射性标记，经过一系列纯化 RNA 并去除非特异性信号的步骤后，通过 TLC 解析核苷酸，以可视化该位点的 A 和 m6A。这些点的量化揭示了感兴趣位点的 m6A 化学计量。 SCARPET 可应用于富含 Poly(A) 的 RNA，或者优选纯化的 mRNA，从而产生更准确的 m6A 化学计量测量。我们证明 SCARPET 的样品处理步骤可以在一天内完成，并可在 mRNA 的特定位点对 m6A 化学计量进行特异性且准确的测量。 SCARPET 将有助于测试特定位点的 m6A 化学计量，并评估 m6A 化学计量在不同条件和细胞环境中如何变化。