Dear Editor,

N6-methyladenosine (m6A) emerges as an abundant chemical modification on RNAs, which enriches in distinct internal regions linked to divergent aspects of RNA fate (Zhao et al., 2017). Transcriptome-wide mapping of m6A sites using high-throughput sequencing enables comparative analysis of cellular m6A dynamics on particular RNAs under both physiological and stress conditions (Dominissini et al., 2012; Meyer et al., 2015), yet is confined to low resolution of coverage and indistinction of adjacent m6A sites. Broad research has focused on developing sensitive and reliable approaches to probe m6A status on individual transcript (Li et al., 2016). A DNA polymerase identified from Thermus thermophilus (Tth pol) is in favor of incorporating thymidine opposite unmodified A over m6A (Harcourt et al., 2013). The specific feature of this enzyme allows determining the locations of m6A in cellular rRNA and mRNA transcribed from exogenous plasmid. SCARLET enables accurate identification and quantification of m6A in endogenous mRNA and long noncoding RNA (lncRNA) (Liu et al., 2013). Nonetheless, these methods require radioisotope labeling that may limit their widespread applicabilities. Recently, two groups screened novel m6A-selective DNA ligases, which were able to accurately determine the m6A locus in mRNA and lncRNA from total RNA or polyA-RNA (Liu et al., 2018; Xiao et al., 2018). Both approaches avoid radioactive labeling but potentially generate unspecific signals induced by other modifications. Here, we present a refined approach termed site-specific monitoring of m6A via reverse transcription (SMART) using fluorescein-labelled dUTP (fluorescein-dUTP), which allows identification of innate m6A status at specific site of both in vitro synthesized and endogenous RNAs without transcript amplification. SMART combines m6A antibody crosslinking with fluorescein-dUTP incorporation during primer extension. In detail, synthesized or isolated RNA is incubated with m6A antibody followed by crosslinking with UV light at 254 nm wavelength. After crosslinking, the antibody‒m6A conjugation could efficiently impede the incorporation of fluorescein-dUTP during reverse transcription (RT). The fluorescent RT products are visualized by the imager after separation on the TBE-urea gel. The low fluorescence intensity of transcribed products at expected size would reflect high methylated fraction of specific adenosine on RNAs (Figure 1A).