As a newly identified mRNA modification, the regulation of ac4C remains largely unexplored. RNA-binding proteins (RBPs) that specifically binds to ac4C modification and mediate downstream cellular activities (readers) have not been reported yet. We synthesized acetylated and non-acetylated RNA probes by in vitro transcription. The sequences of the probes were segments of FUS and 18s rRNA, which contain ac4C sites as reported. A biotin-RNA pulldown assay and mass spectrometry were performed with HEK 293T cell lysates.