Abstract

RNA N4-acetylcytidine (ac4C) and 5-formylcytidine (f5C) regulate translation and RNA fate, yet practical locus-specific assays are limited. We report CATAL, a chemical-assisted ligation-qPCR method for single-nucleotide- resolution analysis of ac4C and f5C. Selective conversion generates a predictable C-to-T outcome at a selected site, which is converted into a ligation-efficiency difference by template-directed probe ligation and quantified by qPCR. CATAL is sequencing-free, uses standard oligonucleotides and widely available qPCR instruments, and bypasses reverse transcription for the primary readout, enabling direct ligation-based detection with low RNA input and tolerance to complex RNA backgrounds. Synthetic RNA mixtures produced calibration curves relating signal to the input ac4C/f5C fraction. We validated CATAL at reported ac4C and f5C sites in rRNA and tRNA from multiple human cell lines. Housekeeping-transcript normalization reduced variability and enabled analysis of NAT10 and ALKBH1 perturbations. Overall, CATAL provides a sequencing-free and ligation-based targeted workflow for site-resolved detection and quantification of RNA modifications that supports mechanistic studies of RNA modification regulation and focused validation in biological samples.