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.
Wang et al. (Thu,) studied this question.
Synapse has enriched 5 closely related papers on similar clinical questions. Consider them for comparative context: