Quantitative and Visual Detection of Pseudouridine at Specific Sites Using Bisulfite-Assisted Rolling Circle Amplification

Pseudouridine (Ψ) is a prevalent RNA modification whose site-resolved quantification is critical for understanding epitranscriptomic regulation. We present pseU-SCOPE, a ligation-assisted rolling circle amplification (RCA) assay that converts a bisulfite-derived Ψ signature into a padlock-probe ligation gate, enabling single-nucleotide resolution without qPCR. The workflow supports fluorescence and gel-based, naked-eye readouts following RCA and operates with low RNA input. We validate pseU-SCOPE on established Ψ sites across human rRNAs and mitochondrial tRNAs, generate calibration curves for absolute quantification, and estimate site stoichiometry. Applied to peripheral blood samples, the assay reveals elevated rRNA pseudouridylation in acute myeloid leukemia relative to healthy donors, suggesting disease relevance. Coupled with the deamination mediated by GLORI (glyoxal and nitrite-mediated deamination of unmethylated adenosines), this strategy is adaptable to other RNA modifications such as <i>N</i>⁶-methyladenosine (m⁶A). Together, pseU-SCOPE provides a simple, rapid, qPCR-free, and visual platform for site-specific detection and quantification of RNA modifications, facilitating functional studies and translational research.