biostudies-literatureUnknownFang XFundamental Research Funds for the Central UniversitiesNational Natural Science Foundation of ChinaNational Science and Technology Major ProjectPseudouridine (Ψ) is one of the most abundant RNA modifications, playing crucial roles in various biological processes. Identifying Ψ sites is vital for understanding their functions. In this study, we proposed a novel method for identifying Ψ sites with an improved signal-to-noise ratio. This method, called RNA exonuclease-assisted identification of pseudouridine sites (RIPS), combines specific CMC-labeling of Ψ sites with an exonuclease-assisted digestion strategy for the detection of Ψ sites. Utilizing exonuclease XRN1 to digest RNA strands not labeled by CMC, RIPS significantly reduces the background signal from unlabeled strands and enhances the positive signal of Ψ sites labeled by CMC, which terminates exonuclease digestion. As a result, we can enrich Ψ sites and identify them at siChemical sciencehttps://www.ebi.ac.uk/biostudies/studies/S-EPMC11515940biostudies-literatureExonuclease-assisted enrichment and base resolution analysis of pseudouridine in single-stranded RNA.PMC1151594022377096217210052042023kfyq052042023kf01189225320292153303221770872023YFC3402200Fang XYang WSun MZhao RLu ZWeng XMo JWang YZhou XfalseExonuclease-assisted enrichment and base resolution analysis of pseudouridine in single-stranded RNA.Pseudouridine (Ψ) is one of the most abundant RNA modifications, playing crucial roles in various biological processes. Identifying Ψ sites is vital for understanding their functions. In this study, we proposed a novel method for identifying Ψ sites with an improved signal-to-noise ratio. This method, called RNA exonuclease-assisted identification of pseudouridine sites (RIPS), combines specific CMC-labeling of Ψ sites with an exonuclease-assisted digestion strategy for the detection of Ψ sites. Utilizing exonuclease XRN1 to digest RNA strands not labeled by CMC, RIPS significantly reduces the background signal from unlabeled strands and enhances the positive signal of Ψ sites labeled by CMC, which terminates exonuclease digestion. As a result, we can enrich Ψ sites and identify them at si2024-01-01T00:00:00Z2024 Oct2026-07-15T14:51:56.684Z2025-04-06T14:23:18.105ZS-EPMC115159403947915910.1039/d4sc03576c