Pseudouridine Detection and Quantification Using Bisulfite Incorporation Hindered Ligation

Pseudouridine (Ψ) is a widespread RNA modification found in various RNA species, including rRNA, tRNA, snRNA, mRNA, and long noncoding RNA (lncRNA). Understanding the function of Ψ in these RNA types requires a robust method for the detection and quantification of the Ψ level at single-nucleotide resolution. A previously used method utilizes Ψ labeling by N-cyclohexyl-N'-β-(4-methylmorpholinium)ethylcarbodiimide (CMC). The quantification of Ψ is based on the stop ratio after reverse transcription. However, the use of CMC followed by strong alkaline treatment causes severe RNA degradation, often requiring a large amount of RNA. The removal of CMC and recovery of RNA by ethanol precipitation are also time-consuming. Here, we introduce a Bisulfite Incorporation Hindered ligation-based method (BIHIND), which can detect and quantify Ψ sites on rRNA, mRNA, and noncoding RNA. BIHIND can be coupled with quantitative PCR (BIHIND-qPCR) for quantitative detection of Ψ fraction at individual modification sites, as well as with next-generation sequencing (BIHIND-seq) for high-throughput sequencing of Ψ without requiring reverse transcription. We validated the robustness of BIHIND with the elucidation of Ψ dynamics following pseudouridine synthase depletion.