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Pseudouridine Detection and Quantification Using Bisulfite Incorporation Hindered Ligation.

Yutao ZhaoXinyuan MaChang YeWenlong LiKinga PajdzikQing DaiHui-Lung SunChuan He
Published in: ACS chemical biology (2024)
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 B isulfite I ncorporation Hind ered 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.
Keyphrases
  • long noncoding rna
  • real time pcr
  • gene expression
  • loop mediated isothermal amplification
  • high throughput sequencing
  • early onset
  • single cell
  • sensitive detection
  • smoking cessation
  • cell free