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PUM-seq: Chemical Labeling Method For Single-Nucleotide Mapping of Pseudouridine Across Species

Interests: Sequencing
Lead Inventor: Chuan He

SUMMARY

Improved single-nucleotide-resolution profiling of pseudouridine across diverse RNA types is offered by a chemical-labeling and mutation-based sequencing method that detects and quantifies this modification under mild conditions

The Unmet Need: RNA pseudouridine mapping remains limited by inefficient and biased detection methods

  • Pseudouridine (Ψ) is a widespread RNA modification whose distribution and biological functions remain incompletely understood. Existing detection approaches can be inefficient or biased, require harsh reaction conditions, or create analytical difficulties, particularly when Ψ sites are closely spaced. 

The Proposed Solution: Chemical labeling and reverse-transcription mutation profiling enable sensitive, single-nucleotide pseudouridine mapping

  • The faculty inventor developed PUM-seq (PseudoUridine-derived Mutation-based sequencing)- a sequencing method that selectively labels Ψ and support cyclization. Following adapter ligation and chemical treatment, reverse transcription produces mutation patterns used to infer modification sites and levels. Compared with existing approaches, the method operates under milder conditions and reduces background, including in regions with closely spaced Ψ sites. The optimized protocol treats samples at 37°C for three hours, achieving near-complete conversion with background noise as low as 0.5%; a faster 70°C, 30-minute protocol achieves similar conversion with slightly more background. Testing on ribosomal RNA and synthetic oligonucleotides supports efficiency and specificity, and analyses in human HEK293T RNA detected Ψ across small-RNA classes and mRNA.

 

ADVANTAGES

  • Single-nucleotide-resolution modification mapping

  • Quantitative site-level Ψ measurement

  • Low background under mild conditions

  • Detection across diverse RNA classes

  • Improved analysis of closely spaced sites

  • Rapid alternative treatment protocol

APPLICATIONS

  • Transcriptome-wide pseudouridine profiling in mRNA

  • Pseudouridine mapping in tRNA and other small RNAs

  • Comparative RNA modification analysis across mammalian species