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

Area of Science:

• Epitranscriptomics
• Molecular Biology
• Biochemistry

Background:

• Pseudouridine (Ψ) is a key RNA modification impacting gene expression.
• Accurate, site-specific quantification of Ψ is essential for understanding its regulatory roles.
• Existing methods for Ψ detection can be complex or lack single-nucleotide resolution.

Purpose of the Study:

• To develop a novel assay, pseU-SCOPE, for sensitive and site-specific quantification of pseudouridine (Ψ).
• To enable visual and qPCR-free detection of RNA modifications.
• To facilitate the study of Ψ in disease contexts.

Main Methods:

• Ligation-assisted rolling circle amplification (RCA) assay.
• Conversion of bisulfite-derived Ψ signature into a padlock-probe ligation gate.
• Development of fluorescence and gel-based, naked-eye readouts.
• Validation on human rRNAs and mitochondrial tRNAs.

Main Results:

• pseU-SCOPE achieves single-nucleotide resolution for Ψ detection without qPCR.
• The assay demonstrates high sensitivity, requiring low RNA input.
• Validation confirmed accuracy across various RNA types.
• Elevated rRNA pseudouridylation was observed in acute myeloid leukemia samples.

Conclusions:

• pseU-SCOPE offers a simple, rapid, and visual platform for site-specific Ψ detection and quantification.
• The assay is adaptable for other RNA modifications like m6A.
• This method facilitates functional epitranscriptomic studies and translational research.