# A bisulfite-assisted and ligation-based qPCR amplification technology for locus-specific pseudouridine detection at base resolution

## TL;DR

This paper introduces a new method called pseU-TRACE for detecting pseudouridine in RNA at base resolution using bisulfite and qPCR.

## Contribution

The novel pseU-TRACE method enables base-resolution detection of pseudouridine in RNA with high sensitivity and specificity.

## Key findings

- pseU-TRACE successfully detects known pseudouridine sites in 28S, 18S, 5.8S, and mRNA.
- The method allows quantification of pseudouridine fractions and analysis of pseudouridine synthase activity.
- pseU-TRACE is a reliable, time-efficient, and sensitive technique for pseudouridine detection.

## Abstract

Over 150 types of chemical modifications have been identified in RNA to date, with pseudouridine (Ψ) being one of the most prevalent modifications in RNA. Ψ plays vital roles in various biological processes, and precise, base-resolution detection methods are fundamental for deep analysis of its distribution and function. In this study, we introduced a novel base-resolution Ψ detection method named pseU-TRACE. pseU-TRACE relied on the fact that RNA containing Ψ underwent a base deletion after treatment of bisulfite (BS) during reverse transcription, which enabled efficient ligation of two probes complementary to the cDNA sequence on either side of the Ψ site and successful amplification in subsequent real-time quantitative PCR (qPCR), thereby achieving selective and accurate Ψ detection. Our method accurately and sensitively detected several known Ψ sites in 28S, 18S, 5.8S, and even mRNA. Moreover, pseU-TRACE could be employed to measure the Ψ fraction in RNA and explore the Ψ metabolism of different pseudouridine synthases (PUSs), providing valuable insights into the function of Ψ. Overall, pseU-TRACE represents a reliable, time-efficient and sensitive Ψ detection method.

Graphical Abstract