Pseudouridine (Ψ), an isomer of uridine (U), is the most common post-transcriptional modification found in RNA (1). Detection and quantification of pseudouridine (Y) in RNA is challenging. The available chemical detection methods are not quantitative. Recently reported mass spectrometry-based methods in this direction include relative quantification of hydrolysis products (base vs nucleoside) in collision-induced dissociation, and pseudouridine-specific SRM (Selected Reaction Monitoring) transition based absolute quantification. These approaches are effective in dealing with modification at a single sequence location. Our goal is to develop an assay that can quantify multiple modifications in a given oligonucleotide. This is done by uridine specific cleavage through hydrazinolysis. Pseudouridine is immune to hydrazine addition compared to uridine. This assay can be used to understand the functional role of multiple contiguous modifications, such as those in the subunit interface and the peptidyltransferase center of 23S rRNA.

Quantification of pseudouridine modifications through uridine specific cleavage of RNA

251st National Meeting (2016)

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