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Oral Sessions
Day 3, June 24（Tue.） 14:40-14:55
Room A (Maesato West)
- 3A-O2-1440
Sequence Characterization for RNA Therapeutics: Leveraging Multiple Ribonucleases and DIA Mass Spectrometry
(1MKI, 2Waters)
oYuki Matsubara1, Akari Ito1, Catalin Doneanu2, Yasuto Yokoi1
The rapid development of RNA therapeutics demands robust analytical techniques for sequence characterization. Liquid chromatography-mass spectrometry (LC-MS) is a powerful tool but struggles with achieving full sequence coverage, especially for long RNA molecules. Here, we present a data-independent acquisition (DIA)-based LC-MS/MS approach, utilizing multiple ribonucleases and advanced bioinformatics, to improve sequence characterization of single-guide RNA (sgRNA) and messenger RNA (mRNA). LC-MS/MS was conducted using a high-resolution QTof system with optimized DPA/HFIP-based separation. Data acquired in MSe mode were analyzed using AQXeNA for DIA deconvolution and sequence mapping. Preliminary results demonstrate that combining multiple ribonucleases with AQXeNA-powered DIA analysis enhances sequence coverage compared to single-enzyme digests. This strategy provides a robust and scalable workflow for improved RNA sequence characterization, supporting the advancement of RNA therapeutics.