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Updated: Jul 14, 2025
Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions
Published on: September 28, 2017
Characterization of synthetic guide ribonucleic acids through hydrophilic interaction chromatography coupled with
Bingchuan Wei1, Jenny Wang1, Lulu Dai1
1Synthetic Molecule Analytical Chemistry, Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, United States of America.
Abstract:
In this study, we aimed to develop a hydrophilic interaction liquid chromatography (HILIC) method for the analysis of single guide ribonucleic acid (sgRNA), a critical reagent used in CRISPR genome editing. Our results showed that effective profiling of sgRNA can be achieved by suppressing the surface charge of the stationary phase in HILIC. We identified hydrogen bonding as the primary retention mechanism with potential weak partitioning in HILIC separation of large oligonucleotides like 100-mer sgRNA. Moreover, we demonstrated that direct coupling of HILIC with mass spectrometry (MS) allows the intact mass analysis of sgRNA and its impurities with minimal adduct present. Finally, we characterized the post peak shown in the low temperature HILIC and identified it as sgRNA aggregates. Our findings provide valuable insight into the characterization of sgRNA and highlight the potential of HILIC-MS as a powerful analytical tool for relatively large oligonucleotide analysis.
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