Quantitative Analysis of Phosphorothioate Isomers in CRISPR sgRNA at Single-Residue Resolution Using Endonuclease Digestion Coupled with Liquid Chromatography Cyclic Ion Mobility Mass Spectrometry (LC/cIMS).

Phosphorothioate (PS) modifications in single-guided RNA (sgRNA) are crucial for genome editing applications using the CRISPR/Cas9 system. These modifications may enhance sgRNA stability, pharmacokinetics, and binding to targets, thereby facilitating the desired genetic alterations. Incorporating multiple PS groups at varying positions may introduce chiral centers into the sgRNA backbone, resulting in a complex mixture of constitutional- and stereoisomers that challenges current analytical capabilities for reliable identification and quantification. In this study, we developed an innovative methodology that combines endonuclease digestion of sgRNA with ion pairing reversed-phase liquid chromatography coupled with cyclic ion mobility mass spectrometry (IPRP-LC/cIMS) to fully distinguish PS-induced isomers in a complex mixture. The relative abundance of each isomer was quantified using a two-step method, wherein the ion abundance was sequentially extracted from an LC/MS ion chromatogram and LC/cIMS two-dimensional ion mobiligram. This quantification method was thoroughly evaluated, demonstrating excellent sensitivity, precision, dynamic range, repeatability, and accuracy. In addition, this method enables the investigation of the kinetics of forming PS to phosphodiester (PO) impurities in sgRNA under oxidative stress conditions, offering unprecedented insights into PS stability at a single-residue resolution. In this context, this method highlights the in-depth characterization of PS, demonstrating its capability to support biomedical research, development, and production of sgRNA products.