Quantitation of Diastereomer Content in PS-Modified Synthetic Oligonucleotides using cIMS-MS

Small interfering RNAs (siRNAs) are promising therapeutics due to their sequence-specific gene silencing and ability to target disease-causing genes. However, susceptibility to nuclease degradation limits their efficiency, prompting modifications to be made to increase stability. A common modification used is phosphorothioate (PS) linkages. These modifications introduce chiral centers, and differences in chiral centers may influence efficacy. While cyclic ion mobility spectrometry-mass spectrometry (cIMS-MS) has proven effective for separating PS diastereomers, its utility for quantitative analysis is still unknown. Herein, we used CID-cIMS-MS to fragment a 22-mer siRNA (UBE) and studied five nucleotide-long fragment ions from both termini (b₅^2- and y₅^2-) containing PS linkages. To assess whether fragment diastereomer relative abundances reflect those of the intact oligonucleotide, 5-mer analogs (shortmers) matching the intact termini were synthesized and analyzed via nESI-cIMS-MS, ion pairing reversed phase liquid chromatography with UV detection (IPRP-LC-UV), and IPRP-LC with IMS detection (IPRP-LC-IMS-MS). Comparison of the fragment ion IMS distributions with their shortmer counterparts showed very similar characteristics, indicating these fragments could be directly compared to the shortmers. By aligning LC retention time with cIMS-MS mobility distributions, the relative abundances of the diastereomers from the fragment ions to those of the shortmers were compared. Our results demonstrate that CID-IMS-MS enables relative quantitation of the PS diastereomers, establishing a framework for PS modified oligonucleotide characterization.