Characterization of antisense oligonucleotide and guide ribonucleic acid diastereomers by hydrophilic interaction liquid chromatography coupled to mass spectrometry

Oligonucleotides have become an essential modality for a variety of therapeutic approaches, including cell and gene therapies. Rapid progress in the field has attracted significant research in designing novel oligonucleotide chemistries and structures. Beyond their polar nature, the length of large RNAs and presence of numerous di-astereomers for phosphorothioate (PS)-modified RNAs pose heightened challenges for their characterization. In this study, the stereochemistry of a fully-modified antisense oligonucleotide (ASO) and partially-modified guide RNAs (gRNAs) was investigated using HILIC and orthogonal techniques. The profiles of three lots of a fully-modified ASO with PS linkages were compared using ion-pairing RPLC (IPRP) and HILIC. Interestingly, three isomer peaks were partially resolved by HILIC for two lots while only one peak was observed on the IPRP profile. Model oligonucleotides having the same sequence of the five nucleotides incorporated to the 3 & PRIME;-end of the gRNA but differing in their number and position of PS linkages were investigated by HILIC, IPRP, ion mobility spec-trometry-mass spectrometry (IM-MS) and nuclear magnetic resonance (NMR). An strategy was ultimately designed to aid in the characterization of gRNA stereochemistry. Ribonuclease (RNase) T1 digestion enabled the characterization of gRNA diastereomers by reducing their number from 32 at the gRNA intact level to 4 or 8 at the fragment level. To our knowledge, this is the first time that HILIC has successfully been utilized for the profiling of diastereomers for various oligonucleotide formats and chemical modifications.

Automated summary

Oligonucleotides have become an important therapeutic modality, but their characterization is challenging due to their unique chemical structure. This study investigates the stereochemistry of fully-modified and partially-modified oligonucleotides using HILIC and other techniques, and proposes a new strategy for characterizing oligonucleotide stereochemistry.