Increasing Oligonucleotide Sequencing Information and Throughput with Ion Mobility Spectrometry-Mass Spectrometry.

Synthetic oligonucleotides, such as antisense oligonucleotides or small interfering RNA, are small chain nucleic acid polymers that can be used therapeutically to control gene expression. Watson-Crick base pair interactions provide the primary mode of interaction between synthetic oligonucleotides and their target molecule, and this binding requires accurate, robust, and rapid sequence verification. The development of high-quality synthetic oligonucleotides and comprehensive analytical workflows for their evaluation is therefore essential. Herein, a platform coupling liquid chromatography, ion mobility spectrometry, collision-induced dissociation, and mass spectrometry (LC-IMS-CID-MS) was applied to facilitate oligonucleotide sequence confirmation. Using IMS, multiple charge states of the same oligonucleotide were mobility separated and analyzed simultaneously. Furthermore, all-ion fragmentation was implemented to provide sequence coverage for each charge state using fewer injections than current targeted multiple injection LC-MS methods. The results demonstrated herein denote sequence coverage is generally inversely proportional to oligonucleotide length (i.e., lower fidelity coverage for longer strands), with observed sequence coverages ranging between 40% and 80% for molecules comprised of 20-40 residues. To ease the burden of spectral interpretation and sequence determination for the LC-IMS-CID-MS data, an analysis workflow using the Pacific Northwest National Laboratories (PNNL) preprocessor and Agilent's BioConfirm software was developed.