Analysis of Oligonucleotide Stability and Fragmentation: Impact of 2'-Modifications, Base Composition, and Charge-States.

RATIONALE: Mass spectrometry-based sequencing of therapeutic oligonucleotides requires controlled fragmentation, but the effects of nucleotide composition, charge state, and chemical modification on collision-induced dissociation are not fully understood. This study systematically evaluates how these parameters influence precursor survival, base loss, and backbone fragmentation to improve and understand analytical characterization and fragmentation behavior of modified oligonucleotides. METHODS: Homopolymeric 15-mer DNA and RNA oligonucleotides containing different nucleobases, 2'-ribose modifications, and phosphorothioate linkages were analyzed by negative electrospray ionization tandem mass spectrometry on a quadrupole time-of-flight instrument. Collision-induced dissociation (CID) was performed over a defined voltage range. Precursor, base-loss, and c-ion intensities were extracted and normalized using an in-house Python workflow to generate fragmentation profiles and breakdown curves. RESULTS: Higher precursor charge states fragmented at lower CID voltages and exhibited narrower optimal fragmentation windows. Distinct differences in precursor depletion and base-loss formation were observed between the investigated 2'-modifications, with chemically modified oligonucleotides generally requiring higher CID voltages than unmodified analogues. In contrast, different nucleobases showed smaller but reproducible differences in fragmentation behavior. The onset and maximum abundance of sequencing-relevant c-ions depend strongly on precursor charge state and CID voltage. CONCLUSIONS: The fragmentation behavior of oligonucleotides is primarily affected by charge state and 2'-ribose modification, whereas nucleobase composition has a smaller influence. The identified CID-voltage-dependent trends provide practical guidance for selecting fragmentation conditions in tandem mass spectrometric characterization and sequencing of modified oligonucleotides.