Advancing the Characterization of Modified Oligonucleotides through an Integrated Infrared Action Spectroscopy-Mass Spectrometry Approach

Therapeutic oligonucleotides (ONs) incorporate subtle chemical modifications to enhance performance, but these small structural differences and related impurities are difficult to identify and characterize with traditional analytical approaches. To address this challenge, we combined infrared (IR) action spectroscopy with mass spectrometry (MS) to characterize a series of modified ONs. To establish the method, we first examined short 6-mer ON model systems, revealing spectral differences arising from variations in nucleobase composition and intramolecular interactions. These effects were further resolved using hydrogen-deuterium exchange (HDX), which enabled distinction between the N-H and O-H stretching modes, allowing for complete vibrational assignment. Building on these insights, we next applied the IR-MS method to therapeutically relevant 16-mer antisense oligonucleotides (ASOs). The broad spectral features of these larger oligonucleotides, caused by conformational diversity and intramolecular interactions, were narrowed by fragmenting them prior to IR irradiation, yielding spectra of the w7 and w5 ions instead. This tandem-MS workflow, together with the 6-mer assignments, enabled characterization of the ASOs. The method further revealed characteristic behavior of locked nucleic acid (LNA) groups, where LNA-induced stabilization decreases fragmentation efficiency and results in lower yield values in the IR action spectra. Interestingly, the LNA-to-DNA ratio indicated that LNA groups cause redshifts, with the magnitude of redshift increasing at higher LNA content. Adenine-specific spectral features were observed upon addition of a second adenine group, demonstrating the specificity of this method. Overall, these methods advance structural characterization of ONs and overcome previous analytical limitations, highlighting their potential for identifying modified ONs.