Advancing the characterization of modified oligonucleotides through an integrated infrared action spectroscopy-mass...

Therapeutic oligonucleotides (ONs) incorporate subtle chemical modifications to enhance performance, but these structural differences and related impurities are difficult to characterize with traditional analytical approaches. To address this challenge, we combined infrared (IR) action spectroscopy with mass spectrometry (MS) to characterize two series of modified ONs. We examined 6-mer ONs, 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 N-H and O-H stretching modes, allowing for complete vibrational assignment. Building on these insights, we applied the IR-MS method to therapeutically relevant 16-mer antisense oligonucleotides (ASOs), representing the first IR action spectroscopy characterization of large ON therapeutics. The broad spectral features of these larger ONs, caused by conformational diversity and intramolecular interactions, were overcome through a novel tandem-MS fragmentation strategy, in which the precursor ions are first fragmented and the IR action spectroscopy spectra of the w7 and w5 ions are recorded instead. Combined with the 6-mer assignments, this workflow enabled detailed characterization of the ASOs. The method revealed characteristic behavior of locked nucleic acid (LNA) groups, where LNA-induced stabilization decreases fragmentation efficiency and thus IR fragmentation yields. 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 demonstrates its potential for identifying modified ONs as an orthogonal technique.