Oligonucleotides comprise a fast-growing group of biologic therapeutics. Since 1998, 21 drugs have been approved by the FDA and/or EMA, with over 2 new drugs being approved per year for the last 5 years. This molecular designation includes multiple modalities and mechanisms of action, such as aptamers, ASOs (anti-sense oligonucleotides), and siRNAs (short interfering RNAs). While the mechanisms used by each modality may be different, characterizing the formation and maintenance of base-pairing and secondary structure in these short oligonucleotide drug substances is essential to a complete understanding of their activity. Nuclear magnetic resonance (NMR) spectroscopy is a non-destructive and data-rich analytical method that can report on higher-order structure, as well as dynamics, of macromolecules in solution at atomic resolution. Thus, it has been applied to the study of oligonucleotides since the 1960s. Here, we show that simple 1D NMR spectra, focusing on the imino resonances, can be leveraged to quantify base-pairing in a series of model short DNA oligonucleotides. By using well-established homonuclear NOESY experiments, the imino resonances can be assigned to provide site-specific information on the solution structures adopted by the oligonucleotides. Additionally, the base-pairing data agrees well with thermal stability data derived from DSC (differential scanning calorimetry).
