Abstract
Elucidating the structure-function relationships of phosphorodiamidate morpholino oligonucleotides (PMOs) is challenging due to limited structural data. We combined surface tension and circular dichroism (CD) spectroscopy with molecular dynamics simulations to investigate how two different PMO molecules interact with Polysorbate 80 and Polysorbate 20. In simulations of 1:1 stoichiometry complexes, we observed strong, staged, concentration-dependent PMO-surfactant interactions, with interaction energies of -60 to -80 kcal/mol for 25-mer conformers and -50 to -70 kcal/mol for 30-mer conformers. Surfactants primarily associate through surface binding but can also insert into PMO structures, preventing unfolding. Structural analysis revealed that position-dependent contacts, especially with hydrophobic surfactant tails, drive these interactions. Importantly, PMO-surfactant binding does not disrupt base pairing, base stacking, or overall chirality, consistent with CD spectra, though it slightly enlarges the tertiary structure. Surfactants reduce solvent exposure of PMO surfaces within complexes, decreasing intermolecular interactions, yet the overall PMO-surfactant complex remains more solvent-exposed. Occasionally, surfactants act in a "chaperone-like" manner, enabling refolding into more compact structures. Together, these findings highlight how surfactants stabilize PMO conformers without disrupting their essential structure. This improved understanding of PMO-surfactant interactions broadens insight into PMO physicochemical behavior and supports the rational design of RNA-mimic therapeutics.