Molecular properties and intramolecular interactions of peptide-conjugated phosphorodiamidate morpholino...

We combined circular dichroism (CD) and viscosity measurements with molecular dynamics (MD) simulations and classification and regression approaches to machine learning to characterize solution structures of 22-mer, 25-mer, and 30-mer peptide- (-GlyArg6) conjugated phosphorodiamidate morpholino oligonucleotides (PPMOs). PPMO molecules form non-canonical folded structures with 1.4- to 1.5-nm radius of gyration, 4-6 base pairs and 5-11 base stacks, characterized by -49 to -71 kcal/mol free energy of folding. The 4.5-6.1 cm³/g intrinsic viscosity and Huggins constant of 4.5-9.7 indicate PPMO-PPMO interactions at higher concentrations. The random-coil 3'-end conjugated -GlyArg₆ portion does not alter molecular properties of phosphorodiamidate morpholino oligonucleotide (PMO) components, which explains why CD spectra, viscosity-concentration profiles, and inhibitor activities of 22-mer, 25-mer, and 30-mer PPMOs and PMOs are similar but the peptide enhances the PPMO cellular uptake. PPMOs' viscosity is lower than PMOs' viscosity, due to PMO-peptide position-dependent interactions, especially in 25-mer PPMO, explaining differences in CD and high-concentration viscosity. These results reiterate the importance of the conformational ensemble view of non-canonical PPMO structures in solution, in agreement with our previous PMO study. The addition of -GlyArg₆ does not alter the structure and molecular properties of the PMO components of the PPMO structures but impacts the viscosity of the PPMO-based aqueous solution formulations.