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

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 cm/g intrinsic viscosity and Huggins constant of 4.5-9.7 indicate PPMO-PPMO interactions at higher concentrations. The random-coil 3'-end conjugated -GlyArg 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 -GlyArg 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.