Molecular Properties and Intramolecular interactions of Peptide-Conjugated Phosphorodiamidate Morpholino Oligonucleotides

We combined Circular Dichroism (CD) and viscosity measurements with 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 1.4-1.5-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 1. 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.