Therapeutic Phosphorodiamidate Morpholino Oligonucleotides, an RNA mimic: Physical Properties, Solution Structures, and Folding Thermodynamics

Elucidating the structure-function relationships for therapeutic RNA mimicking phosphorodiamidate morpholino oligonucleotides (PMOs) is challenging due to the lack of information about their structures. While PMOs have been approved by US FDA for treatment of Duchenne muscular dystrophy, no structural information on these unique, charge neutral and stable molecules is available. We performed Circular Dichroism (CD) and solution viscosity measurements combined with MD simulations and Machine Learning to resolve solution structures of 22-mer, 25-mer, and 30-mer length PMOs. The PMO conformational dynamics are defined by the competition between nonpolar nucleobases and uncharged phosphorodiamidate groups for shielding from solvent exposure. PMO molecules form non-canonical, partially helical, stable folded structures with a small 1.4-1.7-nm radius of gyration, low count of 3-6 base pairs and 6-9 base stacks, characterized by -34-51-kcal/mol free energy, -57-103-kcal/mol enthalpy, and -23-53-kcal/mol entropy for folding. The 4.5-6.2-cm3/g intrinsic viscosity and Huggins constant of 4.5-9.9 are indicative of extended and aggregating systems. The results obtained highlight the importance of the conformational ensemble view of PMO solution structures, thermodynamic stability of their non-canonical structures, and concentration-dependent viscosity properties. These principles form a paradigm to understand the structure-properties-function relationship for therapeutic PMOs to advance the design of new RNA-mimic-based drugs.