Therapeutic phosphorodiamidate morpholino oligonucleotides: 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 the US Food and Drug Administration for treatment of Duchenne muscular dystrophy, no structural information on these unique, charge-neutral, and stable molecules is available. We performed circular dichroism and solution viscosity measurements combined with molecular dynamics 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 non-polar 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- to 1.7-nm radius of gyration, low count of three to six base pairs and six to nine base stacks, characterized by -34 to -51 kcal/mol free energy, -57 to -103 kcal/mol enthalpy, and -23 to -53 kcal/mol entropy for folding. The 4.5- to 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.

A simplified in vivo approach for evaluating the bioabsorbable behavior of candidate stent materials.

Metal stents are commonly used to revascularize occluded arteries. A bioabsorbable metal stent that harmlessly erodes away over time may minimize the normal chronic risks associated with permanent implants. However, there is no simple, low-cost method of introducing candidate materials into the arterial environment. Here, we developed a novel experimental model where a biomaterial wire is implanted into a rat artery lumen (simulating bioabsorbable stent blood contact) or artery wall (simulating bioabsorbable stent matrix contact). We use this model to clarify the corrosion mechanism of iron (≥99.5 wt %), which is a candidate bioabsorbable stent material due to its biocompatibility and mechanical strength. We found that iron wire encapsulation within the arterial wall extracellular matrix resulted in substantial biocorrosion by 22 days, with a voluminous corrosion product retained within the vessel wall at 9 months. In contrast, the blood-contacting luminal implant experienced minimal biocorrosion at 9 months. The importance of arterial blood versus arterial wall contact for regulating biocorrosion was confirmed with magnesium wires. We found that magnesium was highly corroded when placed in the arterial wall but was not corroded when exposed to blood in the arterial lumen for 3 weeks. The results demonstrate the capability of the vascular implantation model to conduct rapid in vivo assessments of vascular biomaterial corrosion behavior and to predict long-term biocorrosion behavior from material analyses. The results also highlight the critical role of the arterial environment (blood vs. matrix contact) in directing the corrosion behavior of biodegradable metals.