Gormley, A. J. | bioRxiv Science

bioRxiv2025

Gene Delivery Mediated by Backbone-Degradable RAFT Copolymers

Cationic polymers present an attractive platform for gene delivery. However, these highly charged macromolecules can also lead to cytotoxicity. Therefore, there is a strong unmet need to develop efficacious polymeric gene delivery vehicles with high biocompatibility. Here, we leveraged recent advances in polymer chemistry to develop backbone-degradable cationic copolymers and evaluate their potential as gene delivery vehicles. Specifically, polycations were prepared via copolymerization with macrocyclic allylic sulfides which can participate in PET-RAFT polymerization via radical ring-opening cascade copolymerization to install degradable backbone segments. A polymer library with varying degradability was prepared and evaluated using a model GFP plasmid to transfect U-2 OS cells. Incorporation of degradable groups into the copolymer backbone improved transfection efficiency 10-fold at low amine/phosphate (N/P) ratios without increasing cytotoxicity, thereby enhancing their value as gene delivery carriers. We hypothesize that degradability may enhance the complexs disassembly kinetics in the cytosol, enabling more efficient payload release.

bioengineering↗

bioRxiv2024

Phosphorylation Toggles the SARS-CoV-2 Nucleocapsid Protein Between Two Membrane-Associated Condensate States

The Nucleocapsid protein (N) of SARS-CoV-2 plays a critical role in the viral lifecycle by regulating RNA replication and packaging the viral genome. N and RNA phase separate to form condensates that may be important for these functions. Both functions occur at membrane surfaces, but how N toggles between these two membrane-associated functional states is unclear. Here, we reveal that phosphorylation switches how N condensates interact with membranes, partly by modulating condensate material properties. Phosphorylated N forms liquid condensates that wet membranes, reminiscent of Ns role in RNA replication. In contrast, unmodified N forms viscoelastic condensates that can be engulfed by membranes, evoking viral genome packaging. These results suggest that phosphorylation serves as a regulatory switch within the viral replication cycle by modulating Ns association with membranes. We gained mechanistic insight through structural analysis and molecular simulations, which suggest phosphorylation induces a conformational change that softens condensate material properties. Our studies also show that phosphorylation alters Ns interaction with viral membrane proteins. Together, our findings uncover a novel aspect of SARS-CoV-2 biology by identifying membrane association as a key feature of N condensates and providing mechanistic insights into the regulatory role of phosphorylation. Understanding this mechanism suggests potential therapeutic targets for COVID infection.

biophysics↗