A universal single-plasmid, single-promoter system for efficient protein display and RNA encapsulation in MS2...

Bacteriophage MS2 virus-like particles (VLPs) represent a promising platform for biomedical applications. However, challenges remain in integrating protein display, RNA encapsulation, and convenient purification methods. Here, we developed a universal single-plasmid single-promoter system (USS) that enables modular assembly of multifunctional VLPs through designated PstI (protein insertion) and KpnI (RNA encapsulation) sites. The USS system achieved: 1) Efficient display of large protein molecules (e.g., EGFP, Nanobody N16) on each VLP surface while encapsulating target RNA; 2) Rapid one-step purification via His-tag affinity chromatography, bypassing ultracentrifugation; 3) The resulting VLPs exhibit thermal stability (60°C for 15 min) and nuclease resistance. Functional validation demonstrated: 1) Diagnostics: CoviN-encapsulated VLPs served as RT-qPCR controls for COVID-19 detection (linear range: 10²-10⁸ copies/μL); 2) Dual functionality for co-delivery: N16-VLPs with shPCSK9 suppressed protein PCSK9 expression by 68 % (p < 0.01) in Hepa1-6 cells and bound PD-L1 with nanomolar affinity (EC50 = 1.6 μg/mL). Crucially, the simplicity of USS eliminated the need for multi-plasmid constructs, providing a streamlined approach to VLPs production that is both efficient and scalable for applications from molecular diagnostics to combination therapies.