Process for Standardizing and Assessing the Parameters Governing MS2 Virus-Like Particle Reassembly around Nucleic Acid Cargo

Abstract MS2 virus-like particles (VLPs) are widely used as protein nanocages for cargo encapsulation, yet in vitro disassembly–reassembly protocols remain poorly standardized, and reassembly yields are reported inconsistently. As a result, the same experiments reported in literature produce widely divergent yields, limiting reproducibility and cross-study comparability. Here, we introduce a cargo-specific, quantitative framework for standardized MS2 VLP reassembly yield determination. We evaluate commonly used disassembly and post-disassembly processing methods and identify practical trade-offs between protein recovery, accessibility, and reproducibility. Reassembly yield is quantified using size exclusion chromatography calibrated against purified VLP standards, enabling robust, cargo-specific yield measurement. Using this framework, we apply a full factorial design of experiments to quantify the individual and combined effects of coat protein concentration, ionic strength, buffer pH, and molecular crowding on reassembly yield. The resulting statistical model explains more than 99% of the explainable variance and its linear fit to the experimental data indicates that optimal reassembly conditions extend beyond those tested to date. Protein concentration and ionic strength dominate reassembly yield, whereas pH and osmolyte concentration contribute more modestly within the tested ranges. Finally, we propose practical guidelines for standardized MS2 VLP disassembly, reassembly, and yield reporting, defining a transferable operating envelope for MS2 VLP reconstruction. While demonstrated here using a single nucleic acid cargo (tr-DNA), the framework is readily extensible to alternative cargos and coat protein variants. Competing Interest Statement The authors have declared no competing interest. Footnotes Supplementary material description: Supplemental Information 1 summarizes literature on MS2 VLP disassembly/reassembly conditions. Supplemental Information 2 compares linearity across protein quantification methods. Supplemental Information 3 provides supporting calculations for statistical design of experiments using reassembly yield as the response. Changes in the Title, Abstract, Introduction, and Conclusion. One final paragraph added in the discussion. Methods and results were not changed. Abbreviations - ANOVA - Analysis of Variance - BCA - Bicinchoninic Acid Assay - CP - Coat Protein - DOE - Design of Experiments - HPLC - High Performance Liquid Chromatography - SDS-PAGE - Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis - SEC - Size Exclusion Chromatography - TEM - Transmission Electron Microscopy - TMAO - Trimethylamine N-Oxide - UV-Vis - Ultraviolet-Visible light spectrum - VLP - Virus-Like Particle