Thermodynamic Modeling of mRNA with the Addition of Precipitants

Tenberg, V.; Walker, P. J.; Inguva, P. K.; Pons Royo, M. d. C.; Acevedo, A. L.; Lin, V. S.; Pang, M.; Wang, Z.-G.; Myerson, A. S.; Braatz, R. D.

doi:10.1101/2025.10.18.683252

bioRxiv · 10.1101/2025.10.18.683252

Thermodynamic Modeling of mRNA with the Addition of Precipitants

Tenberg, V.·Walker, P. J.·Inguva, P. K.·Pons Royo, M. d. C.·Acevedo, A. L.·Lin, V. S.·Pang, M.·Wang, Z.-G.·Myerson, A. S.·Braatz, R. D.

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Abstract

Nucleic acid therapeutics (NATs) have recently emerged as an exciting therapeutic modality for a range of indications, most notably as vaccines for SARS-CoV-2. In many cases, the thermodynamics of a system containing nucleic acids (such as in the downstream purification of mRNA from solution or within the lipid nanoparticle) can significantly influence the properties and efficacy of that system. Consequently, an accurate thermodynamic description of the system is essential for understanding and optimizing that system. In this work, the SAFT-{gamma} Mie equation of state was used to predictively model mRNA solubility. Experimental measurements of the solubility of two different mRNA sequences in various conditions (namely choice of precipitant(s), precipitant concentration, and temperature) were obtained and used to validate the model. Not only was the thermodynamic model able to quantitatively predict the solubility of mRNA in solution under different conditions, it was also able to yield mechanistic insight into the factor driving precipitation, namely the disruption of water-mRNA hydrogen bonding. The developed model can be extended to other mRNA sequences in a range of conditions beyond the experimental data presented in this work.

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Tenberg, V., Walker, P. J., Inguva, P. K., Pons Royo, M. d. C., Acevedo, A. L., Lin, V. S., Pang, M., Wang, Z.-G., Myerson, A. S., Braatz, R. D.. 2025-10-18. Thermodynamic Modeling of mRNA with the Addition of Precipitants. https://doi.org/10.1101/2025.10.18.683252

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