De novo synthesis of infectious bacteriophage MS2 labelled with dyes: Investigation of the electrical properties of its interior

We report the de novo synthesis of infectious MS2 bacteriophage in the presence of acid-base molecular probes designed to enable their incorporation during capsid assembly and to probe the internal electrostatic environment of the virus. Transmission electron microscopy, dynamic light scattering, ζ-potential measurements, UV-Vis spectroscopy, and the apparent pK values analysis were combined with molecular dynamics simulations. Neutral Red incorporated into MS2 exhibited a significant increase in pK value compared to pure water, the so-called medium effect (ΔpK =pK -pK ≈ 3), corresponding to an estimated electrostatic potential of approximately -216 ± 8 mV. This behaviour is consistent with the localisation of the probe in a strongly anionic microenvironment associated with the RNA-containing interior rather than the capsid surface. Comparative analysis with anionic micellar systems, liposomes, DNA solutions, and protein-based media showed that similar spectral shifts occur only in highly anionic environments. Molecular dynamics simulations support these findings and indicate preferential localisation of the probe inside the capsid in proximity to RNA. These results demonstrate that the MS2 interior is characterized by a highly negative electrostatic potential governed by genomic RNA and illustrate the applicability of molecular probes for studying nanoscale electrostatic environments in viral systems.