The effect of oligonucleotides, mononucleotides and their complexes with mannitol on the conformational stability of Interferon α2b

The study aimed to comprehensively investigate the effect of various forms of oligonucleotides (ORNs) (acidic, salt, and complexes with mannitol) and mononucleotides on the conformational stability and aggregation properties of interferon α2b (IFN α2b), a key protein in the antiviral response.

Circular dichroism (CD) was used to analyse the secondary structure of the protein, and fluorescence spectroscopy with thioflavin T was used to assess its tendency to aggregate. CD spectroscopy data clearly demonstrated that protein concentration significantly affects the results of the analysis. At a low concentration of 0.6 mM, the α-helix content was higher (up to 18.4%) than in 1.6 mM IFN α2b samples, where it did not exceed 4.2%. Importantly, during six months of observation with 6 freeze-thaw cycles, no statistically significant changes in the secondary structure of the protein were detected for any of the concentrations, indicating no degradation or significant conformational change of IFN α2b over time.

To assess thermal stability, thermal denaturation experiments were performed. It was found that the addition of the studied ligands (ORN and their complexes with mannitol) leads to an increase in the melting temperature (Tₘ) of the protein from 62 °C to 63.8–64 °C, indicating a slight stabilising effect. This confirms the binding of ligands to the protein and their impact on its conformational state. In addition, fluorescence studies with thioflavin T did not reveal any signs of pathological amyloid aggregate formation under the influence of any of the studied compounds.

Thus, the results of the study indicate that oligonucleotides and their complexes do not cause destabilisation or harmful aggregation of interferon α2b, but rather demonstrate a moderate stabilising effect. The data obtained confirm the potential safety and compatibility of such compounds with protein therapeutics, which may be important for the development of new combined immunomodulatory and antiviral drugs.