Fluorescence-Based Study of Oligonucleotide Interactions With Recombinant Proteins: Insulin, Interferon α2-β, Somatotropin, and Their Receptors
DOI:
https://doi.org/10.20535/ibb.2024.8.3.304238Keywords:
interferon, interferon receptor, insulin, insulin receptor, somatotropin, oligonucleotide, protein-ligand interaction, spectroscopyAbstract
Background. Oligonucleotides (OLNs) can participate in a wide range of protein-ligand interactions and perform numerous cellular functions by forming structures that enable specific interactions with DNA, RNA, and proteins, what is crucial for many biological processes. Advances in understanding these interactions could lead to the development of new technologies for treating various diseases. However, the mechanism of interaction between proteins and OLNs is complex and still requires detailed study. More research is needed to fully elucidate this process and enhance our understanding of these biomolecular interactions.
Objective. The aim of this study was to synthesize, purify, and investigate the interaction of OLNs with recombinant signaling proteins interferon α2-β and insulin with their receptors and somatropin by assessing binding strength using fluorescence spectroscopy.
Methods. The interactions were analyzed using the Stern–Volmer equation in both general and modified forms, as well as the Hill equation. OLNs were synthesized via the solid-phase phosphoramidite method, purified through solid-phase extraction, and subsequently verified with a spectrophotometer.
Results. Fluorometric titration revealed that OLNs bind to proteins within the medium affinity range, forming non-fluorescent complexes, with the most active interactions observed with shorter OLN. Positive cooperative binding of interferon to G20 and T20, and negative cooperative binding of insulin to C20 and A20, were identified. Additionally, negative cooperative binding of somatropin to C20 was observed.
Conclusions. The study demonstrated the interaction between OLNs and recombinant signaling proteins and receptors through various binding mechanisms, which could potentially affect their conformation and biological activity. These findings have implications for the therapeutic use of OLNs in the context of signaling proteins and receptors.
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