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Analysis of DNA thermal stability across a broad range of thionine concentrations

Interest in studying the interaction of small molecules with DNA is caused by the need to develop new, highly effective, and low-toxic drugs for cancer treatment. The strong and highly specific binding of thionine with DNA makes it a promising candidate for use in medicine and pharmacology. In this study, DNA-thionine complexes in aqueous solutions were investigated using UV-Vis absorption spectroscopy. The thermal stability of native DNA was studied in a broad range of thionine concentrations. The mechanisms of thionine binding to DNA, depending on the concentration of thionine, have been established. At low thionine concentrations $([c_{th}] \le 1.5 \text{ mg/L})$, thionine molecules intercalate between the base pairs of the DNA double helix. At a thionine concentration of 1.5-10 mg/L, the groove binding and external electrostatic interaction of positively charged thionine with negatively charged biopolymer phosphate groups of the DNA backbones is preferable. In all cases, the interaction of thionine with DNA leads to an increase in the thermal stability of the polynucleotide. These findings provide valuable insight into the concentration-dependent molecular mechanisms of DNA-small molecule interactions, supporting the rational design of anticancer and antimicrobial agents, as well as exploiting molecular probes for nucleic acid detection, imaging, and other biomedical applications.