Spectroscopic and Theoretical Insights into the Binding of Phenothiazinium Dye Methylene Blue with Triple- and Double-Helical Forms of RNA: A Comparative Study

Higher-ordered nucleic acid structures like triplex and quadruplex forms have been the center of attention of the current research field of medicinal chemistry. The triplex is formed through the sequence-specific association of a single-stranded, triplex-forming oligonucleotide (TFO) with nucleic acid duplex structures. In our present study, we have focused on an RNA triplex (U.A*U) because of its various biological relevance including telomerase activity and movement of ribosomal template in mRNA coding regions. Methylene blue (MB), a cationic dye of phenothiazine families, has diverse pharmacological and clinical applications. Considering the wide range of medicinal uses of MB and the biological importance of higher-ordered structures of RNA, our present study has focused on the elucidation of the comparative binding interaction of methylene blue (MB) with RNA triplex (U.A*U) and duplex (A.U) structures employing various spectroscopic and theoretical tools. MB has been found to show a comparably higher binding affinity toward the triplex form (13.5 × 10⁵ M^-1) than the parent duplex form (2.36 × 10⁵ M^-1). The thermal melting profile revealed that MB substantially stabilized the Watson-Crick base-paired strands of both the RNA triplex and duplex structures; however, it destabilized the Hoogsteen base-paired strand of triple helical RNA. Fluorescence quenching, steady-state anisotropy, and circular dichroic studies all together supported the intercalative mode of binding. Thermodynamic data revealed that the interactions of MB with both RNA triplex and duplex forms were characterized by both negative enthalpy and entropy changes. Ionic strength-dependent study showed that the nonpolyelectrolytic forces are the predominating factors in the binding process, although a minor but certain extent of polyelectrolytic contribution is present. Moreover, theoretical calculations using DFT and TD-DFT methods were explored to validate the experimental spectrophotometric behavior (absorbance) of MB, assigning the energy levels involved in the electronic transition along with the vibrational modes.