Spectroscopic and calorimetric study of the interaction between Nile blue and double-stranded RNA

Nile blue has been widely used in histological staining, fluorescence labeling, and DNA probing, with its intercalation behavior into the DNA helix being well documented. Here, we present a comprehensive investigation to address a current knowledge gap regarding the binding properties of Nile blue to two types of double-stranded RNA (dsRNA): poly(A·U) and poly(I·C), using various biophysical techniques. Absorption and fluorescence spectroscopic studies suggest a significant binding interaction between Nile blue and the two designated dsRNAs, specifically indicating an intercalation binding mode with poly(A·U) and demonstrating a noticeably higher binding affinity compared to poly(I·C). The binding stoichiometry was further determined by Job's plot to be 0.47 for poly(A·U) and 1.0 for poly(I·C). The increased relative viscosity and changes in the circular dichroism (CD) ellipticity of dsRNA after interacting with Nile blue indicate the stacking of Nile blue dyes between the RNA duplexes. These changes suggest a conformational alteration of the dsRNAs and confirm the intercalation mode of binding. The thermal dynamic analysis demonstrates that both binding were favored by negative enthalpy and primarily driven by the hydrophobic effect.