Monomethine cyanine dyes as highly sensitive fluorescent probes for RNA: binding, analytical characterization, and...

Monomethine cyanine dyes (MCDs) are widely used as fluorescent probes for DNA. However, their potential for RNA detection has been studied only sparsely, and quantitative relationships between structure and analytical characteristics remain unclear. This work presents the results of a comprehensive comparative study of the properties of six monomethine cyanine dyes (MCDs), including unsymmetrical derivatives of thiazole orange and oxazole yellow with variable terminal groups, linkers, and side chains, within a single analytical protocol. Steady-state spectrophotometry and fluorescence titration, complemented by DFT and TD-DFT calculations, were used to study the solvatofluorochromism and aggregation of MCDs in aqueous solutions. Interaction with RNA was shown to be accompanied by a multiple (up to 1250-fold) enhancement of fluorescence (the "light-on" effect) due to the suppression of nonradiative deactivation and a shift in the aggregate-monomer equilibrium. Effective binding constants were determined (using the Benesi-Hildebrand, Hill, and Scatchard models), the interaction cooperativity was assessed, and key analytical parameters-the limit of detection (LOD), limit of quantification (LOQ), and linear range-were evaluated. Selectivity of MCDs for RNA with respect to DNA and human serum albumin (HSA) was analyzed. Molecular docking of MCDs with RNA was employed to explain the observed differences in the affinity and sensitivity of the dyes, and the method of spectral moments was used to quantify changes in the dye spectra. The intercalative interaction of the dyes with RNA was confirmed by fluorescent intercalator displacement (FID) method using ethidium bromide as a classical intercalator. Correlation between the structure of substituents, binding energy, and analytical characteristics was demonstrated, paving the way for the rational design of effective RNA sensors.