Spectral-fluorescent study of substituted trimethine cyanine dyes in solutions and in complexes with DNA. Effects of aggregation, moderate heating, and decreasing pH

Abstract

Trimethine cyanine dyes are widely used as probes for the detection, study and quantification of biomolecules. In particular, cationic trimethine cyanines noncovalently interact with DNA with growing fluorescence. However, their use is often limited by the tendency to self-association – to the formation of aggregates. Disubstituted trimethine cyanines with hydrophobic substituents are especially prone to aggregation. In this work, we studied the interaction of a number of substituted trimethine cyanines with DNA (in aqueous buffer solutions) and showed that their aggregation strongly interfered with their use as fluorescent probes for DNA. To eliminate this drawback, preliminary heating of dye solutions with DNA to 60–70 °C was used, followed by cooling to room temperature. Compared to the experiments without heating, an increase in the dye fluorescence intensity was observed due to the partial thermal decomposition of the aggregates and the interaction of the resulting monomers with DNA. To decompose aggregates, another method was also used – protonation of the dyes with amino substituents in buffer solutions with pH 5.0, which also led to growing the dye fluorescence intensity in the presence of DNA. Complexes of the dyes with DNA were modeled using molecular docking. Effective binding constants of the dyes to DNA and detection limits when using the dyes as probes for DNA (LOD and LOQ) were determined. It is shown that dye 3 with heating in neutral buffer and dye 1 in acidic buffer may be recommended as sensitive probes for DNA. It is concluded that the method of preliminary heating may be applied to dyes prone to aggregation, for improving their properties as biomolecular probes. Another possible means to reduce the interfering effects of dye aggregates is to use easily protonated dyes (with amino substituents) in slightly acidic media.