Tuning the DNA binding properties of phenolic hemicyanine dyes for host-guest fluorescent aptasensor applications

Fluorescent dyes that bind specifically to DNA aptamers with turn-on emission serve as powerful tools for biosensor development. DNA three-way junctions (3WJs) consisting of a Y-shaped hydrophobic branch point connecting three double-stranded stems serve as recognition elements for DNA aptasensors for a wide variety of diagnostic applications. Herein, we highlight the tunability of phenolic hemicyanine dyes for host-guest fluorescent aptasensor applications using the cocaine-binding aptamer (MN4) as a representative 3WJ-aptamer substrate. The DNA aptamer MN4 exhibits ligand binding promiscuity and binds the alkaloid quinine with similar to 26-fold greater affinity than its intended cocaine target. Combining the cationic N-methylbenzothiazolium (Btz) acceptor with a 2-fluoro-phenolic (FPhO) donor through a vinyl-linkage creates the FPhOBtz hemicyanine as a universal fluorescent turn-on probe for DNA 3WJs. The FPhOBtz dye binds MN4 with higher affinity than cocaine, but with weaker affinity than quinine. Consequently, FPhOBtz displacement from the 3WJ with turn-off fluorescence is effectively mediated by quinine, but not cocaine, resulting in poor detection of cocaine by the dye-displacement assay. Our current results demonstrate superior cocaine detection by replacing the planar cationic Btz group with the non-planar indolenium (Ind) acceptor to afford FPhOInd as the signaling fluorescent probe. This simple change in dye structure strongly diminishes MN4 binding affinity (19-fold) with cocaine now possessing stronger affinity for the 3WJ of MN4 compared to FPhOInd. Consequently, FPhOInd displacement from MN4 is effectively mediated by cocaine to provide a 13-fold improvement in cocaine detection in undiluted urine using the host-guest biosensing platform.

Automated summary

Fluorescent dyes that bind to DNA aptamers with turn-on emission are useful for biosensor development. In this study, the tunability of phenolic hemicyanine dyes for aptasensor applications was highlighted using the cocaine-binding aptamer (MN4) as a recognition element. The FPhOBtz hemicyanine was found to be a universal fluorescent turn-on probe for DNA three-way junctions (3WJs), but its detection of cocaine was poor due to binding affinity differences with quinine. By replacing the Btz group with the Ind acceptor, the FPhOInd dye showed improved cocaine detection in undiluted urine using the host-guest biosensing platform.