Visible Fluorescent Light-up Probe for DNA Three-Way Junctions Provides Host–Guest Biosensing Applications

Abstract

DNA three-way junctions (3WJs) consist of a Y-shaped hydrophobic branch point connecting three double-stranded stems and are viewed as druggable targets for cancer treatment. They are also important building blocks for the construction of DNA nanostructures and serve as recognition elements for DNA aptasensors for a wide variety of diagnostic applications. However, visible fluorescent light-up probes for specific staining of DNA 3WJs are currently lacking. Herein, we report that a merocyanine containing the N-methylbenzothiazolium (Btz) acceptor vinyl linked to a 2-fluorophenolic (FPhO) donor (FPhOBtz) serves as a universal fluorescent turn-on dye for DNA 3WJs. Our evidence is based on a multifaceted approach to define the specificity and affinity of FPhOBtz for 3WJ DNA aptamers; the cocaine binding aptamer MN4, the cholic acid binding aptamer (CABA), and four steroid aptamers (DOGS.1, DISS.1, BES.1, DCAS.1). FPhOBtz exhibits impressive turn-on (up to 730-fold) fluorescence at 580 nm upon aptamer binding with low micromolar affinity. Direct FPhOBtz displacement from the 3WJ binding domain through competitive alkaloid and steroid binding provides immediate fluorescent read out for host–guest detection strategies in human blood serum in the low micromolar regime. Our results present the first visible light-up fluorescent probe for DNA 3WJ detection strategies.

A modular aldol approach for internal fluorescent molecular rotor chalcone surrogates for DNA biosensing applications

“…48 Our interest in MN4 and other 3WJ DNA aptamers, stems from our recent finding that phenolic merocyanine dyes bind selectively to 3WJs to provide an impressive turn-on fluorescence response. 50 The free dyes can be utilized to detect alkaloid binding through host-guest platforms, in which alkaloid binding displaces the merocyanine dye from the 3WJ to elicit a turn-off fluorescence response. 50,51…”

“…48 Our interest in MN4 and other 3WJ DNA aptamers, stems from our recent nding that phenolic merocyanine dyes bind selectively to 3WJs to provide an impressive turn-on uorescence response. 50 The free dyes can be utilized to detect alkaloid binding through host-guest platforms, in which alkaloid binding displaces the merocyanine dye from the 3WJ to elicit a turn-off uorescence response. 50,51 To employ an FMR chalcone for quinine detection using MN4, the aptamer was split into two fragments by removing three loop residues from stem 2 to afford an 11mer and 22mer sequence.…”

“…50 The free dyes can be utilized to detect alkaloid binding through host-guest platforms, in which alkaloid binding displaces the merocyanine dye from the 3WJ to elicit a turn-off uorescence response. 50,51 To employ an FMR chalcone for quinine detection using MN4, the aptamer was split into two fragments by removing three loop residues from stem 2 to afford an 11mer and 22mer sequence. The An6HI probe was inserted into the G9 position (X) of the 11mer, with anking A and G bases with the probe opposite C (Fig.…”

On-Strand Aldehyde Capture by an Indanone Building Block: A Modular Aldol Approach for Fluorescent Molecular Rotor Nucleobase Surrogates

“…For comparison, free dye assays utilizing hemicyanines and MN4 to observe quinine binding afford a turn-off emission response through dye displacement by the quinine target. 47 Turn-off fluorescent platforms are not advantageous for trace analyte detection due to their inability to provide a detectable signal change in contrast to the high background emission. 48…”

DNA binding by the antimalarial compound artemisinin

“…This aptamer also tightly binds a range of other quinine-based ligands including amodiaquine, chloroquine and mefloquine 9 . Additionally, this aptamer binds levamisole weakly 10 and also binds a fluorescent light-up compound (FPhOBtz) that specifically binds DNA three-way junctions 11 . The MN4 construct of this aptamer (Fig.…”