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

Area of Science:

• Biochemistry
• Molecular Biology
• Chemical Biology

Background:

• DNA three-way junctions (3WJs) are Y-shaped DNA structures with significant roles in cancer biology and nanostructure construction.
• Existing methods lack specific, visible fluorescent probes for detecting DNA 3WJs.
• 3WJs are recognized as potential druggable targets and are utilized in aptasensors for diagnostics.

Purpose of the Study:

• To develop and characterize a novel visible fluorescent light-up probe for specific detection of DNA 3WJs.
• To demonstrate the probe's utility in host-guest detection strategies.

Main Methods:

• Synthesis of a merocyanine-based fluorescent probe (FPhOBtz) comprising N-methylbenzothiazolium (Btz) and 2-fluorophenolic (FPhO) moieties.
• Characterization of FPhOBtz binding affinity and specificity towards various DNA 3WJ aptamers (MN4, CABA, DOGS.1, DISS.1, BES.1, DCAS.1).
• Assessment of FPhOBtz displacement by guest molecules (alkaloids, steroids) for competitive binding assays.

Main Results:

• FPhOBtz acts as a universal fluorescent turn-on dye for DNA 3WJs.
• The probe exhibits up to 730-fold fluorescence enhancement upon binding to 3WJ aptamers with low micromolar affinity.
• Competitive binding assays demonstrated FPhOBtz displacement, enabling low micromolar detection of guest molecules in human blood serum.

Conclusions:

• FPhOBtz is the first visible light-up fluorescent probe specifically designed for DNA 3WJ detection.
• This probe offers a sensitive and specific tool for diagnostic applications and studying 3WJ biology.
• The FPhOBtz probe facilitates novel host-guest detection strategies using DNA 3WJs.