Harnessing a Fluorescent Nucleobase Surrogate for Supramolecular FRET-Aptamer Detection and Target-Site Mapping

Area of Science:

• Biochemistry
• Molecular Biology
• Nanotechnology

Background:

• DNA aptamers offer high-affinity binding to small molecules, crucial for aptasensor development.
• Current aptasensor designs often lack structural insights into ligand binding or have limited sensitivity.
• Fluorescence Resonance Energy Transfer (FRET) offers potential for both detection and structural information, but dual labeling can reduce aptamer affinity.

Purpose of the Study:

• To develop a novel FRET-based strategy for sensitive tetracycline detection with target-site mapping.
• To overcome limitations of existing aptasensor methods by combining structural insight with high sensitivity.
• To establish a new binding model for the tetracycline-aptamer complex.

Main Methods:

• Incorporation of a fluorescent molecular rotor (Th6HI) nucleobase surrogate into the tetracycline aptamer (OTC2).
• Utilizing tetracycline as a donor and Th6HI as an acceptor in a FRET system.
• Employing time-resolved fluorescence anisotropy and indirect FRET excitation for analysis.

Main Results:

• Direct excitation of Th6HI showed minimal response to tetracycline binding due to probe rigidity.
• Indirect excitation via the tetracycline donor yielded site-dependent sensitized fluorescence (Fsen) of the Th6HI acceptor.
• The FRET response enabled sensitive tetracycline detection and target-site mapping, surpassing native fluorescence methods.

Conclusions:

• The developed nucleobase surrogate-ligand FRET strategy successfully combines sensitive target detection with target-site mapping.
• This approach provides structural insights into the tetracycline-aptamer complex, suggesting a three-helical binding model.
• The method offers a significant advancement for aptasensor design, addressing key limitations in sensitivity and structural information.