FRET enabled real time detection of RNA-small molecule binding

Area of Science:

• Biochemistry
• Molecular Biology
• Drug Discovery

Background:

• The bacterial rRNA A-site is a crucial target for antibiotics.
• Developing robust methods to analyze antibiotic binding is essential for drug discovery.

Purpose of the Study:

• To develop a novel platform for analyzing and discovering antibiotics targeting the bacterial rRNA A-site.
• To utilize Förster Resonance Energy Transfer (FRET) for sensitive detection of antibiotic-RNA interactions.

Main Methods:

• Incorporation of an emissive uracil surrogate into the RNA A-site.
• Labeling aminoglycoside antibiotics with a fluorescent acceptor (7-diethylaminocoumarin-3-carboxylic acid).
• Utilizing a FRET pair with an optimal Forster radius (R(0)) of 27 Å for monitoring binding events.

Main Results:

• Demonstrated successful FRET signal generation upon aminoglycoside binding to the emissive A-site construct.
• Quantified binding affinity (EC(50) values) through donor emission quenching and acceptor emission augmentation.
• Successfully detected competitive displacement of aminoglycosides by other A-site binders.

Conclusions:

• The developed FRET-based platform provides a robust method for antibiotic discovery and analysis.
• This approach allows for accurate determination of direct binding and competitive displacement events.
• The system's sensitivity relies on the interaction between two chromophores, not environmental changes of a single fluorophore.