A novel screening method for competitive FRET-aptamers applied to E. coli assay development

Area of Science:

• Biotechnology
• Molecular Biology
• Analytical Chemistry

Background:

• DNA aptamers offer specific molecular recognition capabilities.
• Fluorescence Resonance Energy Transfer (FRET) is a powerful tool for detecting molecular interactions.
• Detecting bacterial pathogens like E. coli requires sensitive and high-throughput methods.

Purpose of the Study:

• To develop a novel high-throughput screening method for identifying DNA aptamers with competitive FRET assay potential.
• To identify and characterize a superior aptamer for the detection of E. coli.
• To determine the binding pocket of the selected aptamer and optimize detection limits.

Main Methods:

• High-throughput screening of DNA aptamers against E. coli outer membrane proteins (OMPs) using PCR with fluorophore-dUTP conjugates.
• Utilizing fluorescence resonance energy transfer (FRET) assays in microtiter plates to assess aptamer potential.
• Competitive displacement FRET assay format with labeled aptamers and bacteria, and verification by fluorescence microscopy.

Main Results:

• A superior competitive FRET-aptamer, EcO 4R, was identified.
• The binding pocket of EcO 4R was localized to specific secondary loop structures.
• Detection of as few as 30 live E. coli per ml was achieved using AF 647-labeled EcO 4R and BHQ-3-labeled bacteria.
• High aptamer affinity did not strongly correlate with competitive FRET potential.

Conclusions:

• The developed high-throughput screening method is effective for identifying aptamers with competitive FRET assay capabilities.
• The identified EcO 4R aptamer enables sensitive and specific detection of E. coli.
• Far-red fluorescence emission facilitates detection in autofluorescent matrices.
• Aptamer affinity is not the sole determinant of FRET assay performance.