High-Affinity Three-Way Junction Aptamers for Sensing Cationic Polymyxin Antibiotics.

Area of Science:

• Biochemistry
• Analytical Chemistry
• Molecular Biology

Background:

• Polymyxins B and E (colistin) are critical last-resort antibiotics against multidrug-resistant Gram-negative bacteria.
• Their narrow therapeutic index and nephrotoxicity demand rapid and accurate detection methods.

Purpose of the Study:

• To isolate DNA aptamers targeting polymyxins B and E.
• To characterize aptamer binding properties and develop a sensor for discriminating between these antibiotics.

Main Methods:

• Capture-SELEX method was used to isolate DNA aptamers against polymyxin B and E mixtures.
• Isothermal titration calorimetry and binding assays under varying ionic strengths were employed for characterization.
• Thioflavin T fluorescence spectroscopy confirmed target binding.
• A sensor array with principal component analysis was developed for discrimination.

Main Results:

• Two aptamer families with three-way junction structures were identified.
• PM-2 aptamers showed low-micromolar affinity for both polymyxins.
• PM-3 aptamers exhibited higher affinity and selectivity for polymyxin B (440 nM Kd) over polymyxin E.
• Electrostatic interactions were found to be crucial for molecular recognition.
• A sensor array successfully discriminated between polymyxin B and E.

Conclusions:

• Novel DNA aptamers capable of specific binding to polymyxins B and E were successfully isolated.
• These aptamers demonstrate potential for developing sensitive and selective detection methods.
• The developed sensor array provides a foundation for clinical monitoring of polymyxin antibiotics.