Do Aptamers Always Bind? The Need for a Multifaceted Analytical Approach When Demonstrating Binding Affinity between...

Area of Science:

• Biochemistry and Molecular Biology
• Analytical Chemistry
• Biotechnology

Background:

• Aptamers are increasingly used as biorecognition elements in various detection strategies.
• Lack of standardized quality control protocols leads to inconsistent and unreliable aptamer performance data in the literature.
• Existing methods for characterizing aptamer binding may not be sufficiently robust for small organic molecules.

Purpose of the Study:

• To critically evaluate the binding capabilities of selected aptamers against their target small molecules using multiple analytical techniques.
• To investigate the reliability of a common colorimetric assay involving gold nanoparticles (AuNPs) for aptamer characterization.
• To establish a comprehensive analytical framework for validating aptamer performance.

Main Methods:

• Comparative analysis of aptamer binding using isothermal titration calorimetry (ITC), native nano-electrospray ionization mass spectrometry (native nESI-MS), and proton nuclear magnetic resonance spectroscopy (¹H NMR).
• Evaluation of ampicillin aptamer binding in solution and in the presence of gold nanoparticles (AuNPs).
• Validation of the analytical approach using well-characterized aptamers for cocaine/quinine (MN4) and l-argininamide (1OLD).

Main Results:

• None of the tested ampicillin aptamers demonstrated specific binding to ampicillin, contrary to previous reports.
• The colorimetric assay using AuNPs yielded conflicting results and did not confirm specific ampicillin binding.
• ITC and ¹H NMR studies, including those with AuNPs, failed to provide evidence of specific ampicillin-aptamer interactions.

Conclusions:

• The study highlights significant discrepancies in reported aptamer binding affinities, particularly for small organic molecules.
• A single analytical method is insufficient; a multifaceted approach combining thermodynamic and structural data is essential for accurate aptamer validation.
• The findings underscore the need for rigorous quality control and validation of aptamers before their application in diagnostic and detection systems.