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Do Aptamers Always Bind? The Need for a Multifaceted Analytical Approach When Demonstrating Binding Affinity between Aptamer and Low Molecular Weight Compounds
journal contribution
posted on 2020-11-09, 21:29 authored by Fabio Bottari, Elise Daems, Anne-Mare de Vries, Pieter Van Wielendaele, Stanislav Trashin, Ronny Blust, Frank Sobott, Annemieke Madder, José C. Martins, Karolien De WaelIn
this manuscript, we compare different analytical methodologies
to validate or disprove the binding capabilities of aptamer sequences.
This was prompted by the lack of a universally accepted and robust
quality control protocol for the characterization of aptamer performances
coupled with the observation of independent yet inconsistent data
sets in the literature. As an example, we chose three aptamers with
a reported affinity in the nanomolar range for ampicillin, a β-lactam
antibiotic, used as biorecognition elements in several detection strategies
described in the literature. Application of a well-known colorimetric
assay based on aggregation of gold nanoparticles (AuNPs) yielded conflicting
results with respect to the original report. Therefore, ampicillin
binding was evaluated in solution using isothermal titration calorimetry
(ITC), native nano-electrospray ionization mass spectrometry (native
nESI-MS), and 1H-nuclear magnetic resonance spectroscopy
(1H NMR). By coupling the thermodynamic data obtained with
ITC with the structural information on the binding event given by
native nESI-MS and 1H NMR we could verify that none of
the ampicillin aptamers show any specific binding with their intended
target. The effect of AuNPs on the binding event was studied by both
ITC and 1H NMR, again without providing positive evidence
of ampicillin binding. To validate the performance of our analytical
approach, we investigated two well-characterized aptamers for cocaine/quinine
(MN4), chosen for its nanomolar range affinity, and l-argininamide
(1OLD) to show the versatility of our approach. The results clearly
indicate the need for a multifaceted analytical approach, to unequivocally
establish the actual detection potential and performance of aptamers
aimed at small organic molecules.