Do Aptamers Always Bind? The Need for a Multifaceted Analytical Approach When Demonstrating Binding Affinity between Aptamer and Low Molecular Weight Compounds

Abstract

Since their inception, DNA aptamers were regarded as the turning point for biochemical sensing in real samples; however up to now their promises are far from being fulfilled. Especially aptamers for small molecules pose a challenge for both selection and characterization. The lack of a universally accepted and robust quality control protocol for the characterization of aptamer performances coupled with the observation of inconsistent data sets in literature, prompted us to address the issue comparing different analytical methodologies to validate (or disprove) the binding capabilities of aptamer sequences. We chose three aptamers for ampicillin, a β-Lactam antibiotic; used several detection strategies described in literature. The colorimetric gold nanoparticles (AuNPs) assay used in the original paper describing the aptamer sequences was repeated with conflicting results. The three sequences were then tested with three different instrumental techniques to assess their Kd and binding mechanism in homogeneous solutions. Coupling the thermodynamic data obtained with Isothermal Titration Calorimetry (ITC) with the structural information on the binding event given by Native Electro Spray Ionization Mass spectrometry (Native ESI-MS) and 1H-NMR it was possible to verify that the three sequences do not show any specific binding with the target ampicillin. To verify the influence of the AuNPs on the binding event, the experiments were repeated in presence of AuNPs both with ITC and 1H-NMR, again without any results. By offering a cross-referenced and robust analitycal approach to aptamer characterization we aim at elucidating the potentialities of aptamer for small organic molecules, especially when ultrasensitive analytical application are involved

Label‐Free Colorimetric Biosensors Based on Aptamers and Gold Nanoparticles: A Critical Review

“…However, truncation cannot be done by simply removing the fixed primer binding regions without confirming binding. We recently found that the truncated chloramphenicol aptamer cannot bind chloramphenicol, [119] and Bottari et al confirmed that the ampicillin aptamer cannot bind the intended target either [120] . Based on sequence alignment, mutation of one or a few critical nucleotides should fully abolish binding without changing the overall secondary structure of aptamers.…”

Comparison of Two DNA Aptamers for Dopamine Using Homogeneous Binding Assays

“…Since Apt2 is shorter and has reliable binding, and structure‐switching property, it is recommended for future use. Recently, a number of aptamers were reported to be unable to bind their targets after careful binding assays, such as those for As(III), [38] ampicillin, [39] and chloramphenicol [40] . The Apt1 for dopamine was also studied on electrode surface for binding [20] with negative results.…”

Aptamer-based assays: strategies in the use of aptamers conjugated to magnetic micro- and nanobeads as recognition elements in food control

“…As recently argued by Wu et al [ 42 ], there is an urgent need to go beyond the “proof of concept,” exploring the real potentialities and limitations of aptamers as bioreceptors when facing analytical problems. Although thousands of papers have been published on aptamers, a rigorous multifaceted characterization of aptamer–target structures and the binding mechanism is still lacking, which would be critical for assessing the reliability of aptamers as bioreceptors [ 42 , 43 ]. The scientific community is urgently called to fill the gap between aptamer selection and biosensing strategies, since unfortunately the risk to misinterpret the real performance of an aptamer sequence is high.…”

“…In many cases, aptamer binding assays are not straightforward and can be prone to artifacts [ 19 ]. In this regard, it should be noted that two very recent studies disproved that some oligonucleotide sequences, widely used in literature for the development of aptasensors, do not actually bind to their expected target [ 43 , 44 ]. A multi-technique platform used to characterize the aptamer–target interaction allowed demonstrating that arsenic-binding aptamer, previously used in more than two dozen papers, did not provide a specific binding of As(III), and the same binding trends were observed with a random DNA [ 44 ].…”