Assays to Estimate the Binding Affinity of Aptamers

Aptamers have become coming-of-age molecular recognition elements in both diagnostic and therapeutic applications. Generated by SELEX, the ‘quality control’ of aptamers, which involves the validation of their binding affinity against their respective targets is pivotal to ascertain their potency prior to use in any downstream assays or applications. Several aptamers have been isolated thus far, however, the usage of inappropriate validation assays renders some of these aptamers dubitable in terms of their binding capabilities. Driven by this need, we provide an up-to-date critical review of the various strategies used to determine the aptamer-target binding affinity with the aim of providing researchers a better comprehension of the different analytical approaches in respect to the molecular properties of aptamers and their intended targets. The techniques reported have been classified as label-based techniques such as fluorescence intensity, fluorescence anisotropy, filter-binding assays, gel shift assays, ELISA; and label-free techniques such as UV–Vis spectroscopy, circular dichroism, isothermal titration calorimetry, native electrospray ionization-mass spectrometry, quartz crystal microbalance, surface plasmon resonance, NECEEM, backscattering interferometry, capillary electrophoresis, HPLC, and nanoparticle aggregation assays. Hybrid strategies combining the characteristics of both categories such as microscale thermophoresis have been also additionally emphasized. The fundamental principles, complexity, benefits, and challenges under each technique are elaborated in detail.