Comprehensive Analytical Comparison of Strategies Used for Small Molecule Aptamer Evaluation

Abstract

Nucleic acid aptamers are versatile molecular recognition agents that bind to their targets with high selectivity and affinity. The past few years have seen a dramatic increase in aptamer development and interest for diagnostic and therapeutic applications. As the applications for aptamers expand, the need for a more standardized, stringent, and informative characterization and validation methodology increases. Here we performed a comprehensive analysis of a panel of conventional affinity binding assays using a suite of aptamers for the small molecule target ochratoxin A (OTA). Our results highlight inconsistency between conventional affinity assays and the need for multiple characterization strategies. To mitigate some of the challenges revealed in our head-to-head comparison of aptamer binding assays, we further developed and evaluated a set of novel strategies that facilitate efficient screening and characterization of aptamers in solution. Finally, we provide a workflow that permits rapid and robust screening, characterization, and functional verification of aptamers thus improving their development and integration into novel applications.

Comparison of In-Solution Biorecognition Properties of Aptamers against Ochratoxin A

“…From our comparison, we can clearly conclude that each of these aptamers binds with high affinity (nanomolar range) in-solution (Figure 2). Furthermore, these data compare favorably with previous reports [25,26,27,29] and confirm that all of these aptamers are good candidates for in-solution OTA biorecognition assays. Comparing these aptamer families in more detail, A08min (the A08 minimal aptamer sequence) recognizes OTA with the highest affinity and therefore may be the best aptamer choice for improving the sensitivity of OTA detection assays.…”

“…Given the positive results obtained by incorporating a fluorescein modification on the aptamers used for microscale thermophoresis assay, we repeated both assays using H8 and H12 aptamers with a 5′ fluorescein amidite modifier; however, there was no change in absorbance (AuNP assay) or fluorescence (SG assay). We confirmed this finding using two previously reported solution-based assays, i.e., ultrafiltration and equilibrium dialysis [25,29]. As a result, we proposed that this family of aptamers is not amenable to solution-based detection assays where the aptamer interacts with a competitive dye or nanoparticle surface.…”

“…Previous work highlighted the importance of measuring aptamer affinity using multiple binding assays prior to integration within an application platform [29]. One important conclusion of that work was that for utilizing aptamers in common solution-based biosensing applications, the measured interaction between the aptamer and the target free in-solution is required for assessing successful aptamer performance.…”

“…However, there is precedence that a given aptamer may have limitations or opportunities that make it better suited for certain molecular recognition schemes or applications. Indeed, a recent report suggests that there may be important differences beyond binding affinity (measured as the K D ) between these three families [29]. Thus, the development of aptamer-based technology for OTA determination requires a thorough understanding of the opportunities and limitations for each aptamer candidate.…”

Reagentless, Structure-Switching, Electrochemical Aptamer-Based Sensors

“…In addition to environmental conditions, the performance of a given aptamer sequence is dramatically altered based on the signal transduction methodology employed. A study recently published by McKeague et al highlights inconsistencies in quantification of aptamer binding parameters as a result of different assays used to perform the characterization (74). …”