Many published aptamer sequences selected and characterized by surface-based methods fail to show quantifiable binding in solution, indicating that their true equilibrium dissociation constants (Kd) are far higher than those originally reported. This discrepancy raises fundamental concerns about the reliability of quantitative binding studies that underpin the aptamer field. Surface-based assays enable high-throughput screening but are prone to non-specific binding that can be mistaken for true molecular recognition. Therefore, reliable Kd determination should be anchored in solution-based techniques that avoid such artifacts. A growing consensus also holds that Kd values should be verified by at least two orthogonal methods, since these constants are not measured directly but are inferred from quantitative observables in the absence of reference standards. Here, we compare the principal solution-based approaches for aptamer interactions with small-molecule and protein targets, focusing on quantitative accuracy, universality, material requirements, and feasibility. This analysis proposes a best-practice roadmap for method selection, aiming to reduce experimental workload while supporting defensible Kd determination through orthogonal confirmation. We hope this roadmap encourages community discussion and convergence toward shared practices for solution-based Kd determination.
Le et al. (2026) studied this question.