A widely used CRP aptamer does not bind CRP in solution, suggesting potential streptavidin-driven artifacts in aptasensors

Aptamer sequences are often incorporated into diagnostics without independent confirmation of their binding activity, although modern standards require rigorous, quantitative validation across multiple orthogonal methods. Here, we re-examined a DNA sequence reported in 2010 to bind C-reactive protein (CRP) with a Kd of 3.5 nM determined by surface plasmon resonance (SPR). Since its publication, this sequence has been repeatedly and “successfully” incorporated into diverse sensing formats by many research groups, yet its affinity to CRP has never been independently verified. We assessed the reported sequence for CRP binding using nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) and fluorescence anisotropy, each implemented in both direct and competitive modes, with the competitive assays employing a validated CRP-binding SOMAmer as a tracer. In every configuration, we observed no detectable binding of the reported aptamer to native CRP. These results are consistent with the aptamer having no meaningful affinity for CRP, with a Kd exceeding the micromolar range. Replication of the original 2010 SPR assay, in which the biotinylated DNA sequence was immobilized on a streptavidin-coated sensor surface, revealed that CRP binds with low-nanomolar apparent Kd to the streptavidin surface itself, while immobilization of the DNA sequence does not measurably alter the response. These findings indicate that the originally reported 3.5 nM Kd most likely reflects CRP binding to streptavidin rather than to the DNA sequence. This case illustrates how a non-binding sequence can acquire the status of a canonical reagent through surface artifacts and citation cascades. It reinforces the need to treat solution-based validation as the primary standard for establishing aptamer-target interactions in analytical or diagnostic applications. Our findings also raise a broader concern for aptasensors intended for use with blood-derived samples: because streptavidin is widely used to immobilize biotinylated DNA sequences and streptavidin-coated surfaces are prone to nonspecific adsorption of abundant serum proteins, many streptavidin-based aptasensors may be vulnerable to surface-driven artifacts when applied to complex clinical matrices.