Systematic functional screening of switchable aptamer beacon probes.

Immunoassays using affinity binders such as antibodies and aptamers are crucial for molecular biology. However, the advancement of analytical methods based on these affinity probes is often hampered by complex operational steps that can introduce errors, particularly in intricate environments such as intracellular settings and microfluidic systems. There is growing interest in developing molecular probes for wash-free assays that activate signals upon target detection. Here we report a systematic functional screening platform for switchable aptamer beacon probes that can achieve target-responsive detection. A stem-loop, hairpin-shaped beacon library was constructed on microbeads and screened using target-responsive fluorescence-activated sorting. The selected aptamer beacons exhibit strong affinities, triggering fluorescence only upon binding, thus enabling wash-free immunoassays for the detection of intracellular and membrane proteins. Computational modelling offers insights into aptamer binding and structural switching mechanisms, revealing how specific protein-aptamer interactions drive stem-loop unwinding and postbinding conformational changes critical for functional activation. This approach establishes a standardized platform for generating switchable aptameric tools, supporting their potential in advanced diagnostics and research.