Extending Small-Molecule Aptamer Discovery Principles to Protein Targets: Selection and Characterization of Green...

Since the advent of systematic evolution of ligands by exponential enrichment (SELEX), numerous protein-binding aptamers have been reported, yet their binding characterizations are often less developed than those of small-molecule aptamers, limiting mechanistic understanding and practical applications. Here, we establish green fluorescent protein (GFP) variants as a tractable model system for studying protein-binding aptamers using strategies commonly applied to small-molecule aptamers. Independent SELEX experiments against mGreenLantern GFP (mGL-GFP), superfolder GFP (sfGFP), and GFPuv yielded closely related aptamers with distinct specificity profiles. Sequence analysis, truncation studies, circular dichroism spectroscopy, and 2D nuclear magnetic resonance spectroscopy results are consistent with differences in epitopes among the GFP variants. Divalent metal ions strongly influence selection outcomes: aptamers isolated with Ca²⁺ require Ca²⁺ for binding, whereas Ca^{2 +}-free selection produced a distinct family of Mg²⁺-dependent aptamers with potential intracellular utility. Together, these results establish GFP variants as a reliable platform for dissecting protein-aptamer recognition and provide a roadmap for achieving small-molecule-level mechanistic insight into protein-binding aptamers.