Fluorescence Quenching and Polarization Analysis of DNA Aptamers Binding to Green Fluorescent Protein: A Model System for Protein-Binding Aptamers and Assay Development

Abstract Protein-binding aptamers are widely used in biosensing and therapeutics, yet reliable solution-phase methods for validating aptamer binding remain limited. Here, we establish green fluorescent protein (GFP) as a self-reporting benchmark system for homogeneous analysis of aptamer-protein interactions. A previously identified GFP-binding DNA aptamer, GFP-1, was modified at the 3′ terminus with a fluorescence quencher, generating a dual-readout probe that simultaneously reports binding through fluorescence quenching and fluorescence polarization. The quencher modification preserved nanomolar affinity for GFP while producing a substantially more sensitive change than polarization alone. Using this probe, we developed a homogeneous competitive displacement assay that revealed overlapping binding sites among three distinct GFP aptamers. We further implemented a reverse-chase kinetic format which enabled the direct determination of apparent dissociation rate constants (koff) from solution-phase fluorescence traces. The kinetic ranking placed GFP-1 with the slowest dissociation and GFP-3 with the quickest. Both affinity and kinetic parameters were preserved in 10% human serum, demonstrating assay robustness in complex biological media. This fluorescence-based platform operates entirely in solution and provides a practical framework for validating aptamer-target interactions, measuring dissociation kinetics, and benchmarking aptamer assays under physiologically relevant conditions.