Engineering 3D Hydrogel Microenvironments for Advanced Aptamer Discovery

Aptamers are synthetic nucleic acid ligands capable of recognizing diverse biological targets with high affinity and specificity. Although these are generally identified through systematic evolution of ligands by exponential enrichment (SELEX), conventional selection is often performed under simplified twodimensional (2D) or solution-based conditions that do not adequately reproduce the native microenvironment of complex biological targets. Here, we engineered threedimensional (3D) hydrogel microenvironments as advanced platforms for aptamer discovery. By tailoring hydrogel composition, architecture, and target presentation, we developed 3D selection systems applicable to both cellular and extracellular targets, including tumor cells and cancer-derived exosomes. These engineered microenvironments provide spatially organized and biologically relevant conditions for molecular recognition during iterative aptamer selection, while allowing the selection process to be adapted toward different downstream functions. Our studies highlight 3D hydrogels not simply as passive scaffolds, but as tunable polymeric platforms that actively define the context of molecular selection. Engineering the selection microenvironment may therefore provide a versatile strategy for discovering aptamers with improved biological relevance and potential applications in targeted therapy and biosensing.