Computational Analysis Uncovering Contrasts in G-Quadruplex Propensity and Aptamer Enrichment in SELEX-Derived Libraries

G-quadruplexes (G4s) are noncanonical nucleic acid structures with biological and therapeutic significance, and they are found in many aptamer sequences. Using c-kit 1 G4 DNA as a target, systematic evolution of ligands by exponential enrichment (SELEX) has been carried out using an RNA library, resulting in G4-rich aptamers. Herein, this article investigates the relationship between predicted G4-forming potential and aptamer enrichment by analyzing high-throughput SELEX libraries using three G4 prediction tools: G4NN, G4Hunter, and QGRS Mapper. While the tools demonstrate strong internal consistency and overlap in identifying G4-prone sequences, their predictions show limited concordance with experimental abundance and enrichment trends across SELEX rounds. Only a small fraction of sequences display both high G4 scores and consistent enrichment. Experimental validation using electrophoretic mobility shift assays confirmed that strong predicted G4-forming sequences often lack strong binding activity, whereas highly enriched aptamers with strong binding activities may show weaker G4 signatures computationally. These findings suggest that current G4-prediction tools alone are insufficient for aptamer candidate selection and highlight the need for integrative evaluation strategies that combine structural prediction with empirical performance data. © 2025 The Author(s). ChemBioChem published by Wiley-VCH GmbH.