2025 ICIEST

The increasing prevalence of pathogenic Escherichia coli (E. coli) in water and food sources poses a significant threat to public health, necessitating the development of rapid and accurate biosensor detection methods such as aptamerbased biosensors due to their high specificity and sensitivity. Aptamers are nucleic acids that can bind with high affinity and specificity to a range of target molecules. In this study, 1,000 shuffled variants of a known aptamer (PDB ID: 2AU4) were generated and evaluated for stability using RNAfold and RNALfoldz based on minimum free energy (MFE). The five most stable sequences were selected and analyzed for their secondary and tertiary structures using RNAComposer. The target protein, Shiga toxin (Stx, PDB ID: 1C48), was modeled with AlphaFold 3 and validated through Ramachandran plot analysis. Molecular docking using the HDOCK server revealed aptamer-protein binding interactions, offering insights into the structural features that influence binding specificity and stability. In conclusion, this research bridges theory for future applications, thereby establishing a theoretical framework to support the future development of aptamer-based biosensors targeting E. coli O157:H7.