Functional Aptamers In Vitro Evolution for Protein–Protein Interaction Blockage

Abstract

As aptamer development progresses, their applications have expanded significantly beyond high affinity to include functional capabilities. Currently, the identification of functional aptamers relies on traditional SELEX techniques, followed by functional validation and computer-assisted redesign of high-affinity aptamers. However, high affinity does not guarantee optimal functionality, making the search for functional aptamers from binding pools time-consuming and labor-intensive. Addressing this challenge, we introduce functional aptamers in vitro evolution (FAIVE), a novel screening method that links sequence functionality to fluorescence intensity. We demonstrated the effectiveness of FAIVE by obtaining modified DNA aptamers capable of disrupting the interaction between the SARS-CoV-2 spike receptor-binding domain (RBD) and hACE2, targeting protein–protein interaction inhibition. Furthermore, we investigated the criteria for validating the quality of the bead library generated for selection by modeling the emulsion PCR process, providing theoretical insights for future applications. The concept of incorporating fluorescent signal reporting of aptamer functionality into the aptamer selection process holds the potential to facilitate the identification of aptamers with diverse functionalities and is readily adaptable to various research contexts.