Heterobivalent fusion aptamers that target a single protein show significant promise for studying protein-protein interactions. However, a major challenge is finding two distinct aptamers that can simultaneously recognize the same protein. In this study, we used a novel technique called Aptamer-Assisted DNA SELEX (AADS) to isolate two distinct aptamers capable of recognizing different sites on the programmed death-ligand 1 (PD-L1) protein. Initially, Aptamer 1 (P1C2) was identified by using conventional DNA SELEX targeting the PD-L1 protein. Subsequently, Aptamer 2 (P1CSC) was obtained via AADS, which was designed to bind to the PD-L1/P1C2 complex. After confirming that both aptamers could simultaneously recognize the PD-L1 protein, we engineered fusion aptamers by optimizing their orientation and linker sequences, resulting in the creation of the optimized fusion aptamer, P1CSC-T7-P1C1. Our fusion aptamer targeting PD-L1 demonstrated remarkable specificity and affinity, effectively inhibiting PD-1/PD-L1 interactions at both the protein and cellular levels. These findings highlight the potential of fusion aptamers via AADS as powerful tools for targeting the PD-L1 protein and cancer cells (A549 cells). This represents a significant advancement in aptamer-based molecular recognition and has the potential to drive innovation as a versatile method for targeting a wide range of proteins.
