Construction of Agonistic Bivalent Double-Stranded Aptamers Targeting c-MET

Aptamers, single-stranded oligonucleotides selected via SELEX technology, exhibit high-affinity and selective binding to target molecules by folding into specific intramolecular tertiary structures. Importantly, aptamers can inhibit target biological functions when their binding disrupts the interaction between the target and its natural ligand. However, aptamers capable of activating target molecule functions remain rare. In this study, we performed molecular engineering on the c-MET aptamer HF3-58, previously identified as biologically inert. Through rational design, we successfully developed a series of bivalent double-stranded aptamers (BVDSApts) with enhanced c-MET binding, while their single-stranded counterparts failed to bind c-MET. By optimizing the central duplex length to 18, 20 and 22 base pairs (bp), these aptamers potently induced c-MET dimerization, phosphorylation, and downstream protein phosphorylation, while significantly enhancing cell migration and dispersion. The process of reconstructing biologically inert aptamers to obtain those with agonistic activity demonstrates that, with a thorough understanding of the binding mechanisms, it is possible to design new aptamers with novel functions through sequence engineering. Additionally, the BVDSApts obtained provide precursor molecules for the further development of HGF substitutes.