Abstract

As a key component of the FGF19–FGFR4 signaling axis, fibroblast growth factor receptor 4 (FGFR4) plays a crucial role in the development and progression of various cancers, including hepatocellular carcinoma, breast cancer, and pancreatic cancer, serving as a critical driver of tumor proliferation, invasion, and drug resistance. This study, utilizing the SELEX technique with the FGFR4 extracellular domain as the target, successfully obtained multiple aptamers exhibiting high affinity and specificity for FGFR4 through positive and negative selection. By combining high-throughput sequencing and core sequence structure analysis, the aptamers were further structurally optimized, yielding truncated sequences with even higher binding affinity. Results from flow cytometry, surface plasmon resonance (SPR), and pull-down assays demonstrated that the optimized aptamer, termed FGFR4zxh-11b, achieves highly specific target binding at both the protein and cellular levels. Stability in serum-containing environments was significantly enhanced following 3′-inverted dT modification and terminal methoxy capping. Functional studies further confirmed that FGFR4zxh-11bm effectively and competitively blocks FGF19–FGFR4 interaction, significantly inhibiting downstream FRS2 phosphorylation and FGF19-induced migration of HUH-7 cells. This study presents the first systematic construction and validation of a class of high-affinity aptamers targeting FGFR4, providing a versatile molecular recognition tool for investigating the FGF19–FGFR4 signaling pathway and offering new opportunities for the targeted intervention of FGFR4-dependent malignancies, such as hepatocellular carcinoma.