Aptamers Structure Flexibility Is Crucial to EGFR Inhibition Function.

Background/

Objectives

Glioblastoma is an aggressive brain tumour with a poor prognosis and limited therapy. The truncated aptamer GR20, derived from aptamer U31, was developed to retain EGFR binding while reducing size. This study evaluated how aptamer truncation, while maintaining its tertiary structure, affects its antiproliferative activity against human glioma cells.

Methods

Five transplantable human glioblastoma cell cultures (BU73, G22, G23, G01, Sus/fP2) were used. Aptamer binding was assessed by flow cytofluorimetry, intracellular accumulation was tracked by confocal microscopy. Changes in stemness and malignancy gene expression were evaluated by real-time PCR. Protein expression levels were assessed by immunocytochemistry. Proliferative activity was measured using the MTS assay.

Results

GR20 retained EGFR binding and demonstrated more than two-fold higher binding efficiency to glioblastoma cells than antibodies or the U31 aptamer. Both aptamers accumulated in cells after 2 h of incubation. GR20 significantly reduced the expression of stemness genes (CD133, OCT4) and malignancy genes (EGFR, PDGFRα, TP53) but showed modest antiproliferative effects (9-11% reduction). U31 exhibited stronger antiproliferative activity, reducing Sus/fP2 viability by 31% and 58%. Molecular dynamics showed that U31 maintains a "multi-loop" conformational state absent in GR20.

Conclusions

Although the truncated GR20 still binds to the EGFR and is taken up by cells, its antiproliferative activity is reduced compared to U31. The multi-loop conformation present in U31 but missing in GR20 appears to enhance activity, identifying this motif as a promising target for future aptamer optimization.