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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, PDGFR\u03b1, TP53) but showed modest antiproliferative effects (9\u201311% reduction). U31 exhibited stronger antiproliferative activity, reducing Sus\/fP2 viability by 31% and 58%. Molecular dynamics showed that U31 maintains a \u201cmulti-loop\u201d 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.<\/jats:p>","DOI":"10.3390\/ph19081246","type":"journal-article","created":{"date-parts":[[2026,8,7]],"date-time":"2026-08-07T16:00:43Z","timestamp":1786118443000},"page":"1246","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Aptamers Structure Flexibility Is Crucial to EGFR Inhibition Function"],"prefix":"10.3390","volume":"19","author":[{"ORCID":"https:\/\/orcid.org\/0009-0008-1266-5145","authenticated-orcid":false,"given":"Yahya","family":"Sliman","sequence":"first","affiliation":[{"name":"Institution N. N. Burdenko National Medical Research Center of Neurosurgery of the Ministry of Health of the Russian Federation, 125047 Moscow, Russia"},{"name":"Moscow Institute of Physics and Technology, National Research University, 141701 Dolgoprudny, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0005-1146-6548","authenticated-orcid":false,"given":"Valeria","family":"Ivko","sequence":"additional","affiliation":[{"name":"Institution N. N. Burdenko National Medical Research Center of Neurosurgery of the Ministry of Health of the Russian Federation, 125047 Moscow, Russia"},{"name":"Belozersky Research Institute of Physical Chemical Biology, Lomonosov Moscow State University, 119991 Moscow, Russia"},{"name":"Department of Chemistry, Lomonosov Moscow State University, 119991 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lika","family":"Fab","sequence":"additional","affiliation":[{"name":"Institute of Higher Nervous Activity and Neurophysiology, Russian Academy of Sciences, 117485 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2884-6142","authenticated-orcid":false,"given":"Fatima","family":"Dzarieva","sequence":"additional","affiliation":[{"name":"Institution N. N. Burdenko National Medical Research Center of Neurosurgery of the Ministry of Health of the Russian Federation, 125047 Moscow, Russia"},{"name":"Institute of Higher Nervous Activity and Neurophysiology, Russian Academy of Sciences, 117485 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nadezhda","family":"Samoylenkova","sequence":"additional","affiliation":[{"name":"Institution N. N. Burdenko National Medical Research Center of Neurosurgery of the Ministry of Health of the Russian Federation, 125047 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ekaterina","family":"Savchenko","sequence":"additional","affiliation":[{"name":"Institution N. N. Burdenko National Medical Research Center of Neurosurgery of the Ministry of Health of the Russian Federation, 125047 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7496-4310","authenticated-orcid":false,"given":"Sergey","family":"Drozd","sequence":"additional","affiliation":[{"name":"Institution N. N. Burdenko National Medical Research Center of Neurosurgery of the Ministry of Health of the Russian Federation, 125047 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6685-8056","authenticated-orcid":false,"given":"Olga","family":"Kalennik","sequence":"additional","affiliation":[{"name":"Institution N. N. Burdenko National Medical Research Center of Neurosurgery of the Ministry of Health of the Russian Federation, 125047 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8908-4268","authenticated-orcid":false,"given":"Andrey","family":"Golovin","sequence":"additional","affiliation":[{"name":"Belozersky Research Institute of Physical Chemical Biology, Lomonosov Moscow State University, 119991 Moscow, Russia"},{"name":"Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, GSP-1, Leninskiye Gory, 1-73, 119234 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0962-8905","authenticated-orcid":false,"given":"Alexey","family":"Kopylov","sequence":"additional","affiliation":[{"name":"Institution N. N. Burdenko National Medical Research Center of Neurosurgery of the Ministry of Health of the Russian Federation, 125047 Moscow, Russia"},{"name":"Belozersky Research Institute of Physical Chemical Biology, Lomonosov Moscow State University, 119991 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1520-5650","authenticated-orcid":false,"given":"Dmitry","family":"Usachev","sequence":"additional","affiliation":[{"name":"Institution N. N. Burdenko National Medical Research Center of Neurosurgery of the Ministry of Health of the Russian Federation, 125047 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6885-6601","authenticated-orcid":false,"given":"Galina","family":"Pavlova","sequence":"additional","affiliation":[{"name":"Institution N. N. Burdenko National Medical Research Center of Neurosurgery of the Ministry of Health of the Russian Federation, 125047 Moscow, Russia"},{"name":"Institute of Higher Nervous Activity and Neurophysiology, Russian Academy of Sciences, 117485 Moscow, Russia"},{"name":"Department of Medical Genetics and Postgenomic Technologies, Sechenov First Moscow State Medical University, 119991 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,8,7]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1231","DOI":"10.1093\/neuonc\/noab106","article-title":"The 2021 WHO Classification of Tumors of the Central Nervous System: A Summary","volume":"23","author":"Louis","year":"2021","journal-title":"Neuro. Oncol."},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Brown, N.F., Ottaviani, D., Tazare, J., Gregson, J., Kitchen, N., Brandner, S., Fersht, N., and Mulholland, P. (2022). 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