Nucleic acid aptamers are generally accepted as promising elements for the specific and high-affinity binding of various biomolecules. It has been shown for a number of aptamers that the complexes with several related proteins may possess a similar affinity. An outstanding example is the G-quadruplex DNA aptamer RHA0385, which binds to the hemagglutinins of various influenza A virus strains. These hemagglutinins have homologous tertiary structures but moderate-to-low amino acid sequence identities. Here, the experiment was inverted, targeting the same protein using a set of related, parallel G-quadruplexes. The 5′- and 3′-flanking sequences of RHA0385 were truncated to yield parallel G-quadruplex with three propeller loops that were 7, 1, and 1 nucleotides in length. Next, a set of minimal, parallel G-quadruplexes with three single-nucleotide loops was tested. These G-quadruplexes were characterized both structurally and functionally. All parallel G-quadruplexes had affinities for both recombinant hemagglutinin and influenza virions. In summary, the parallel G-quadruplex represents a minimal core structure with functional activity that binds influenza A hemagglutinin. The flanking sequences and loops represent additional features that can be used to modulate the affinity. Thus, the RHA0385–hemagglutinin complex serves as an excellent example of the hypothesis of a core structure that is decorated with additional recognizing elements capable of improving the binding properties of the aptamer.

The current work presents comparative assessment of affinity of the designed DNA aptamers for extracellular domain of the human epidermal growth factor receptor (EGFR*). The affinity data of the 20 previously published aptamers are summarized. Diversity of the aptamer selection methods and techniques requires unification of the comparison algorithms, which is also necessary for designing aptamers used in the post-selection fitting to the target EGFR* protein. In this study affinities of the DNA aptamers from two families, U31 and U2, previously obtained by Wu et al. from the same selection [Wu et al. (2014) PLoS One, 9, e90752] and their derivatives – GR20, U2s, and Gol1 obtained by us through rational design, were compared. Affinity of the aptamers to EGFR* was measured by two different methods: a solution-phase technique – fluorescence polarization of FAM-labeled aptamers, and by a kinetic method using biolayer interferometry technique with aptamers immobilized on the surface. Unlike the values of equilibrium dissociation constants obtained through titration and expressed in units of protein concentration, analysis of the titration curve profiles themselves and kinetics of interaction proved to be more informative. This allowed us to identify how even subtle changes in the aptamers and their structures affect affinity. Hypotheses regarding the “structure–function” relationships and recognition mechanisms were formulated. The data obtained for the set of aptamer constructs are critical for moving forward to examination of aptamer interactions with EGFR on the cell surface.

Theranostics combines diagnostics and therapeutic exposure. Regarding glioblastomas, theranostics solves the problem of detecting and destroying tumor stem cells resistant to irradiation and chemotherapy and causing tumor recurrence. Transmembrane surface antigen CD133 is considered as a potential marker of tumor stem cells. Objective. To detect CD133 in patient-derived glioblastoma continuous cell cultures using fluorescence microscopy and modified aptamers (molecular recognition elements) anti-CD133. Material and methods. To detect CD133, we used mousey fluorescence monoclonal antibodies anti-CD133 MA1-219, FAM-modified DNA aptamers anti-CD133 AP-1-M and Cs5. Non-aptamer DNA oligonucleotide NADO was used as a negative control. Detection was performed for three samples of patient-derived glioblastoma continuous cell cultures coded as 1548, 1721 and 1793. Results. MA1-219 antibodies brightly stained cell culture 1548, to a lesser extent — 1721. There was diffuse staining of cell culture 1793. Cs5-FAM aptamer stained cells in a similar way, but much weaker. AP-1-M-FAM aptamer interacted with cells even weaker and diffusely stained only cell culture 1793. Non-aptamer NADO did not stain cell culture 1548 and very weakly diffusely stained cell culture 1793. Conclusion. For both molecular recognition elements (MA1-219 antibody and Cs5 aptamer), 3 cell culture samples can be arranged in the following order possibly reflecting CD133 status decrease: strong signal for cell culture 1548, much weaker for 1721, even weaker for 1793. Only cell culture 1548 can be considered CD133 positive with combination of Cs5+ and NADO signals. Cell culture 1793 is CD133 false positive with combination of Cs5+ and NADO+ signals.

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.

The WHO considers the Epidermal Growth Factor Receptor (EGFR) one of the key biomarkers of glioblastoma (GB). EGFR can be identified and targeted using molecular recognition elements (MoREs), like aptamers and aptamer–drug conjugates (ApDCs). Understanding the kinetics of anti-EGFR ApDC interactions with EGFR as well as the kinetics of their internalization into the cells is a crucial step for the further development of anti-EGFR ApDCs. For the first time, a novel approach was implemented to study real-time kinetics by measuring the cellular index (CI) using impedance (xCELLigence). Doxorubicin (DOX) was used as an indicator drug. Because DOX intercalates into the DNA double helix, aptamer–DOX non-covalent complexes were obtained. For the anti-EGFR DNA aptamer GR20, an additional duplex was constructed by synthesizing the extra region (GR20h) and via hybridization with the complementary oligonucleotide (h’) to form a duplex (hh’), thus creating the aptamer construct with complementary oligonucleotide (ACCO) GR20hh’. The original HPLC method quantified the assembly efficiency of an ACCO. The ACCO GR20hh’ retained affinity for the recombinant extracellular domain of EGFR, as measured using Biolayer Interferometry (BLI). According to cytofluorimetry, the ACCO GR20hh’ interacts with cells of continuous culture from GB patient (CCGBP) surgical samples. The DOX–ACCO GR20hh’ complexes are more efficiently internalized by EGFR+ cells lines A-431 and CCGBP 107 than DOX alone.

Glioblastoma or grade IV glioma is characterized by extremely poor prognosis. Resistance to radio- / chemotherapy and recurrences are associated with tumor stem cells. Transmembrane protein CD133 is considered a potential marker of tumor stem cells. To overcome the limitations of detecting cellular CD133 by antibodies, potential of aptamers (nucleic acid-based molecular recognition elements) is being explored. To obtain reproducible results, it is necessary to study the interaction of fluorescent aptamers to CD133 with standard cell lines and cell cultures derived from patient tumors using various methods. Objective. To detect the CD133 marker in continuous cell cultures of patient-derived GB cells using fluorescent DNA aptamers by flow cytometry. Material and methods. Fluorescein (FAM)-labeled DNA aptamers of the Cs and Ap series were used to detect the CD133 marker. Three continuous cell cultures from the Burdenko Neurosurgical Center Biobank were tested using flow cytometry. Burdenko: BU881 (107), G01, and Sus (harvested from postoperative GB tissues). Results. CD133 DNA aptamers at a concentration of 1 μM yield positive signals for GB cell cultures. Their magnitude correlates with CD133 gene transcription. Fluorescence signal shift (∆MFI) for FAM DNA aptamers of the Cs and Ap series is greater for BU881 cells (1942 and 5449 c.u., respectively) than for G01 cells (1469 and 1817 c.u., respectively). This is consistent with CD133 gene transcription. There is no fluorescence signal shift for Sus cells with minimal CD133 gene transcription. Conclusion. Fluorescein-labeled DNA aptamers of the Cs and Ap series at a concentration of 1 μM enable detection of the CD133 marker in continuous cell cultures of GB through flow cytometry.