GR20

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.

Targeted delivery of chemotherapeutic agents with aptamers is a very effective method increasing therapeutic index compared to non-targeted drugs. Objective. To study the effectiveness of in vitro therapeutic effect of covalently conjugated GR20 DNA aptamer with doxorubicin on glioblastoma cells compared to reference culture of human fibroblasts. Material and methods. A Sus/fP2 cell culture was obtained from glioblastoma tissue sample to analyze the effectiveness of conjugate. A linear culture of human dermal fibroblasts (mesenchymal stem cells) DF1 was used as a control. To assess antiproliferative activity of covalently conjugated GR20 aptamer with doxorubicin, we used the MTS test. The Cell Index was measured using the xCelligence S16 cell analyzer assessing viability of cell cultures by recording changes in real time. Results. Human glioblastoma Sus/fP2 cells reduce own proliferative potential by 80% when exposed to doxorubicin (0.5 µM, 72 hours, MTS test), by 9% when exposed to GR20 aptamer (10 µM, 72 hours, MTS test) and by 26% when exposed to covalently conjugated DOX-GR20 (0.5 µM, 72 hours, MTS test). A long-term study of proliferative potential of Sus/fP2 cells on the xCelligence S16 analyzer revealed a significant decrease in the number of cells under the effect of doxorubicin and covalently conjugated DOX-GR20. Effectiveness of covalently conjugated DOX-GR20 is halved. GR20 aptamer at a concentration of 10 μM and its conjugate with doxorubicin DOX-GR20 at a concentration of 1 μM have no negative effect on cells of the control culture of DF1 fibroblasts, while doxorubicin is toxic for these cells. MTS test and xCelligence S16 cell analyzer found no decrease in metabolic activity of DF1 cells and their ability to proliferate. Conclusion. We established obvious antiproliferative effect of covalent conjugate DOX-GR20 on continuous human glioblastoma cell culture Sus/fP2 without toxic effect on the reference culture (dermal fibroblasts DF1).