Post-Selection Design of Aptamers: Comparative Study of Affinity of the DNA Aptamers to Recombinant Extracellular Domain of Human Epidermal Growth Factor Receptors

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Valeria L. Moiseenko^1,2,a*, Olga M. Antipova^1,3, Aleksandra A. Rybina¹, Liliya I. Mukhametova¹, Sergei A. Eremin¹, Galina V. Pavlova^2,3, Alexey M. Kopylov^1,2

¹Faculty of Chemistry, Lomonosov Moscow State University, 119991 Moscow, Russia

²Burdenko National Medical Research Institute for Neurosurgery, 125047 Moscow, Russia

³Institute of Higher Nervous Activity and Neurophysiology, Russian Academy of Sciences, 117485 Moscow, Russia

^* To whom correspondence should be addressed.

Received: September 24, 2024; Revised: October 15, 2024; Accepted: October 16, 2024

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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.

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KEY WORDS: aptamer, EGFR, affinity, biolayer interferometry, fluorescence polarization

DOI: 10.1134/S0006297924120071

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