Post-selection design of aptamers: comparative study of the DNA aptamers affinity to the recombinant extracellular domain of human epidermal growth factor receptors

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The current work provides a comparative assessment of the designed DNA aptamers affinity for the extracellular domain of the human epidermal growth factor receptor (EGFR*). The affinity data of the 20 previously published aptamers are summarized. The diversity of aptamer selection methods and techniques requires the unification of comparison algorithms, which is also necessary for designing aptamers used in post-selection fitting to the target protein EGFR*. In this study the affinity of 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 development, was compared. The aptamer affinity for 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, the analysis of the titration profiles themselves and the kinetics of interaction proved to be more informative. This allowed us to identify how even subtle changes in the aptamers and their structures affect the affinity. Hypotheses regarding the “structure–function” relationships and recognition mechanisms were formulated. The data obtained for the set of aptamer constructions are critical for moving forward to examination of aptamer interactions with EGFR on the cell surface.

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V. Moiseenko

Lomonosov Moscow State University; Burdenko National Medical Research Institute for Neurosurgery

编辑信件的主要联系方式.
Email: valerian.moiseenko@gmail.com
俄罗斯联邦, 119991 Moscow; 125047 Moscow

O. Antipova

Lomonosov Moscow State University; Institute of Higher Nervous Activity and Neurophysiology, Russian Academy of Sciences

Email: valerian.moiseenko@gmail.com
俄罗斯联邦, 119991 Moscow; 117485 Moscow

A. Rybina

Lomonosov Moscow State University

Email: valerian.moiseenko@gmail.com
俄罗斯联邦, 119991 Moscow

L. Mukhametova

Lomonosov Moscow State University

Email: valerian.moiseenko@gmail.com
俄罗斯联邦, 119991 Moscow

S. Eremin

Lomonosov Moscow State University

Email: valerian.moiseenko@gmail.com
俄罗斯联邦, 119991 Moscow

G. Pavlova

Burdenko National Medical Research Institute for Neurosurgery; Institute of Higher Nervous Activity and Neurophysiology, Russian Academy of Sciences

Email: valerian.moiseenko@gmail.com
俄罗斯联邦, 125047 Moscow; 117485 Moscow

A. Kopylov

Lomonosov Moscow State University; Burdenko National Medical Research Institute for Neurosurgery

Email: valerian.moiseenko@gmail.com
俄罗斯联邦, 119991 Moscow; 125047 Moscow

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2. Fig. 1. Possible secondary structures of anti-EGFR DNA aptamers U31, U2 and their derivatives. The structures were obtained based on the analysis of the results obtained using The ViennaRNA Package service and visualized using the VARNA Java applet: a – U31; b – GR20; c – U2; d – U2s; d – Gol1. The red and purple contours in the structures of the U31 (a) and GR20 (b) aptamers highlight two blocks of nucleotides found in the corresponding loops of aptamers of both families. In the structures of the U2 series aptamers (c–d), nucleotides in identical regions of the P-2 loops and the Sh-2 hairpin are highlighted in green, and repeating regions in the P-3 loops are highlighted in blue. The blue dots indicate bonds in non-canonical base pairs.

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3. Fig. 2. Concentration dependences of the change in fluorescence polarization upon binding of FAM-labeled DNA aptamers of the U31 (a and b) and U2 (c–d) families to EGFR*. a – U31; b – GR20; c – U2; g – U2s, d – Gol1; R2 > 0.98. The measurements were carried out in triplicate. The calculated kKd are indicated.

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4. Fig. 3. Sensograms obtained by the IBS method for immobilized biotinylated DNA aptamers upon binding to EGFR*. a – U31; b – GR20; c – U2; d – U2s; d – Gol1

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5. Appendix
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