Abstract
A method of thermal dissociation of DNA–protein conjugates was developed for the selection of aptamers to human alpha-fetoprotein. The method is based on the identification of the DNA oligonucleotides from the combinatorial DNA libraries with the highest specificity to targets, which remained within the conjugate after two stages: a) washing of unbound sequences; and b) removal of the sequences bound to the DNA but displaying a low affinity to it. The undestroyed most stable complexes were subjected to the thermal dissociation with the involvement of the corresponding oligonucleotides in the next rounds of selection. A comparison of the proposed method with the standard washing demonstrated a more intensive enrichment of the library. Due to the tighter conditions for washing of the target-bound DNA oligonucleotides the use of thermal dissociation can increase the efficiency of selection of highly specific aptamers to their molecular targets.
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Funding
The work was supported by the Federal program “Research and Development in the Prioritized Areas of the Elaboration of the Research and Technological Complex of Russia for 2014–2020” (agreement no. 14.576.21.0096; project unique identifier RFMEFI57617X0096).
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All the experiments were performed in vitro. Humans or animals were not involved as subjects of studies.
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Translated by E. Shirokova
Abbreviations: ATD, aptamer thermal dissociation; AFP, alpha-fetoprotein; NHS, N-hydroxysuccineimide; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; MES, 2‑(N-morpholino)ethanesulfonic acid.
Corresponding author: phone: +7 (495) 135-9800; fax: +7 (495) 135-1405; e-mail: lapa@biochip.ru.
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Lapa, S.A., Shershov, V.E., Krasnov, G.S. et al. The Use of Thermal Dissociation for Selection of DNA Aptamers. Russ J Bioorg Chem 46, 551–556 (2020). https://doi.org/10.1134/S106816202004010X
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DOI: https://doi.org/10.1134/S106816202004010X