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Abstract <p><b>Objective:</b> A method to increase the efficiency of cell-SELEX-based selection of aptamers to cellular receptors, in particular, to the receptor tyrosine kinase c-KIT, was presented. <b>Methods:</b> Direct monitoring of each aptamer selection cycle by analyzing the shift of the melting curve towards higher temperatures allows monitoring the enrichment of a combinatorial oligonucleotide library due to the correlation of increasing GC content and melting temperature with an increase of aptamer affinity for the corresponding target. <b>Results and Discussion:</b> The use of non-ionic surfactants (Tween 20) in the composition of buffer solutions, as well as trypsinolysis of surface proteins at the stage of elution of the oligonucleotide library bound to the cell surface, significantly reduce non-specific sorption. Non-ionic surfactants in low concentrations can be incorporated into the lipid bilayer, forming polar defects that change the physical properties of cell membranes and lead to their reversible solubilization, which causes a decrease in non-specific sorption. Trypsin, an enzyme of the hydrolase class, possessing proteolytic activity, cleaves peptide bonds of proteins on the cell surface without compromising the integrity of the membrane and DNA aptamer. <b>Conclusions:</b> We presented a method that allows increasing the specificity of aptamers and decreasing non-specific sorption, according to the results of fluorescence microscopy, thermofluorimetric analysis, and high-precision sequencing.</p>

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A Method for Increasing the Efficiency of Selection of Aptamers to Cellular Receptors

  • 期刊论文

  • 发表日期：

    2025年6月3日

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  • V. E. Kuznetsova,
  • T. D. Lebedev,
  • V. E. Shershov,
  • G. F. Shtylev,
  • I. Yu. Shishkin,
  • R. A. Miftakhov,
  • V. I. Butvilovskaya,
  • I. V. Grechishnikova,
  • O. A. Zasedateleva,
  • A. V. Chudinov

摘要

Abstract

Objective: A method to increase the efficiency of cell-SELEX-based selection of aptamers to cellular receptors, in particular, to the receptor tyrosine kinase c-KIT, was presented. Methods: Direct monitoring of each aptamer selection cycle by analyzing the shift of the melting curve towards higher temperatures allows monitoring the enrichment of a combinatorial oligonucleotide library due to the correlation of increasing GC content and melting temperature with an increase of aptamer affinity for the corresponding target. Results and Discussion: The use of non-ionic surfactants (Tween 20) in the composition of buffer solutions, as well as trypsinolysis of surface proteins at the stage of elution of the oligonucleotide library bound to the cell surface, significantly reduce non-specific sorption. Non-ionic surfactants in low concentrations can be incorporated into the lipid bilayer, forming polar defects that change the physical properties of cell membranes and lead to their reversible solubilization, which causes a decrease in non-specific sorption. Trypsin, an enzyme of the hydrolase class, possessing proteolytic activity, cleaves peptide bonds of proteins on the cell surface without compromising the integrity of the membrane and DNA aptamer. Conclusions: We presented a method that allows increasing the specificity of aptamers and decreasing non-specific sorption, according to the results of fluorescence microscopy, thermofluorimetric analysis, and high-precision sequencing.