A method for increasing the efficiency of selection of aptamers to cellular receptors

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Abstract

A method has been proposed to increase the efficiency of selection of aptamers to cellular receptors by the cell-Selex method, in particular to the receptor tyrosine kinase c-KIT. The use of Tween 20 in buffer solutions in concentrations not exceeding 0.01%, as well as trypsinolysis of surface proteins at the stage of elution of the combinatorial library of oligonucleotides bound to the cell surface, led to an increase in the specificity of aptamers and a decrease in nonspecific sorption according to the results of fluorescence microscopy, thermofluorimetric analysis and high-precision sequencing.

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About the authors

V. Е. Kuznetsova

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Author for correspondence.
Email: kuzneimb@gmail.com
Russian Federation, ul. Vavilova 32, Moscow, 119991

T. D. Lebedev

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: kuzneimb@gmail.com
Russian Federation, ul. Vavilova 32, Moscow, 119991

V. Е. Shershov

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: kuzneimb@gmail.com
Russian Federation, ul. Vavilova 32, Moscow, 119991

G. F. Shtylev

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: kuzneimb@gmail.com
Russian Federation, ul. Vavilova 32, Moscow, 119991

I. Yu. Shishkin

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: kuzneimb@gmail.com
Russian Federation, ul. Vavilova 32, Moscow, 119991

R. A. Miftahov

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: kuzneimb@gmail.com
Russian Federation, ul. Vavilova 32, Moscow, 119991

V. I. Butvilovskaya

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: kuzneimb@gmail.com
Russian Federation, ul. Vavilova 32, Moscow, 119991

I. V. Grechishnikova

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: kuzneimb@gmail.com
Russian Federation, ul. Vavilova 32, Moscow, 119991

О. А. Zasedateleva

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: kuzneimb@gmail.com
Russian Federation, ul. Vavilova 32, Moscow, 119991

А. V. Chudinov

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: kuzneimb@gmail.com
Russian Federation, ul. Vavilova 32, Moscow, 119991

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Supplementary files

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2. Fig. 1. Electrophoretic separation of primer extension reaction products in a reaction with Kod XL DNA polymerase. Denaturing 15% PAAG (acrylamide-bisacrylamide ratio 19:1), 50°C, registration in the fluorescence range of the Cy3 dye.

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3. Fig. 2. Melting temperature curves of the amplification product after each selection cycle.

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4. Fig. 3. Non-specific sorption using the example of one round of selection (a) and using Tween 20 as part of buffer solutions (b).

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5. Fig. 4. Evaluation of the effect of Tween 20 on the viability of FDC-P1 cells using fluorescence microscopy (DAPI dye).

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6. Fig. 5. Images of non-specific sorption of a fluorescently labeled DNA library during heat treatment of cells in a buffer solution (a) and trypsinolysis of aptamers bound to the cell surface (b), obtained using fluorescence microscopy, using the example of one round of selection.

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