The Use of Thermal Dissociation for Selection of DNA Aptamers
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Use of Thermal Dissociation for Selection of DNA Aptamers S. A. Lapaa, 1, V. E. Shershova, G. S. Krasnova, O. S. Volkovaa, V. E. Kuznetsovaa, S. P. Radkob, A. S. Zasedateleva, and A. V. Chudinova, b aEngelhardt
Institute of Molecular Biology, Russian Academy of Sciences, Moscow, 119991 Russia bIBMC-EcoBioPharm Ltd., Moscow, 119121 Russia
Received December 18, 2019; revised December 25, 2019; accepted December 29, 2019
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. Keywords: DNA aptamers, human alpha-fetoprotein, thermal dissociation of aptamers DOI: 10.1134/S106816202004010X
INTRODUCTION Aptamers are single-stranded DNA or RNA oligonucleotides with the affinity to a wide spectrum of biomolecules. Due to this property they can be regarded as functional analogs of monoclonal antibodies. Aptamers are used in clinical diagnostics. Currently, the attempts are being made to use them as individual therapeutic agents [1, 2]. The process of selection of target-specific aptamers from the primary combinatorial library is called SELEX (Systematic Evolution of Ligands by EXponential Enrichment) and is a series of cycles of oligonucleotide binding to the target, isolation of the bound fraction, and amplification of the bound fraction [3, 4]. In case of DNA libraries each cycle of the SELEX procedure can be divided into several standard steps: —Interaction of the DNA library representing a combination of many individual DNA oligonucleotides with the molecular target. —Washing of target-unbound DNA oligonucleotides. —Removal of the bound DNA oligonucleotides from the target molecule. Abbreviations: ATD, aptamer thermal dissociation; AFP, alphafetoprotein; NHS, N-hydroxysuccineimide; EDC, 1-ethyl-3(3-dimethylaminopropyl)carbodiimide hydrochloride; MES, 2-(N-morpholino)ethanesulfonic acid. 1 Corresponding author: phone: +7 (495) 135-9800; fax: +7 (495) 135-1405; e-mail: [email protected].
—Amplification of the bound DNA oligonucleotides. —Repeated involvement of the bound DNA oligonucleotides in the selection rounds with the molecular target with the goal of enriching the combinatorial DNA library with oligonucleotide sequences dis
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