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Single-Strand DNA-Like Oligonucleotide Aptamer Against Proprotein Convertase Subtilisin/Kexin 9 Using CE-SELEX: PCSK9 Targeting Selection

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Abstract

Background

Proprotein convertase subtilisin/kexin 9 (PCSK9) serves a key regulatory function in the metabolism of low-density lipoprotein (LDL)-cholesterol (LDL-C) through interaction with the LDL receptor (LDLR) followed by its destruction that results in the elevation of the plasma levels of LDL-C. The aims of the present study were to separate and select a number of single-stranded DNA (ssDNA) aptamers against PCSK9 from a library pool (n > 1012) followed by their characterization.

Methods

The aptamers obtained from the DNA-PCSK9 complexes which presented the highest affinity against PCSK9 were separated and selected using capillary electrophoresis evolution of ligands by exponential enrichment (CE-SELEX). The selected aptamers were amplified and cloned into a T/A vector. The plasmids from the positive clones were extracted and sequenced. The Mfold web server was used to predict the secondary structure of the aptamers.

Results

Following three rounds of CE-SELEX, the identified anti-PCSK9 ssDNA aptamers, namely aptamer 1 (AP-1) and aptamer 2 (AP-2), presented half maximal inhibitory concentrations of 325 and 327 nM, lowest dissociation constants of 294 and 323 nM, and most negative Gibbs free energy values of − 9.17 and − 8.28 kcal/mol, respectively.

Conclusion

The results indicated that the selected aptamers (AP-1 and AP-2) induced potent inhibitory effects against PCSK9. Further in vivo studies demand to find out AP-1 and AP-2 aptamers as suitable candidates, instead of antibodies, for using in therapeutic purposes in patients with hypercholesterolemia and cardiovascular disease.

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Data Availability

The datasets used and/or analyzed during the current study are included in this published article.

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Acknowledgments

The authors acknowledge the Isfahan University of Medical Sciences for providing academic and laboratory supports.

Funding

The present study was supported by the Iran National Science Foundation (INSF grant no. 93016087) and the Isfahan University of Medical Sciences (project no. 395902).

Author information

Authors and Affiliations

  1. Department of Clinical Biochemistry, Faculty of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Hezar Jerib Street, Isfahan, 817467346, Iran

    Roohollah Sattari & Abbasali Palizban

  2. Department of Molecular Biology and Genetics, Faculty of Medicine, Isfahan University of Medical Sciences, Isfahan, 8174673461, Iran

    Hossein Khanahmad

Authors
  1. Roohollah Sattari
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  2. Abbasali Palizban
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  3. Hossein Khanahmad
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Contributions

AAP conceived the study, designed the experiments, performed the capillary electrophoresis evolution of ligands by exponential enrichment, and analyzed the data. RS prepared the samples for capillary electrophoresis and performed the experiments. HK facilitated the molecular biology experiments.

Corresponding author

Correspondence to Abbasali Palizban.

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The authors declare that they have no conflict interests.

Ethics Approval and Consent to Participate

This study was approved by the Ethics Committee (no. 395902) of the Isfahan University of Medical Sciences, Iran.

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Not applicable.

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Sattari, R., Palizban, A. & Khanahmad, H. Single-Strand DNA-Like Oligonucleotide Aptamer Against Proprotein Convertase Subtilisin/Kexin 9 Using CE-SELEX: PCSK9 Targeting Selection. Cardiovasc Drugs Ther 34, 475–485 (2020). https://doi.org/10.1007/s10557-020-06986-y

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  • DOI: https://doi.org/10.1007/s10557-020-06986-y

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