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

Abstract

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. 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. 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. 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.