Department of Life and Environmental Sciences, Integrated Graduate School of Medicine, Engineering, and Agricultural Sciences, University of Yamanashi, 4-4-37 Takeda, Kofu, Yamanashi 400-8510, Japan.
Department of Integrated Applied Life Science, Integrated Graduate School of Medicine, Engineering, and Agricultural Sciences, University of Yamanashi, 4-4-37 Takeda, Kofu, Yamanashi 400-8510, Japan.
J Biosci Bioeng. 2021 Mar;131(3):326-332. doi: 10.1016/j.jbiosc.2020.10.009. Epub 2020 Nov 9.
Proprotein convertase subtilisin/kexin type 9 (PCSK9) induces low-density lipoprotein (LDL)-receptor (LDLR) degradation, increasing plasma LDL-cholesterol levels and causing hypercholesterolemia. Therefore, inhibition of PCSK9-LDLR interaction is an attractive therapeutic target for hypercholesterolemia treatment. In this study, we have identified a novel RNA aptamer that binds specifically to PCSK9 by in vitro selection, also known as systematic evolution of ligands by exponential enrichment (SELEX). The binding kinetics of the PCSK9-binding RNA aptamer was measured by biolayer interferometry assay, showing that the aptamer has higher affinity compared to PCSK9-LDLR interaction. Competitive inhibition assay using chemiluminescence detection revealed that the RNA aptamer inhibits PCSK9-LDLR interaction. In cellular LDL-uptake assays with HepG2 cells, the RNA aptamer recovered LDL uptake in the PCSK9-treated cells, demonstrating its anti-PCSK9 antagonistic activity. These results indicated that the PCSK9-binding RNA aptamer has the potential to be an affinity reagent for PCSK9 protein analysis and a therapeutic reagent for hypercholesterolemia treatment.
前蛋白转化酶枯草溶菌素 9（PCSK9）诱导低密度脂蛋白（LDL）受体（LDLR）降解，增加血浆 LDL-胆固醇水平，导致高胆固醇血症。因此，抑制 PCSK9-LDLR 相互作用是治疗高胆固醇血症的一个有吸引力的治疗靶点。在这项研究中，我们通过体外选择（也称为指数富集的配体系统进化（SELEX））鉴定了一种与 PCSK9 特异性结合的新型 RNA 适体。通过生物层干涉测定法测量 PCSK9 结合 RNA 适体的结合动力学，表明与 PCSK9-LDLR 相互作用相比，该适体具有更高的亲和力。使用化学发光检测的竞争性抑制测定法显示 RNA 适体抑制 PCSK9-LDLR 相互作用。在 HepG2 细胞的细胞 LDL 摄取测定中，RNA 适体恢复了 PCSK9 处理细胞中的 LDL 摄取，证明了其抗 PCSK9 拮抗活性。这些结果表明，PCSK9 结合 RNA 适体有可能成为 PCSK9 蛋白分析的亲和试剂和高胆固醇血症治疗的治疗试剂。
