Identification of Podoplanin Aptamers by SELEX for Protein Detection and Inhibition of Platelet Aggregation Stimulated by C-Type Lectin-like Receptor 2.

IF 4.9 3区 工程技术 Q1 CHEMISTRY, ANALYTICAL Biosensors-Basel Pub Date : 2024-09-27 DOI:10.3390/bios14100464
Hui-Ju Tsai, Kai-Wen Cheng, Jou-Chen Li, Tsai-Xiang Ruan, Ting-Hsin Chang, Jin-Ru Wang, Ching-Ping Tseng
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Abstract

Tumor cell-induced platelet aggregation (TCIPA) is a mechanism for the protection of tumor cells in the bloodstream and the promotion of tumor progression and metastases. The platelet C-type lectin-like receptor 2 (CLEC-2) can bind podoplanin (PDPN) on a cancer cell surface to facilitate TCIPA. Selective blockage of PDPN-mediated platelet-tumor cell interaction is a plausible strategy for inhibiting metastases. In this study, we aimed to screen for aptamers, which are the single-stranded DNA oligonucleotides that form a specific three-dimensional structure, bind to specific molecular targets with high affinity and specificity, bind to PDPN, and interfere with PDPN/CLEC-2 interactions. The systematic evolution of ligands by exponential enrichment (SELEX) was employed to enrich aptamers that recognize PDPN. The initial characterization of ssDNA pools enriched by SELEX revealed a PDPN aptamer designated as A1 displaying parallel-type G-quadruplexes and long stem-and-loop structures and binding PDPN with a material with a dissociation constant (Kd) of 1.3 ± 1.2 nM. The A1 aptamer recognized both the native and denatured form of PDPN. Notably, the A1 aptamer was able to quantitatively detect PDPN proteins in Western blot analysis. The A1 aptamer could interfere with the interaction between PDPN and CLEC-2 and inhibit PDPN-induced platelet aggregation in a concentration-dependent manner. These findings indicated that the A1 aptamer is a candidate for the development of biosensors in detecting the levels of PDPN expression. The action by A1 aptamer could result in the prevention of tumor cell metastases, and if so, could become an effective pharmacological agent in treating cancer patients.

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用 SELEX 法鉴定 Podoplanin 短链肽,用于蛋白质检测和抑制由 C 型凝集素样受体 2 刺激的血小板聚集。
肿瘤细胞诱导的血小板聚集(TCIPA)是血液中保护肿瘤细胞、促进肿瘤进展和转移的一种机制。血小板 C 型凝集素样受体 2(CLEC-2)能与癌细胞表面的 podoplanin(PDPN)结合,从而促进 TCIPA。选择性阻断 PDPN 介导的血小板-肿瘤细胞相互作用是抑制转移的一种可行策略。在这项研究中,我们的目标是筛选出能形成特定三维结构、以高亲和力和特异性与特定分子靶标结合、与 PDPN 结合并干扰 PDPN/CLEC-2 相互作用的单链 DNA 寡核苷酸配体。通过指数富集配体的系统进化(SELEX)来富集识别 PDPN 的适配体。通过对 SELEX 富集的 ssDNA 池进行初步表征,发现了一种被命名为 A1 的 PDPN 类似物,它具有平行型 G-四重链和长茎环结构,能与 PDPN 结合,其解离常数(Kd)为 1.3 ± 1.2 nM。A1 合体既能识别原生形式的 PDPN,也能识别变性形式的 PDPN。值得注意的是,A1 合体能够在 Western 印迹分析中定量检测 PDPN 蛋白。A1 合体能干扰 PDPN 与 CLEC-2 之间的相互作用,并以浓度依赖性的方式抑制 PDPN 诱导的血小板聚集。这些研究结果表明,A1 拟合物是开发检测 PDPN 表达水平的生物传感器的候选材料。A1 aptamer 的作用可以防止肿瘤细胞转移,因此可以成为治疗癌症患者的有效药剂。
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来源期刊
Biosensors-Basel
Biosensors-Basel Biochemistry, Genetics and Molecular Biology-Clinical Biochemistry
CiteScore
6.60
自引率
14.80%
发文量
983
审稿时长
11 weeks
期刊介绍: Biosensors (ISSN 2079-6374) provides an advanced forum for studies related to the science and technology of biosensors and biosensing. It publishes original research papers, comprehensive reviews and communications. Our aim is to encourage scientists to publish their experimental and theoretical results in as much detail as possible. There is no restriction on the length of the papers. The full experimental details must be provided so that the results can be reproduced. Electronic files and software regarding the full details of the calculation or experimental procedure, if unable to be published in a normal way, can be deposited as supplementary electronic material.
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