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

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.

Keywords

• aptamer
• C-type lectin-like receptor 2
• platelet aggregation
• podoplanin
• systematic evolution of ligands by exponential enrichment