Screening an aptamer against Amphidinium carterae by WC-SELEX coupled with optimization by G-quadruplex-forming mutation

Amphidinium carterae, as a commonly distributed harmful algal species, poses a severe threat to marine ecosystems and the sustainable development of fishery industry. Although the current detection methods for A. carterae are accurate, they are usually time-consuming and dependent on special instruments, greatly limiting their practical use. Therefore, there is an urgent need for the development of more efficient and convenient detection methods. This study utilized whole cell-systematic evolution of ligands by exponential enrichment (WC-SELEX) coupled with optimization by G-quadruplex-forming mutation to obtain a novel aptamer as a cell probe targeting A. carterae. The WC-SELEX involved designing a random ssDNA library, optimizing PCR conditions for the preparation of secondary library, introducing counter-SELEX, and generating high-quality ssDNA library using a long-short-strand method. After 20 rounds of screening, four candidate aptamers (Apt-2, Apt-6, Apt-7 and Apt-20) were identified, among which Apt-2 exhibited the highest affinity (K_D = 100.0 nM ± 9.7 nM). To further enhance the affinity of Apt-2, a strategy of G-quadruplex-forming mutation was employed to obtain a new aptamer, i.e., gApt-2-2. gApt-2-2 demonstrated a significantly greater binding affinity (K_D = 61.1 ± 9.7 nM) compared to Apt-2, while maintaining good specificity. In conclusion, a new cell-targeting probe for A. carterae was obtained in this study, laying a basis for the development of a rapid and quantitative detection method for A. carterae. In addition, it also provides a valuable reference for the screening and optimization of aptamer targeting other harmful marine microalgae.