In vitro selection and characterization of a DNA aptamer targeted to Prorocentrum minimum-A common harmful algae

Abstract

Prorocentrum minimum is a common diarrhetic shellfish toxins-producing marine microalga that may seriously endanger marine resources and cause great economic losses. The development of a novel rapid detection technique is of great importance for the prevention and control of the damage caused by P. minimum. In this study, the aptamer against P. minimum was for the first time generated from an artificially synthesized single-stranded DNA library by systematic evolution of ligand by exponential enrichment (SELEX), using P. minimum and P. minimum-related species, including Prorocentrum donghaiense, Prorocentrum lima and Prorocentrum micans as target and counter-screening species, respectively. The aptamer library was successfully obtained at the end of 18 rounds of SELEX-screening by continuously monitoring the binding ratio of the resultant ssDNA from each round. Three sequences (Apt 1, Apt 2 and Apt 3) with the highest frequency in the aptamer library resulted from high-throughput sequencing were first selected as candidate aptamers. The secondary structure of these sequences was predicted and analyzed. In addition, the specificity and affinity of these candidate aptamers were determined by flow cytometry analysis. The results indicated that these aptamers had high specificity and affinity, with a KD of (224.6 ± 8.8) nM (Apt 1), (286.6 ± 13.9) nM (Apt 2) and (388.5 ± 44.6) nM (Apt 3), respectively. Apt 1 was therefore chosen as the best aptamer against P. minimum. Finally, the fluorescence microscopic examination further confirmed that Apt 1 can well bind to P. minimum. In summary, Apt 1 may be promising for being used as a novel molecular recognition element for P. minimum.