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

Highlights

• We obtained aptamer against P. minimum by cell-SELEX in vitro.

• The specificity and affinity of these candidate aptamers were analyzed.

• Aptamer Apt 1 was finally chosen because it had the highest binding affinity.

• This study laid a foundation for the development of novel detection of P. minimum.

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 K_D 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.

Introduction

Prorocentrum minimum is a common toxic dinoflagellate, which can generate diarrhetic shellfish toxins, causing potential hazards to fish, shells and even humans. Despite that the components of toxins produced by P. minimum are uncertain, they have been usually correlated to oxygen depletion in seawater and the death of marine organisms (Rodríguez et al., 2017; Klanjšček et al., 2016). P. minimum, known as a mixed-nutrient microalga, has a strong ability to utilize nutrients, which enables it to adapt rapidly to variable environments and consequently expands its geographical distribution and occupies new ecological niches. P. minimum lives mainly in marine waters of temperate and subtropical zones and has been found worldwide, including the western coast of the United States, Japan, UK, Australia, the Mediterranean Sea, Norway and China (Cai et al., 2006; Sierra-Beltrán et al., 2005). The frequency and geographical extent of harmful algal blooms (HABs) caused by P. minimum have been increasing over the last few decades (Zhang et al., 2014; Heil et al., 2005). P. minimum, with a cell concentration exceeding 10³ cells L⁻¹, may adversely affect the growth or survival of other surrounding aquatic organisms. On the other hand, the cell concentrations of P. minimum-forming HABs can usually reach up to 10⁹ cells L⁻¹ (Tas and Okus, 2011).

The main detection technology for microalgae is currently microscopic examination of target cells based on morphological taxonomy. Unfortunately, most microalgae are not able to be easily distinguished without professional taxonomic due to variable cellular shape and tiny size (Barkallah et al., 2020; Rodríguez-Ramos et al., 2014). Although absorption spectrometry assay and fluorescence spectrometry assay can allow for accurate classification of algal species at the phylum level, they however have a poor resolution to distinguish different microalgal species at the genus and species level (Liu et al., 2020b; Sanchini and Grosjean, 2020; Zieger et al., 2018). In recent years, the emergence of novel molecular biology techniques has provided a new idea for the detection of harmful algae species. To date, several molecular detection methods for harmful algae have been established, including sandwich hybridization assay (SHA) (Kang et al., 2020; Xu and Zhen, 2018), quantitative PCR (qPCR) (Barkallah et al., 2020; Ruvindy et al., 2018) and isothermal amplification (Liu et al., 2021; Fu et al., 2019; Qin et al., 2019). Although the previously reported molecular detection methods can accurately and specifically detect target microalgal species in natural samples, their detection processes are too complex to meet the need for rapid analysis of field samples in large numbers. Therefore, more efforts should be made to develop novel methods aimed at detecting and monitoring harmful microalgae more effectively and accurately.

Aptamer, typically consisting of 10–100 nt, is a single-stranded DNA (ssDNA) or RNA capable of binding to a specific target with high affinity and specificity. Aptamer is generally selected from a random library through systematic evolution of ligands by exponential enrichment (SELEX) (Röthlisberger and Hollenstein, 2018; Ellington and Szostak, 1990; Tuerk and Gold, 1990). The presence of a target can induce aptamer to form a stable secondary structure that can bind to it following the principle of complementary base pairing, such as hairpin, pseudoknot, step loop, G-quadruplex, etc. (Xing et al., 2015). Aptamer, also known as a “chemical antibody”, mainly has the following advantages compared with the traditional antibody (Lee et al., 2010): (1) Aptamer can be directly obtained by in vitro screening; (2) Aptamer is stable and is characterized with good thermal renaturation and wide-range pH tolerance (Kinghorn et al., 2017); (3) The synthesis of aptamer is easy, with low batch variability and good reproducibility; (4) Aptamer can easily be functionally modified to further enhance its stability (Elskens et al., 2020); (5) The composition of aptamer exclusively consisting of four bases (adenine, thymine, guanine and cytosine) is relatively simple and aptamer can bind a wide range of targets, including ions, small molecules, proteins, cells, bacteria and viruses (Lu et al., 2017; Zhou and Rossi, 2017).

Aptamer has received a great deal of attention from the scientific community due to its unique technical advantages. Various aptamers against different targets have been successively reported, including Vibrio parahaemolyticus (Duan et al., 2012), hepatocellular carcinoma (Xie et al., 2014), Shigella sonnei (Song et al., 2017), Streptococcus pyogenes (Huang et al., 2018), furaneol (Komarova et al., 2018), ochratoxin A (Hou et al., 2019), methicillin-resistant Staphylococcus aureus (A Ocsoy et al., 2021; Ocsoy et al., 2017; Turek et al., 2013), tramadol hydrochloride (Hedayati et al., 2021) and RNase H2 from Clostridium difficile (Li et al., 2021). To our knowledge, however, the aptamer targeting harmful algal species has not been reported. Thus, in this study, the aptamer against P. minimum was generated from the random library by cell-SELEX, in which P. minimum and P. minimum-closely related species, including Prorocentrum donghaiense, Prorocentrum lima and Prorocentrum micans, were used as target and counter-screening cells, respectively. The sequences with high frequency in the high-throughput sequenced ssDNA library that was obtained in the final round of SELEX screening were first selected. Then, the secondary structures of these sequences were predicted and analyzed. Next, the affinity and specificity of the selected aptamer were verified. Finally, the binding of the aptamer to P. minimum was confirmed by fluorescence microscope examination. The obtained aptamers could bind to P. minimum with high specificity and affinity, which may be used as a novel recognition element of P. minimum and can be employed to establish a new detection method for the harmful algae in future studies. More importantly, this study is the first report for screening aptamer for harmful algae, providing a reference for screening aptamer targeting other harmful algae.