In vitro selection and characterization of a DNA aptamer targeted to Prorocentrum minimum-A common harmful algae
Graphical abstract
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.
Section snippets
Algal culture
All of the microalgal cultures used in this study and their geographical origin are summarized in Table S1. The microalgae were cultured using f/2 or f/2-silicon medium in 500 mL triangular flasks (Guillard, 1975). The natural seawater that was harvested from Jinhai Bay, Weihai, Shandong Province was filtered and autoclaved to prepare the medium, which the pore size of the membrane used to filter seawater is 0.45 μm. The culture conditions were as follows: light intensity, 3000–8000 lx;
Optimization of conditions of preparative PCR
To obtain a specific and optimal amplification signal, the main PCR conditions for the preparation of the secondary library including annealing temperature and primer concentration ratio (Fp to Rp) were successively optimized by fixing other factors. The electrophoresis analysis of PCR products from different annealing temperatures and primer concentration ratios are shown in Fig. S1. All the PCR products from different annealing temperatures displayed a similar band pattern. However, the
Discussion
As a toxin-producing harmful alga, P. minimum is also one of the most common HABs-forming species. To date, a variety of detection techniques have been developed for P. minimum, including fluorescence in situ hybridization (FISH) (Hou et al., 2009), SHA (Zhu et al., 2012; Cai et al., 2006), qPCR (Zhang et al., 2014), rolling circle amplification (RCA) (Liu et al., 2020a) and recombinase polymerase amplification (RPA) (Fu et al., 2020). Although FISH can directly detect target cells by employing
Conclusion
Aptamer is an ssDNA or RNA that can able to bind to a specific target (small molecules, proteins and cells) with high affinity and specificity. In the study, we used the cell-SELEX and obtained the aptamer against P. minimum, common harmful algae which can endanger marine organisms and human health because it can produce toxins. Obtained ssDNA pools were sequenced and divided into different groups based on their primary sequence homology and secondary structure. The sequences with the highest
CRediT authorship contribution statement
Fuguo Liu: Conceptualization, Methodology, Formal analysis, Writing - Original Draft
Chunyun Zhang: Conceptualization, Supervision, Writing - Review & Editing
Yu Duan: Data Curation, Formal analysis, Investigation
Jinju Ma: Data Curation, Formal analysis, Conceptualization
Yuanyuan Wang: Conceptualization, Formal analysis, Writing - Review & Editing
Guofu Chen: Resources, Supervision, Formal analysis, Project administration, Funding acquisition
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
This work was supported by Shandong Provincial Natural Science Foundation, China (ZR2020MD081); the National Science Foundation of China (No. 31600309, 41476086); HIT Scientific Research Innovation Fund/the Fundamental Research Funds for the Central Universities (No. HIT.NSRIF.201702 and HIT.NSRIF.201709); and HIT Environment and Ecology Innovation Special Funds (No. HSCJ201622).
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