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Screen and Optimization of an Aptamer for Alexandrium tamarense—A Common Toxin-Producing Harmful Alga

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Abstract

Among all the paralytic shellfish toxins (PSTs)-producing algae, Alexandrium tamarense is one of the most widespread harmful species posing a serious threat to marine resources and human health. Therefore, it is extremely important to establish a rapid and accurate monitoring method for A. tamarense that can provide early warnings of harmful algal blooms (HABs) caused by this alga and limit the contamination due to PSTs. In this study, an ssDNA library was first obtained by whole cell systematic evolution of ligands by exponential enrichment after 18 consecutive rounds of iterative screening. After sequencing in combination with subsequent multiple alignment of sequences and secondary structure simulation, the library could be classified into 2 families, namely, Family1 and Family2, according to sequence similarity. Flow cytometry was used to test the affinity and cross-reactivity of Ata19, Ata6, Ata25 and Ata29 belonging to Family2. Ata19 was selected to be modified by truncation, through which a new resultant aptamer named as Ata19-1-1 was obtained. Ata19-1-1 with a KD of 75.16 ± 11.10 nM displayed a much higher affinity than Ata19. The specificity test showed that Ata19-1-1 has the same discrimination ability as Ata19 and can at least distinguish the target microalga from other microalgae. The observation under a fluorescence microscopy showed that the A. tamarense cells labeled with Ata19-1-1 are exhibiting bright green fluorescence and could be easily identified, factually confirming the binding of the aptamer with target cells. In summary, the aptamer Ata19-1-1 produced in this study may serve as an ideal molecular recognition element for A. tamarense, which has the potential to be developed into a novel detection method for this harmful alga in the future.

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References

  • Alshehri MA (2020) Identification of algae species using advanced molecular techniques. Int J Pharm Res Allied Sci 9:142–159

    Google Scholar 

  • Amorim CA, Moura ADN (2021) Ecological impacts of freshwater algal blooms on water quality, plankton biodiversity, structure, and ecosystem functioning. Sci Total Environ 758:143605

    Article  CAS  PubMed  Google Scholar 

  • Avci-Adali M, Paul A, Wilhelm N, Ziemer G, Wendel HP (2010) Upgrading SELEX technology by using lambda exonuclease digestion for single-stranded DNA generation. Molecules 15:1–11

    Article  CAS  Google Scholar 

  • Bakhtiari H, Palizban AA, Khanahmad H, Mofid MR (2021) Novel approach to overcome defects of Cell-SELEX in developing aptamers against aspartate β−hydroxylase. ACS Omega 6:11005–11014

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Balaji-Prasath B, Wang Y, Su YP, Hamilton DP, Lin H, Zheng L, Zhang Y (2022) Methods to control harmful algal blooms: a review. Environ Chem Lett 20:3133–3152

    Article  CAS  Google Scholar 

  • Bazzoni AM, Cangini M, Mudadu AG, Lorenzoni G, Arras I, Sanna G, Pino F, Milandri A, Virgilio S (2020) Recent findings of paralytic shellfish toxins linked to the genus Alexandrium halim in mediterranean mollusc production areas. Toxicon 174:48–56

    Article  CAS  PubMed  Google Scholar 

  • Bianchi VA, Bickmeyer U, Tillmann U, Krock B, Müller A, Abele D (2021) In vitro effects of paralytic shellfish toxins and lytic extracellular compounds produced by Alexandrium strains on hemocyte integrity and function in mytilus edulis. Toxins 13:544

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Blind M, Blank M (2015) Aptamer selection technology and recent advances. Mol Ther Nucleic Acids 4:223

    Article  Google Scholar 

  • Chinnappan R, Alzabn R, Mir TA, Bader M, Zourob M (2019) Fluorometric determination of okadaic acid using a truncated aptamer. Microchim Acta 186:406

    Article  Google Scholar 

  • Cowperthwaite MC, Ellington AD (2008) Bioinformatic analysis of the contribution of primer sequences to aptamer structures. J Mol Evol 67:95–102

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • de Bustos A, Figueroa RI, Sixto M, Bravo I, Cuadrado Á (2020) The 5S rRNA genes in Alexandrium: their use as a FISH chromosomal marker in studies of the diversity, cell cycle and sexuality of dinoflagellates. Harmful Algae 98:101903

    Article  PubMed  Google Scholar 

  • de Melo MIA, Da Silva Cunha P, de Miranda MC, Barbosa JL, Faria JAQA, Rodrigues MA, de Goes AM, Gomes DA (2021) Selection of DNA aptamers for differentiation of human adipose−derived mesenchymal stem cells from fibroblasts. Appl Biochem Biotechnol 193:3704–3718

    Article  PubMed  Google Scholar 

  • Diercks S, Medlin LK, Metfies K (2008) Colorimetric detection of the toxic dinoflagellate Alexandrium minutum using sandwich hybridization in a microtiter plate assay. Harmful Algae 7:137–145

    Article  CAS  Google Scholar 

  • Du YP, Liu D, Wang M, Guo FK, Lin JS (2021) Preparation of DNA aptamer and development of lateral flow aptasensor combining recombinase polymerase amplification for detection of erythromycin. Biosens Bioelectron 181:113157

    Article  CAS  PubMed  Google Scholar 

  • Dyhrman ST, Erdner D, Du JL, Galac M, Anderson DM (2006) Molecular quantification of toxic Alexandrium fundyense in the Gulf of Maine using real-time PCR. Harmful Algae 5:242–250

    Article  CAS  Google Scholar 

  • Ellington AD, Szostak JW (1990) In vitro selection of RNA molecules that bind specific ligands. Nature 346:818–822

    Article  CAS  PubMed  Google Scholar 

  • Gu HF, Wu YR, Lü SH, Lu DT, Tang YZ, Qi YZ (2022) Emerging harmful algal bloom species over the last four decades in China. Harmful Algae 111:102059

    Article  PubMed  Google Scholar 

  • Guillard RRL (1975) Culture of phytoplankton for feeding marine invertebrates. In culture of marine invertebrate animals 29–60. Springer

  • Guo WF, Zhang CX, Ma TT, Liu XY, Chen Z, Li S, Deng Y (2021) Advances in aptamer screening and aptasensors’ detection of heavy metal ions. J Nanobiotechnol 19:166

    Article  Google Scholar 

  • Helander KG (1999) Formaldehyde binding in brain and kidney: a kinetic study of fixation. J Histotechnol 22:317–318

    Article  Google Scholar 

  • Huang J, Chen XX, Fu XS, Li Z, Huang YH, Liang CL (2021) Advances in aptamer−based biomarker discovery. Front Cell Dev Biol 9:659760

    Article  PubMed  PubMed Central  Google Scholar 

  • Jia M, Sha J, Li Z, Wang W, Zhang H (2020) High affinity truncated aptamers for ultra−sensitive colorimetric detection of bisphenol a with label−free aptasensor. Food Chem 317:126459

    Article  CAS  PubMed  Google Scholar 

  • Khan RM, Salehi B, Mahdianpari M, Mohammadimanesh F, Mountrakis G, Quackenbush LJ (2021) A meta−analysis on harmful algal bloom (HAB) detection and monitoring: a remote sensing perspective. Remote Sens Basel 13:4347

    Article  Google Scholar 

  • Kim C, Sako Y (2005) Molecular identification of toxic Alexandrium tamiyavanichii (Dinophyceae) using two DNA probes. Harmful Algae 4:984–991

    Article  CAS  Google Scholar 

  • Kong QQ, Yue FL, Liu MY, Huang JC, Yang FZ, Liu JJ, Li JS, Li FL, Sun X, Guo YM, Zhu YL (2022) Non−immobilized GO-SELEX of aptamers for label−free detection of thiamethoxam in vegetables. Anal Chim Acta 1202:339677

    Article  CAS  PubMed  Google Scholar 

  • Li DL, Liu LY, Huang QL, Tong T, Zhou Y, Li ZY, Bai QQ, Liang H, Chen LL (2021) Recent advances on aptamer−based biosensors for detection of pathogenic bacteria. World J Microbiol Biotechnol 37:45

    Article  PubMed  Google Scholar 

  • Li YN, Liu XX, Hou YY, Wu Q, Hou JZ (2022) A higher affinity melamine binding aptamer mutant for more sensitive detection. ChemistrySelect 7:30

  • Li Z, Hu B, Zhou R, Zhang XJ, Wang RY, Gao Y, Sun MJ, Jiao BH, Wang LH (2020) Selection and application of aptamers with high affinity and high specificity against dinophysistoxin−1. Rsc Adv 10:8181–8189

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Liang CL, Li FF, Wang LY, Zhang ZC, Wang C, He B, Li J, Chen Z, Shaikh AB, Liu J, Wu X, Peng S, Dang L, Guo B, He X, Au DWT, Lu C, Zhu HL, Zhang G (2017) Tumor cell-targeted delivery of CRISPR/Cas9 by aptamer−functionalized lipopolymer for therapeutic genome editing of VEGFA in osteosarcoma. Biomaterials 147:68–85

    Article  CAS  PubMed  Google Scholar 

  • Liu FG, Zhang CY, Duan Y, Ma JJ, Wang YY, Chen GF (2022a) In vitro selection and characterization of a DNA aptamer targeted to Prorocentrum minimum−a common harmful algae. Sci Total Environ 830:154771

    Article  CAS  PubMed  Google Scholar 

  • Liu S, Xu YY, Jiang X, Tan H, Wu BY (2022b) Translation of aptamers toward clinical diagnosis and commercialization. Biosens Bioelectron 208:114168

    Article  CAS  PubMed  Google Scholar 

  • Lokesh GL, Wang H, Lam CH, Thiviyanathan V, Ward N, Gorenstein DG, Volk DE (2017) X−aptamer selection and validation. Methods Mol Biol 1632:151–174

    Article  CAS  PubMed  Google Scholar 

  • Ma X, Song L, Zhou N (2017) A novel aptasensor for the colorimetric detection of S. typhimurium based on gold nanoparticles. Int J Food Microbiol 245:1–5

    Article  CAS  PubMed  Google Scholar 

  • Maxam AM, Gilbert W (1977) A new method for sequencing DNA. Proc Natl Acad Sci 74:560–564

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Mirian M, Kouhpayeh S, Shariati L, Boshtam M, Rahimmanesh I, Darzi L, Taghizadeh R, Jahanian-Najafabadi A, Khanahmad H (2021) Generation of HBsAg DNA aptamer using modified cell-based SELEX strategy. Mol Biol Rep 48:139–146

    Article  CAS  PubMed  Google Scholar 

  • Morris KN, Jensen KB, Julin CM, Weil M, Gold L (1998) High affinity ligands from in vitro selection: complex targets. Proc Natl Acad Sci 95:2902–2907

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Musumeci D, Montesarchio D (2012) Polyvalent nucleic acid aptamers and modulation of their activity: a focus on the thrombin binding aptamer. Pharmacol Ther 136:202–215

    Article  CAS  PubMed  Google Scholar 

  • Ni S, Zhuo Z, Pan Y, Yu Y, Li F, Liu J, Wang L, Wu X, Li D, Wan Y, Zhang L, Yang Z, Zhang B, Lu A, Zhang G (2021) Recent progress in aptamer discoveries and modifications for therapeutic applications. Acs Appl Mater Inter 13:9500–9519

    Article  CAS  Google Scholar 

  • Ninomiya K, Kaneda K, Kawashima S, Miyachi Y, Ogino C, Shimizu N (2013) Cell-SELEX based selection and characterization of DNA aptamer recognizing human hepatocarcinoma. Bioorg Med Chem Lett 23:1797–1802

    Article  CAS  PubMed  Google Scholar 

  • Nooranian S, Mohammadinejad A, Mohajeri T, Aleyaghoob G, Kazemi Oskuee R (2021) Biosensors based on aptamer−conjugated gold nanoparticles: a review. Biotechnol Appl Bioc 69:1517–1534

    Article  Google Scholar 

  • Pagratis NC, Bell C, Chang YF, Jennings S, Fitzwater T, Jellinek D, Dang C (1997) Potent 2’−amino−, and 2’−fluoro−2’−deoxyribonucleotide RNA inhibitors of keratinocyte growth factor. Nat Biotechnol 15:68–73

    Article  CAS  PubMed  Google Scholar 

  • Paredes-Mella J, Mardones JI, Norambuena L, Fuenzalida G, Labra G, Espinoza-González O, Guzmán L (2021) Toxic Alexandrium catenella expanding northward along the Chilean coast: new risk of paralytic shellfish poisoning off the Bío−Bío region (36° S). Mar Pollut Bull 172:112783

    Article  CAS  PubMed  Google Scholar 

  • Paul A, Avci-Adali M, Ziemer G, Wendel HP (2009) Streptavidin−coated magnetic beads for DNA strand separation implicate a multitude of problems during Cell-SELEX. Oligonucleotides 19:243–254

    Article  CAS  PubMed  Google Scholar 

  • Roy D, Pascher A, Juratli MA, Sporn JC (2021) The potential of aptamer−mediated liquid biopsy for early detection of cancer. Int J Mol Sci 22:5601

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Saito S (2021) SELEX-based DNA aptamer selection: a perspective from the advancement of separation techniques. Anal Sci 37:17–26

    Article  CAS  PubMed  Google Scholar 

  • Sajid M, Kawde AN, Daud M (2015) Designs, formats and applications of lateral flow assay: a literature review. J Saudi Chem Soc 19:689–705

    Article  Google Scholar 

  • Sari AK, Gaffar S, Hartati YW (2022) A review on the development of aptamer immobilization techniques in aptamer−based electrochemical biosensors for viruses detection. Anal Bioanal Electrochem 14:127–143

    CAS  Google Scholar 

  • Sefah K, Shangguan DH, Xiong X, Tan WH (2010) Development of DNA aptamers using Cell-SELEX. Nat Protoc 5:1169–1185

    Article  CAS  PubMed  Google Scholar 

  • Tang WJ, Lin ZL, Zhang QC, Geng HX, Sun HX, Tang XX, Yu RC (2022) An investigation on bloom dynamics of Alexandrium catenella and A. pacificum and toxin accumulation in shellfish along the coast of Qinhuangdao, China. Mar Pollut Bull 183:114058

  • Tapp M, Dennis P, Naik RR, Milam VT (2021) Competition−enhanced ligand selection to screen for DNA aptamers for spherical gold nanoparticles. Langmuir 37:9043–9052

    Article  CAS  PubMed  Google Scholar 

  • Thevendran R, Citartan M (2022) Assays to estimate the binding affinity of aptamers. Talanta 238:122971

    Article  CAS  PubMed  Google Scholar 

  • Tuerk C, Gold L (1990) Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 249:505–510

    Article  CAS  PubMed  Google Scholar 

  • Wang T, Chen CY, Larcher LM, Barrero RA, Veedu RN (2019) Three decades of nucleic acid aptamer technologies: lessons learned, progress and opportunities on aptamer development. Biotechnol Adv 37:28–50

    Article  CAS  PubMed  Google Scholar 

  • Xiang DX, Shigdar S, Qiao G, Wang T, Kouzani AZ, Zhou S, Kong L, Li Y, Pu C, Duan W (2015) Nucleic acid aptamer−guided cancer therapeutics and diagnostics: the next generation of cancer medicine. Theranostics 5:23–42

    Article  PubMed  PubMed Central  Google Scholar 

  • Xing LG, Zhao Y, Gong M, Liu X, Zhang Y, Li D, He Z, Yan P, Yang J (2021) Graphene oxide and lambda exonuclease assisted screening of L−carnitine aptamers and the site−directed mutagenesis design of C−rich structure aptamer. Biochem Bioph Res Co 545:171–176

    Article  CAS  Google Scholar 

  • Yadavalli T, Volety I, Shukla D (2021) Aptamers in virology−a consolidated review of the most recent advancements in diagnosis and therapy. Pharmaceutics 13:1646

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ye H, Yang ZX, Khan IM, Niazi S, Guo YX, Wang ZP (2022) Split aptamer acquisition mechanisms and current application in antibiotics detection: a short review. Crit Rev Food Sci Nutr 62:1–12

    PubMed  Google Scholar 

  • Yeoh TS, Anna A, Tang T-H, Citartan M (2022) Development of an optimization pipeline of asymmetric PCR towards the generation of DNA aptamers: a guide for beginners. World J Microbiol Biotechnol 38:31

    Article  CAS  PubMed  Google Scholar 

  • Yu HX, Alkhamis O, Canoura J, Liu YZ, Xiao Y (2021a) Advances and challenges in small−molecule DNA aptamer isolation, characterization, and sensor development. Angew Chem Int Edit 60:16800–16823

    Article  CAS  Google Scholar 

  • Yu RC, Zhang QC, Liu Y, Chen ZF, Geng HX, Dai L, Lin ZR, Tang WJ, Kong FZ, Yan T, Zhou MJ (2021b) The dinoflagellate Alexandrium catenella producing only carbamate toxins may account for the seafood poisonings in Qinhuangdao. China Harmful Algae 103:101980

  • Zhong Y, Zhao JY, Li JZ, Liao X, Chen FL (2020) Advances of aptamers screened by Cell-SELEX in selection procedure, cancer diagnostics and therapeutics. Anal Biochem 598:113620

  • Zhou J, Soontornworajit B, Snipes MP, Wang Y (2011) Structural prediction and binding analysis of hybridized aptamers. J Mol Recognit 24:119–126

    Article  CAS  PubMed  Google Scholar 

  • Zou XR, Wu J, Gu JQ, Shen L, Mao LX (2021) DNA aptamer against EV−A71 VP1 protein: selection and application. Virol J 18:164

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Funding

This work was supported by Shandong Provincial Natural Science Foundation, China (ZR202204170002, ZR2020MD081); HIT Scientific Research Innovation Fund (No. 2022KYCXJJ07); National Science Foundation of China (No. 42377422); and “Double First Class” Discipline Construction Fundation of HIT (2023SYLHY06).

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Authors and Affiliations

  1. School of Marine Science and Technology, Harbin Institute of Technology (Weihai), Wenhua West Road, 2#, Weihai, 264209, People’s Republic of China

    Yu Duan, Fuguo Liu, Chunyun Zhang, Yuanyuan Wang & Guofu Chen

  2. School of Environment, Harbin Institute of Technology, Harbin, 150001, People’s Republic of China

    Yu Duan, Fuguo Liu & Guofu Chen

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  1. Yu Duan
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  2. Fuguo Liu
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  3. Chunyun Zhang
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  4. Yuanyuan Wang
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  5. Guofu Chen
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Contributions

Yu Duan: Conceptualization, Methodology, Formal analysis, Writing-Original Draft. Fuguo Liu: Data Curation, Formal analysis, Conceptualization. Chunyun Zhang: Conceptualization, Supervision, Writing-Review & Editing. Yuanyuan Wang: Conceptualization, Formal analysis, Writing-Review & Editing. Guofu Chen: Resources, Supervision, Project administration, Funding acquisition.

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Correspondence to Chunyun Zhang or Guofu Chen.

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Duan, Y., Liu, F., Zhang, C. et al. Screen and Optimization of an Aptamer for Alexandrium tamarense—A Common Toxin-Producing Harmful Alga. Mar Biotechnol 25, 935–950 (2023). https://doi.org/10.1007/s10126-023-10251-2

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