The identification of single strand DNA aptamers which specifically bind to platelets using cell-SELEX technique

Document Type : Research Article


1 Division of Biotechnology, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran.

2 Immunology Research Center, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.

3 Division of Biotechnology, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran & Research Institute of Biotechnology, Ferdowsi University of Mashhad, Mashhad, Iran.

4 Department of Pathobiology, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran & Research Institute of Biotechnology, Ferdowsi University of Mashhad, Mashhad, Iran.


        Aptamers are oligonucleotides that can be easily synthesized and bind to their targets with high affinity and specificity. Several aptamers specific to soluble factors of coagulation cascade have been produced, however, aptamers specific to platelet cell membrane molecules have not been reported yet. We aimed to discover DNA aptamers that specifically bind to human platelets. The cell-SELEX method was used for aptamer discovery. Synthetic 79 nucleotides length single-strand oligonucleotides were used as a library. Ultra-pure platelets were prepared using differential centrifugation steps and magnetic-bead-assisted removal of contaminating cells. The FITC-labeled forward primer was used for amplification of the selected oligonucleotides by PCR, and Lambda exonuclease was used for digestion of the lagging strand. After 12 rounds of cell-SELEX, selected oligos were amplified and cloned to pTG19-T vector, transfected into E. coli  (TOP10) and sequenced. Sequences of aptamers from 200 individual positive colonies were aligned and seven clusters were identified. Representative aptamers were amplified and their affinity, specificity, and digestibility of their targets were evaluated. Interferences of the aptamers to two platelet function tests were also investigated. Affinity (KD) of the representative aptamers were between 109 and 340 nM. Trypsin exposure of the platelets completely abolished the binding of the 7 aptamers to the targets. The binding of the four aptamers fully protected their target molecules from digestion. No one of the aptamers changed the parameters of the platelet function tests. Seven aptamers specific to platelets were identified and characterized. These aptamers may have potentially diverse applications in the diagnosis or treatment of platelet disorders.


1.    Ni X, Castanares M, Mukherjee A, Lupold SE. Nucleic acid aptamers: clinical applications and promising new horizons. Curr Med Chem. 2011;18(27):4206-14.
2.    Xiang D, Zheng C, Zhou SF, Qiao S, Tran PH, Pu C, et al. Superior Performance of Aptamer in Tumor Penetration over Antibody: Implication of Aptamer-Based Theranostics in Solid Tumors. Theranostics. 2015;5(10):1083-97.
3.    Xiang D, Shigdar S, Qiao G, Wang T, Kouzani AZ, Zhou SF, et al. Nucleic acid aptamer-guided cancer therapeutics and diagnostics: the next generation of cancer medicine. Theranostics. 2015;5(1):23-42.
4.    Sefah K, Meng L, Lopez-Colon D, Jimenez E, Liu C, Tan W. DNA aptamers as molecular probes for colorectal cancer study. PLoS One. 2010;5(12):e14269.
5.    Sefah K, Shangguan D, Xiong X, O'Donoghue MB, Tan W. Development of DNA aptamers using Cell-SELEX. Nat Protoc. 2010;5(6):1169-85.
6.    Ellington AD, Szostak JW. Selection in vitro of single-stranded DNA molecules that fold into specific ligand-binding structures. Nature. 1992;355(6363):850-2.
7.    Chen M, Yu Y, Jiang F, Zhou J, Li Y, Liang C, et al. Development of Cell-SELEX Technology and Its Application in Cancer Diagnosis and Therapy. Int J Mol Sci. 2016;17(12).
8.    Ringquist S, Jones T, Snyder EE, Gibson T, Boni I, Gold L. High-affinity RNA ligands to Escherichia coli ribosomes and ribosomal protein S1: comparison of natural and unnatural binding sites. Biochemistry. 1995;34(11):3640-8.
9.    Morris KN, Jensen KB, Julin CM, Weil M, Gold L. High affinity ligands from in vitro selection: complex targets. Proc Natl Acad Sci U S A. 1998;95(6):2902-7.
10.    Shangguan D, Li Y, Tang Z, Cao ZC, Chen HW, Mallikaratchy P, et al. Aptamers evolved from live cells as effective molecular probes for cancer study. Proc Natl Acad Sci U S A. 2006;103(32):11838-43.
11.    Gold L, Ayers D, Bertino J, Bock C, Bock A, Brody EN, et al. Aptamer-based multiplexed proteomic technology for biomarker discovery. PLoS One. 2010;5(12):e15004.
12.    Yang M, Jiang G, Li W, Qiu K, Zhang M, Carter CM, et al. Developing aptamer probes for acute myelogenous leukemia detection and surface protein biomarker discovery. J Hematol Oncol. 2014;7:5.
13.    Nimjee SM, Lohrmann JD, Wang H, Snyder DJ, Cummings TJ, Becker RC, et al. Rapidly regulating platelet activity in vivo with an antidote controlled platelet inhibitor. Mol Ther. 2012;20(2):391-7.
14.    Chang YM, Donovan MJ, Tan W. Using aptamers for cancer biomarker discovery. J Nucleic Acids. 2013; 817350.
15.    Forier C, Boschetti E, Ouhammouch M, Cibiel A, Duconge F, Nogre M, et al. DNA aptamer affinity ligands for highly selective purification of human plasma-related proteins from multiple sources. J Chromatogr A. 2017;1489:39-50.
16.    Sypabekova M, Bekmurzayeva A, Wang R, Li Y, Nogues C, Kanayeva D. Selection, characterization, and application of DNA aptamers for detection of Mycobacterium tuberculosis secreted protein MPT64. Tuberculosis (Edinb). 2017;104:70-8.
17.    Wu Q, Wu L, Wang Y, Zhu Z, Song Y, Tan Y, et al. Evolution of DNA aptamers for malignant brain tumor gliosarcoma cell recognition and clinical tissue imaging. Biosens Bioelectron. 2016;80:1-8.
18.    Mehennaoui S, Poorahong S, Jimenez GC, Siaj M. Selection of high affinity aptamer-ligand for dexamethasone and its electrochemical biosensor. Sci Rep. 2019;9(1):6600.
19.    Floege J, Ostendorf T, Janssen U, Burg M, Radeke HH, Vargeese C, et al. Novel approach to specific growth factor inhibition in vivo: antagonism of platelet-derived growth factor in glomerulonephritis by aptamers. Am J Pathol. 1999;154(1):169-79.
20.    Zheng X, Hu B, Gao SX, Liu DJ, Sun MJ, Jiao BH, et al. A saxitoxin-binding aptamer with higher affinity and inhibitory activity optimized by rational site-directed mutagenesis and truncation. Toxicon. 2015;101:41-7.
21.    Krishnegowda M, Rajashekaraiah V. Platelet disorders: an overview. Blood Coagul Fibrinolysis. 2015;26(5):479-91.
22.    Lam FW, Vijayan KV, Rumbaut RE. Platelets and Their Interactions with Other Immune Cells. Compr Physiol. 2015;5(3):1265-80.
23.    Liu M, Zaman K, Fortenberry YM. Overview of the Therapeutic Potential of Aptamers Targeting Coagulation Factors. Int J Mol Sci. 2021;22(8).
24.    Amisten S. A rapid and efficient platelet purification protocol for platelet gene expression studies. Methods Mol Biol. 2012;788:155-72.
25.    Wrzyszcz A, Urbaniak J, Sapa A, Wozniak M. An efficient method for isolation of representative and contamination-free population of blood platelets for proteomic studies. Platelets. 2017;28(1):43-53.
26.    Schror K. Aspirin and platelets: the antiplatelet action of aspirin and its role in thrombosis treatment and prophylaxis. Semin Thromb Hemost. 1997;23(4):349-56.
27.    Friedman EA, Ogletree ML, Haddad EV, Boutaud O. Understanding the role of prostaglandin E2 in regulating human platelet activity in health and disease. Thromb Res. 2015;136(3):493-503.
28.    Zhuo Z, Yu Y, Wang M, Li J, Zhang Z, Liu J, et al. Recent Advances in SELEX Technology and Aptamer Applications in Biomedicine. Int J Mol Sci. 2017;18(10).
29.    Ohuchi S. Cell-SELEX Technology. Biores Open Access. 2012;1(6):265-72.
30.    Thon JN, Italiano JE. Platelet formation. Semin Hematol. 2010;47(3):220-6.
31.    Saboor M, Ayub Q, Ilyas S, Moinuddin. Platelet receptors; an instrumental of platelet physiology. Pak J Med Sci. 2013;29(3):891-6.
32.    Bury L, Malara A, Momi S, Petito E, Balduini A, Gresele P. Mechanisms of thrombocytopenia in platelet-type von Willebrand disease. Haematologica. 2019;104(7):1473-81.
33.    Ayabe K, Goto S. Is there a 'therapeutic window' for antiplatelet therapy? Eur Heart J Cardiovasc Pharmacother. 2017;3(1):18-20.
34.    Marginean A, Banescu C, Scridon A, Dobreanu M. Anti-platelet Therapy Resistance - Concept, Mechanisms and Platelet Function Tests in Intensive Care Facilities. J Crit Care Med (Targu Mures). 2016;2(1):6-15.
35.    Oney S, Nimjee SM, Layzer J, Que-Gewirth N, Ginsburg D, Becker RC, et al. Antidote-controlled platelet inhibition targeting von Willebrand factor with aptamers. Oligonucleotides. 2007;17(3):265-74.
36.    McKeague M, Derosa MC. Challenges and opportunities for small molecule aptamer development. J Nucleic Acids. 2012;2012:748913.
37.    Bibby DF, Gill AC, Kirby L, Farquhar CF, Bruce ME, Garson JA. Application of a novel in vitro selection technique to isolate and characterise high affinity DNA aptamers binding mammalian prion proteins. J Virol Methods. 2008;151(1):107-15.
38.    Chen Z, Liu H, Jain A, Zhang L, Liu C, Cheng K. Discovery of Aptamer Ligands for Hepatic Stellate Cells Using SELEX. Theranostics. 2017;7(12):2982-95.
39.    Duan Y, Wang L, Gao Z, Wang H, Zhang H, Li H. An aptamer-based effective method for highly sensitive detection of chloramphenicol residues in animal-sourced food using real-time fluorescent quantitative PCR. Talanta. 2017;165:671-6.
40.    Tang XL, Wu SM, Xie Y, Song N, Guan Q, Yuan C, et al. Generation and application of ssDNA aptamers against glycolipid antigen ManLAM of Mycobacterium tuberculosis for TB diagnosis. J Infect. 2016;72(5):573-86.
41.    Berg K, Lange T, Mittelberger F, Schumacher U, Hahn U. Selection and Characterization of an alpha6beta4 Integrin blocking DNA Aptamer. Mol Ther Nucleic Acids. 2016;5:e294.
42.    Zhou L, Li P, Yang M, Yu Y, Huang Y, Wei J, et al. Generation and characterization of novel DNA aptamers against coat protein of grouper nervous necrosis virus (GNNV) with antiviral activities and delivery potential in grouper cells. Antiviral Res. 2016;129:104-14.
43.    Spiga FM, Maietta P, Guiducci C. More DNA-Aptamers for Small Drugs: A Capture-SELEX Coupled with Surface Plasmon Resonance and High-Throughput Sequencing. ACS Comb Sci. 2015;17(5):326-33.
44.    Baig IA, Moon JY, Lee SC, Ryoo SW, Yoon MY. Development of ssDNA aptamers as potent inhibitors of Mycobacterium tuberculosis acetohydroxyacid synthase. Biochim Biophys Acta. 2015;1854(10 Pt A):1338-50.
45.    Moon J, Kim G, Park SB, Lim J, Mo C. Comparison of whole-cell SELEX methods for the identification of Staphylococcus aureus-specific DNA aptamers. Sensors (Basel). 2015;15(4):8884-97.
46.    Moosavian SA, Jaafari MR, Taghdisi SM, Mosaffa F, Badiee A, Abnous K. Development of RNA aptamers as molecular probes for HER2(+) breast cancer study using cell-SELEX. Iran J Basic Med Sci. 2015;18(6):576-86.
47.    Mozioglu E, Gokmen O, Tamerler C, Kocagoz ZT, Akgoz M. Selection of Nucleic Acid Aptamers Specific for Mycobacterium tuberculosis. Appl Biochem Biotechnol. 2016;178(4):849-64.
48.    Mayer G, Hover T. In vitro selection of ssDNA aptamers using biotinylated target proteins. Methods Mol Biol. 2009;535:19-32.
49.    Jin YR, Ryu CK, Moon CK, Cho MR, Yun YP. Inhibitory effects of J78, a newly synthesized 1,4-naphthoquinone derivative, on experimental thrombosis and platelet aggregation. Pharmacology. 2004;70(4):195-200.
50.    Trowbridge IS, Johnson P, Ostergaard H, Hole N. Structure and function of CD45: a leukocyte-specific protein tyrosine phosphatase. Adv Exp Med Biol. 1992;323:29-37.
51.    Ramstrom S, O'Neill S, Dunne E, Kenny D. Annexin V binding to platelets is agonist, time and temperature dependent. Platelets. 2010;21(4):289-96.
52.    Gaillard C, Strauss F. Ethanol precipitation of DNA with linear polyacrylamide as carrier. Nucleic Acids Res. 1990;18(2):378.