In Vitro Selection of a DNA Aptamer by Cell-SELEX as a Molecular Probe for Cervical Cancer Recognition and Imaging

  • Jine Wang
  • Tian Gao
  • Yu Luo
  • Zhili Wang
  • Yajie Zhang
  • Ye Zhang
  • Yuanyuan ZhangEmail author
  • Renjun PeiEmail author
Original Article


Aptamers have become the most promising recognition reagents in terms of early diagnosis and effective treatment of cancers. In this study, using cervical cancer as a model, we have identified a DNA aptamer specifically binding to cervical cancer cells with high affinity using the cell-SELEX (systematic evolution of ligands by exponential enrichment) method, in which a negative selection was carried out using normal epithelial cells as control. The binding abilities of 6 selected truncated aptamers were determined by laser confocal fluorescence microscopy and flow cytometry, while most of them only recognize the target cells and do not bind the control cells, and the aptamer C-9S with 51-mer shows the best binding affinity to Ca Ski cells (target cells) with a dissociation constant value of 19.3 ± 2.9 nM. Moreover, at physiological temperature, C-9S remains its specific recognition capability to Ca Ski cells as well. Meanwhile, C-9S shows a similar binding ability to another cervical cancer cells (HeLa). Therefore, on the basis of its excellent targeting properties and inherent functional versatility of aptamer, C-9S holds great potential to be a molecular probe for early detection, in vivo imaging, and targeted delivery for further researches in cancer.

Graphical Abstract


Aptamer Cell-SELEX Molecular probe Cervical cancer 



The authors would like to thank the National Natural Science Foundation of China (Nos. 21775160, 21575154), the International Partnership Program of Chinese Academy of Sciences (No. 121E32KYSB20170025), the Science Foundation of Jiangsu Province (Nos. BE2016680, BK20161262, BE2018665, EK20180250), and the Jiangsu Province Six Talent Peaks program for financial support.

Supplementary material

239_2019_9886_MOESM1_ESM.docx (645 kb)
Supplementary material 1 (DOCX 644 KB)


  1. Boshart M, Gissmann L, Ikenberg H, Kleinheinz A, Scheurlen W, zur Hausen H (1984) A new type of papillomavirus DNA, its presence in genital cancer biopsies and in cell lines derived from cervical cancer. EMBO J 3:1151–1157PubMedPubMedCentralCrossRefGoogle Scholar
  2. Cerchia L, Duconge F, Pestourie C, Boulay J, Aissouni Y, Gombert K, Tavitian B, de Franciscis V, Libri D (2005) Neutralizing aptamers from whole-cell SELEX inhibit the RET receptor tyrosine kinase. PLoS Biol 3:e123PubMedPubMedCentralCrossRefGoogle Scholar
  3. Chang YM, Donovan MJ, Tan W (2013) Using aptamers for cancer biomarker discovery. J Nucleic Acids 2013: 817350PubMedPubMedCentralCrossRefGoogle Scholar
  4. Chen HW, Medley CD, Sefah K, Shangguan D, Tang Z, Meng L, Smith JE, Tan W (2008) Molecular recognition of small-cell lung cancer cells using aptamers. ChemMedChem 3:991–1001PubMedPubMedCentralCrossRefGoogle Scholar
  5. Daniels DA, Chen H, Hicke BJ, Swiderek KM, Gold L (2003) A tenascin-C aptamer identified by tumor cell SELEX: systematic evolution of ligands by exponential enrichment. Proc Natl Acad Sci USA 100:15416–15421PubMedCrossRefGoogle Scholar
  6. Ellington AD, Szostak JW (1990) In vitro selection of RNA molecules that bind specific ligands. Nature 346:818–822PubMedCrossRefGoogle Scholar
  7. Engelberg S, Modrejewski J, Walter JG, Livney YD, Assaraf YG (2018) Cancer cell-selective, clathrin-mediated endocytosis of aptamer decorated nanoparticles. Oncotarget 9:20993–21006PubMedPubMedCentralCrossRefGoogle Scholar
  8. Esposito CL, Catuogno S, de Franciscis V, Cerchia L (2011) New insight into clinical development of nucleic acid aptamers. Discov Med 11:487–496PubMedGoogle Scholar
  9. Ferrandina G, Macchia G, Legge F, Deodatod F, Fornib F, Digesud C, Caronec V, Morgantid AG, Scambia G (2008) Squamous cell carcinoma antigen in patients with locally advanced cervical carcinoma undergoing preoperative radiochemotherapy: association with pathological response to treatment and clinical outcome. Oncology 74:42–49PubMedCrossRefGoogle Scholar
  10. Gong P, Sun L, Wang F, Liu X, Yan Z, Wang M, Zhang L, Tian Z, Liu Z, You J (2019) Highly fluorescent N-doped carbon dots with two-photon emission for ultrasensitive detection of tumor marker and visual monitor anticancer drug loading and delivery. Chem Eng J 356:994–1002CrossRefGoogle Scholar
  11. Graham JC, Zarbl H (2012) Use of cell-SELEX to generate DNA aptamers as molecular probes of HPV-associated cervical cancer cells. PLoS ONE 7(4):e36103PubMedPubMedCentralCrossRefGoogle Scholar
  12. Grubisic G (2007) Limitations of colposcopy in early invasive cervical cancer detection. Coll Antropol 31:135–138PubMedGoogle Scholar
  13. Ireson CR, Kelland LR (2006) Discovery and development of anticancer aptamers. Mol Cancer Ther 5:2957–2962PubMedCrossRefGoogle Scholar
  14. Jayasena SD (1999) Aptamers: an emerging class of molecules that rival antibodies in diagnostics. Clin Chem 45:1628–1650PubMedGoogle Scholar
  15. Li X, Zhang W, Liu L, Zhu Z, Ouyang G, An Y, Zhao C, Yang CJ (2014) In vitro selection of DNA aptamers for metastatic breast cancer cell recognition and tissue imaging. Anal Chem 86:6596–6603PubMedCrossRefGoogle Scholar
  16. Li X, An Y, Jin J, Zhu Z, Hao L, Liu L, Shi Y, Fan D, Ji T, Yang CJ (2015) Evolution of DNA aptamers through in vitro metastatic-cell-based systematic evolution of ligands by exponential enrichment for metastatic cancer recognition and imaging. Anal Chem 87:4941–4948PubMedCrossRefGoogle Scholar
  17. Liu K, Lin B, Lan X (2013) Aptamers: a promising tool for cancer imaging, diagnosis, and therapy. J Cell Biochem 114:250–255PubMedCrossRefGoogle Scholar
  18. Lyu Y, Chen G, Shangguan D, Zhang L, Wan S, Wu Y, Zhang H, Duan L, Liu C, You M, Wang J, Tan W (2016) Generating cell targeting aptamers for nanotheranostics using cell-SELEX. Theranostics 6:1440–1452PubMedPubMedCentralCrossRefGoogle Scholar
  19. Meng H-M, Fu T, Zhang X-B, Tan W (2015) Cell-SELEX-based aptamer-conjugated nanomaterials for cancer diagnosis and therapy. Natl Sci Rev 2:71–84CrossRefGoogle Scholar
  20. Mitchell DG, Snyder B, Coakley F, Reinhold C, Thomas G, Amendola M, Schwartz LH, Woodward P, Pannu H, Hricak H (2006) Early invasive cervical cancer: tumor delineation by magnetic resonance imaging, computed tomography, and clinical examination, verified by pathologic results, in the ACRIN 6651/GOG 183 Intergroup Study. J Clin Oncol 24:5687–5694PubMedCrossRefGoogle Scholar
  21. Ohuchi SP, Ohtsu T, Nakamura Y (2006) Selection of RNA aptamers against recombinant transforming growth factor-β type III receptor displayed on cell surface. Biochimie 88:897–904PubMedCrossRefGoogle Scholar
  22. Pang X, Cui C, Wan S, Jiang Y, Zhang L, Xia L, Li L, Li X, Tan W (2018) Bioapplications of cell-SELEX-generated aptamers in cancer diagnostics, therapeutics, theranostics and biomarker discovery: a comprehensive review. Cancers 10:47PubMedCentralCrossRefGoogle Scholar
  23. Sefah K, Shangguan D, Xiong X, O’Donoghue MB, Tan W (2010) Development of DNA aptamers using Cell-SELEX. Nat Protoc 5:1169–1185PubMedCrossRefGoogle Scholar
  24. Shangguan D, Li Y, Tang Z, Cao ZC, Chen HW, Mallikaratchy P, Sefah K, Yang CJ, Tan W (2006) Aptamers evolved from live cells as effective molecular probes for cancer study. Proc Nat Acad Sci USA 103:11838–11843PubMedCrossRefGoogle Scholar
  25. Shangguan D, Tang Z, Mallikaratchy P, Xiao Z, Tan W (2007) Optimization and modifications of aptamers selected from live cancer cell lines. ChemBioChem 8:603–606PubMedCrossRefGoogle Scholar
  26. Shangguan D, Cao Z, Meng L, Mallikaratchy P, Sefah K, Wang H, Li Y, Tan W (2008a) Cell-specific aptamer probes for membrane protein elucidation in cancer cells. J Proteome Res 7:2133–2139PubMedPubMedCentralCrossRefGoogle Scholar
  27. Shangguan D, Meng L, Cao ZC, Xiao Z, Fang X, Li Y, Cardona D, Witek RP, Liu C, Tan W (2008b) Identification of liver cancer-specific aptamers using whole live cells. Anal Chem 80:721–728PubMedCrossRefGoogle Scholar
  28. Shi Y, Zhang J, He J, Liu D, Meng X, Huang T, He H (2019) A method of detecting two tumor markers (p-hydroxybenzoic acid and pcresol) in human urine using a porous magnetic β-cyclodextrine polymer as solid phase extractant, an alternative for early gastric cancer diagnosis. Talanta 191: 133–140.PubMedCrossRefGoogle Scholar
  29. Siegel RL, Miller KD, Jemal A (2015) Cancer statistics, 2015. CA Cancer J Clin 65:5–29PubMedCrossRefGoogle Scholar
  30. Song KM, Lee S, Ban C (2012) Aptamers and their biological applications. Sensors 12:612–631PubMedCrossRefGoogle Scholar
  31. Sun Y, Wang Y, Lau C, Chen G, Lu J (2018) Hybridization-initiated exonuclease resistance strategy for simultaneous detection of multiple microRNAs. Talanta 190:248–254PubMedCrossRefGoogle Scholar
  32. Tang Z, Parekh P, Turner P, Moyer RW, Tan W (2009) Generating aptamers for recognition of virus-infected cells. Clin Chem 55:813–822PubMedCrossRefGoogle Scholar
  33. Tuerk C, Gold L (1990) Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 249:505–510PubMedCrossRefGoogle Scholar
  34. Wang F, Rong Y, Fang M, Yuan J, Peng C, Liu S, Li Y (2013) Recognition and capture of metastatic hepatocellular carcinoma cells using aptamer conjugated quantum dots and magnetic particles. Biomaterials 34:3816–3827PubMedCrossRefGoogle Scholar
  35. Wang J, Zhang Y, Chen Y, Hong S, Sun Y, Sun N, Pei R (2017) In vitro selection of DNA aptamers against renal cell carcinoma using living cell-SELEX. Talanta 175:235–242PubMedCrossRefGoogle Scholar
  36. Wu X, Zhao Z, Bai H, Fu T, Yang C, Hu X, Liu Q, Champanhac C, Teng IT, Ye M, Tan W (2015) DNA aptamer selected against pancreatic ductal adenocarcinoma for in vivo imaging and clinical tissue recognition. Theranostics 5:985–994PubMedPubMedCentralCrossRefGoogle Scholar
  37. Xu J, Teng IT, Zhang L, Delgado S, Champanhac C, Cansiz S, Wu C, Shan H, Tan W (2015) Molecular recognition of human liver cancer cells using DNA aptamers generated via Cell-SELEX. PLoS ONE 10:e0125863PubMedPubMedCentralCrossRefGoogle Scholar
  38. Zhang Y, Chen Y, Han D, Ocsoy I, Tan W (2010) Aptamers selected by cell-SELEX for application in cancer studies. Bioanalysis 2:907–918PubMedPubMedCentralCrossRefGoogle Scholar
  39. Zhou J, Rossi J (2017) Aptamers as targeted therapeutics: current potential and challenges. Nat Rev Drug Discov 16:181–202PubMedCrossRefGoogle Scholar
  40. Zuker M (2003) M-fold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31:3406–3415PubMedPubMedCentralCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.CAS Key Laboratory of Nano-Bio Interface, Suzhou Institute of Nano-Tech and Nano-BionicsChinese Academy of SciencesSuzhouChina
  2. 2.School of Nano Technology and Nano BionicsUniversity of Science and Technology of ChinaHefeiChina
  3. 3.School of Life ScienceAnhui Medical UniversityHefeiChina

Personalised recommendations