Analytical and Bioanalytical Chemistry

, Volume 409, Issue 21, pp 5081–5089 | Cite as

A general double library SELEX strategy for aptamer selection using unmodified nonimmobilized targets

  • Kyung Hyun Lee
  • Huaqiang Zeng
Research Paper


Aptamer discovery for unmodified nonimmobilized targets has been constantly presenting itself as a significant challenge to the research community. We demonstrate here a novel double library (DL) SELEX strategy and its usefulness and generality toward discovering both ssDNA- and RNA-based aptamers with nanomolar binding affinities toward unmodified targets of both small (e.g., doxycycline) and large (e.g., VEGF165) sizes. The same selection strategy further allows for concurrent selection of an aptamer pair, recognizing discrete epitopes on the same protein, from the same selection cycles for the sandwich aptamer pair-based biosensor development (e.g., one aptamer for the recognition and the other for the signal transduction). These results establish the DL-SELEX method developed here as a valuable and highly accessible selection strategy for aptamer discovery, especially when chemical modifications of target molecules are not preferred or simply impossible.


Aptamer SELEX Nucleic acids Nonimmobilization Label-free 



This work was supported by the Institute of Bioengineering and Nanotechnology (Biomedical Research Council, Agency for Science, Technology and Research, Singapore).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

216_2017_454_MOESM1_ESM.pdf (2.3 mb)
ESM 1 (PDF 2382 kb)


  1. 1.
    Tuerk C, Gold L. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science. 1990;249(4968):505–10.CrossRefGoogle Scholar
  2. 2.
    Ellington AD, Szostak JW. In vitro selection of RNA molecules that bind specific ligands. Nature. 1990;346(6287):818–22. doi: 10.1038/346818a0.CrossRefGoogle Scholar
  3. 3.
    Liu J, Cao Z, Lu Y. Functional nucleic acid sensors. Chem Rev. 2009;109(5):1948–98. doi: 10.1021/cr030183i.CrossRefGoogle Scholar
  4. 4.
    Keefe AD, Pai S, Ellington A. Aptamers as therapeutics. Nat Rev Drug Discov. 2010;9(7):537–50. doi: 10.1038/nrd3141.CrossRefGoogle Scholar
  5. 5.
    Li D, Song S, Fan C. Target-responsive structural switching for nucleic acid-based sensors. Acc Chem Res. 2010;43(5):631–41. doi: 10.1021/ar900245u.CrossRefGoogle Scholar
  6. 6.
    Famulok M, Mayer G. Aptamer modules as sensors and detectors. Acc Chem Res. 2011;44(12):1349–58. doi: 10.1021/ar2000293.CrossRefGoogle Scholar
  7. 7.
    Xing H, Wong NY, Xiang Y, Lu Y. DNA aptamer functionalized nanomaterials for intracellular analysis, cancer cell imaging and drug delivery. Curr Opin Chem Biol. 2012;16(3–4):429–35. doi: 10.1016/j.cbpa.2012.03.016.CrossRefGoogle Scholar
  8. 8.
    Tan W, Donovan MJ, Jiang J. Aptamers from cell-based selection for bioanalytical applications. Chem Rev. 2013;113(4):2842–62. doi: 10.1021/cr300468w.CrossRefGoogle Scholar
  9. 9.
    Shen J, Li Y, Gu H, Xia F, Zuo X. Recent development of sandwich assay based on the nanobiotechnologies for proteins, nucleic acids, small molecules, and ions. Chem Rev. 2014;114(15):7631–77. doi: 10.1021/cr300248x.CrossRefGoogle Scholar
  10. 10.
    Famulok M, Mayer G. Aptamers and SELEX in chemistry & biology. Chem Biol. 2014;21(9):1055–8. doi: 10.1016/j.chembiol.2014.08.003.CrossRefGoogle Scholar
  11. 11.
    Ma H, Liu J, Ali MM, Mahmood MA, Labanieh L, Lu M, et al. Nucleic acid aptamers in cancer research, diagnosis and therapy. Chem Soc Rev. 2015;44(5):1240–56. doi: 10.1039/c4cs00357h.CrossRefGoogle Scholar
  12. 12.
    Toh SY, Citartan M, Gopinath SC, Tang TH. Aptamers as a replacement for antibodies in enzyme-linked immunosorbent assay. Biosens Bioelectron. 2015;64:392–403. doi: 10.1016/j.bios.2014.09.026.CrossRefGoogle Scholar
  13. 13.
    Li F, Zhang H, Wang Z, Newbigging AM, Reid MS, Li XF, et al. Aptamers facilitating amplified detection of biomolecules. Anal Chem. 2015;87(1):274–92. doi: 10.1021/ac5037236.CrossRefGoogle Scholar
  14. 14.
    Ozer A, Pagano JM, Lis JT. New technologies provide quantum changes in the scale, speed, and success of SELEX methods and aptamer characterization. Mol Ther Nucleic Acids. 2014;3:e183. doi: 10.1038/mtna.2014.34.CrossRefGoogle Scholar
  15. 15.
    Park JW, Tatavarty R, Kim DW, Jung HT, Gu MB. Immobilization-free screening of aptamers assisted by graphene oxide. Chem Commun (Camb). 2012;48(15):2071–3. doi: 10.1039/c2cc16473f.CrossRefGoogle Scholar
  16. 16.
    Tsai RY, Reed RR. Identification of DNA recognition sequences and protein interaction domains of the multiple-Zn-finger protein Roaz. Mol Cell Biol. 1998;18(11):6447–56.CrossRefGoogle Scholar
  17. 17.
    Mendonsa SD, Bowser MT. In vitro selection of high-affinity DNA ligands for human IgE using capillary electrophoresis. Anal Chem. 2004;76(18):5387–92. doi: 10.1021/ac049857v.CrossRefGoogle Scholar
  18. 18.
    Nutiu R, Li Y. In vitro selection of structure-switching signaling aptamers. Angew Chem Int Ed Engl. 2005;44(7):1061–5. doi: 10.1002/anie.200461848.CrossRefGoogle Scholar
  19. 19.
    Yang KA, Barbu M, Halim M, Pallavi P, Kim B, Kolpashchikov DM, et al. Recognition and sensing of low-epitope targets via ternary complexes with oligonucleotides and synthetic receptors. Nat Chem. 2014;6(11):1003–8. doi: 10.1038/nchem.2058.CrossRefGoogle Scholar
  20. 20.
    Peng L, Stephens BJ, Bonin K, Cubicciotti R, Guthold M. A combined atomic force/fluorescence microscopy technique to select aptamers in a single cycle from a small pool of random oligonucleotides. Microsc Res Tech. 2007;70(4):372–81. doi: 10.1002/jemt.20421.CrossRefGoogle Scholar
  21. 21.
    Platt M, Rowe W, Wedge DC, Kell DB, Knowles J, Day PJ. Aptamer evolution for array-based diagnostics. Anal Biochem. 2009;390(2):203–5. doi: 10.1016/j.ab.2009.04.013.CrossRefGoogle Scholar
  22. 22.
    Qu H, Csordas AT, Wang J, Oh SS, Eisenstein MS, Soh HT. Rapid and label-free strategy to isolate aptamers for metal ions. ACS Nano. 2016;10(8):7558–65. doi: 10.1021/acsnano.6b02558.CrossRefGoogle Scholar
  23. 23.
    Bae H, Ren S, Kang J, Kim M, Jiang Y, Jin MM, et al. Sol-gel SELEX circumventing chemical conjugation of low molecular weight metabolites discovers aptamers selective to xanthine. Nucleic Acid Ther. 2013;23(6):443–9. doi: 10.1089/nat.2013.0437.CrossRefGoogle Scholar
  24. 24.
    Tang Z, Mallikaratchy P, Yang R, Kim Y, Zhu Z, Wang H, et al. Aptamer switch probe based on intramolecular displacement. J Am Chem Soc. 2008;130(34):11268–9. doi: 10.1021/ja804119s.CrossRefGoogle Scholar
  25. 25.
    Jeon J, Lee KH, Rao J. A strategy to enhance the binding affinity of fluorophore-aptamer pairs for RNA tagging with neomycin conjugation. Chem Commun (Camb). 2012;48(80):10034–6. doi: 10.1039/c2cc34498j.CrossRefGoogle Scholar
  26. 26.
    Zuker M. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 2003;31(13):3406–15.CrossRefGoogle Scholar
  27. 27.
    Niazi JH, Lee SJ, Gu MB. Single-stranded DNA aptamers specific for antibiotics tetracyclines. Bioorg Med Chem. 2008;16(15):7245–53. doi: 10.1016/j.bmc.2008.06.033.CrossRefGoogle Scholar
  28. 28.
    Berens C, Thain A, Schroeder R. A tetracycline-binding RNA aptamer. Bioorg Med Chem. 2001;9(10):2549–56.CrossRefGoogle Scholar
  29. 29.
    Potty AS, Kourentzi K, Fang H, Jackson GW, Zhang X, Legge GB, et al. Biophysical characterization of DNA aptamer interactions with vascular endothelial growth factor. Biopolymers. 2009;91(2):145–56. doi: 10.1002/bip.21097.CrossRefGoogle Scholar
  30. 30.
    Hasegawa H, Sode K, Ikebukuro K. Selection of DNA aptamers against VEGF165 using a protein competitor and the aptamer blotting method. Biotechnol Lett. 2008;30(5):829–34. doi: 10.1007/s10529-007-9629-6.CrossRefGoogle Scholar
  31. 31.
    Kimoto M, Yamashige R, Matsunaga K, Yokoyama S, Hirao I. Generation of high-affinity DNA aptamers using an expanded genetic alphabet. Nat Biotechnol. 2013;31(5):453–7. doi: 10.1038/nbt.2556.CrossRefGoogle Scholar
  32. 32.
    Ahmad Raston NH, Gu MB. Highly amplified detection of visceral adipose tissue-derived serpin (vaspin) using a cognate aptamer duo. Biosens Bioelectron. 2015;70:261–7. doi: 10.1016/j.bios.2015.03.042.CrossRefGoogle Scholar
  33. 33.
    Baker M. Reproducibility crisis: blame it on the antibodies. Nature. 2015;521(7552):274–6. doi: 10.1038/521274a.CrossRefGoogle Scholar
  34. 34.
    Edwards KA, Wang Y, Baeumner AJ. Aptamer sandwich assays: human alpha-thrombin detection using liposome enhancement. Anal Bioanal Chem. 2010;398(6):2645–54. doi: 10.1007/s00216-010-3920-4.CrossRefGoogle Scholar
  35. 35.
    Nonaka Y, Sode K, Ikebukuro K. Screening and improvement of an anti-VEGF DNA aptamer. Molecules. 2010;15(1):215–25. doi: 10.3390/molecules15010215.CrossRefGoogle Scholar
  36. 36.
    Xiao SJ, Hu PP, Wu XD, Zou YL, Chen LQ, Peng L, et al. Sensitive discrimination and detection of prion disease-associated isoform with a dual-aptamer strategy by developing a sandwich structure of magnetic microparticles and quantum dots. Anal Chem. 2010;82(23):9736–42. doi: 10.1021/ac101865s.CrossRefGoogle Scholar
  37. 37.
    Shi H, Fan X, Sevilimedu A, Lis JT. RNA aptamers directed to discrete functional sites on a single protein structural domain. Proc Natl Acad Sci U S A. 2007;104(10):3742–6. doi: 10.1073/pnas.0607805104.CrossRefGoogle Scholar
  38. 38.
    Gong Q, Wang J, Ahmad KM, Csordas AT, Zhou J, Nie J, et al. Selection strategy to generate aptamer pairs that bind to distinct sites on protein targets. Anal Chem. 2012;84(12):5365–71. doi: 10.1021/ac300873p.CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany 2017

Authors and Affiliations

  1. 1.Institute of Bioengineering and NanotechnologySingaporeSingapore

Personalised recommendations