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

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Abstract

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.

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References

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  3. Liu J, Cao Z, Lu Y. Functional nucleic acid sensors. Chem Rev. 2009;109(5):1948–98. doi:10.1021/cr030183i.

    Article  CAS  Google Scholar 

  4. Keefe AD, Pai S, Ellington A. Aptamers as therapeutics. Nat Rev Drug Discov. 2010;9(7):537–50. doi:10.1038/nrd3141.

    Article  CAS  Google Scholar 

  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.

    Article  Google Scholar 

  6. Famulok M, Mayer G. Aptamer modules as sensors and detectors. Acc Chem Res. 2011;44(12):1349–58. doi:10.1021/ar2000293.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  26. Zuker M. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 2003;31(13):3406–15.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  28. Berens C, Thain A, Schroeder R. A tetracycline-binding RNA aptamer. Bioorg Med Chem. 2001;9(10):2549–56.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  33. Baker M. Reproducibility crisis: blame it on the antibodies. Nature. 2015;521(7552):274–6. doi:10.1038/521274a.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

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Acknowledgements

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

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  1. Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos, Singapore, 138669, Singapore

    Kyung Hyun Lee & Huaqiang Zeng

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  1. Kyung Hyun Lee
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  2. Huaqiang Zeng
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Correspondence to Huaqiang Zeng.

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Lee, K.H., Zeng, H. A general double library SELEX strategy for aptamer selection using unmodified nonimmobilized targets. Anal Bioanal Chem 409, 5081–5089 (2017). https://doi.org/10.1007/s00216-017-0454-z

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  • DOI: https://doi.org/10.1007/s00216-017-0454-z

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