Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Fluorescence-activated cell sorting for aptamer SELEX with cell mixtures

Nature Protocols volume 5, pages 1993–2004 (2010)Cite this article

Subjects

Abstract

Aptamers that target a specific cell subpopulation within composite mixtures represent invaluable tools in biomedical research and in the development of cell-specific therapeutics. Here we describe a detailed protocol for a modular and generally applicable scheme to select aptamers that target the subpopulations of cells in which you are interested. A fluorescence-activated cell-sorting device is used to simultaneously differentiate and separate those subpopulations of cells having bound and unbound aptamers. There are fewer false positives when using this approach in comparison with other cell-selection approaches in which unspecific binding of nucleic acids to cells with reduced membrane integrity or their unselective uptake by dead cells occurs more often. The protocol provides a state-of-the-art approach for identifying aptamers that selectively target virtually any cell type under investigation. As an example, we provide the step-by-step protocol targeting CD19+ Burkitt's lymphoma cells, starting from the pre-SELEX (systematic evolution of ligands by exponential amplification) measurements to establish suitable SELEX conditions and ending at completion of the SELEX procedure, which reveals the enriched single-stranded DNA library.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Selection scheme based on FACS.
Figure 2: Workflow for setting up and executing a FACS-SELEX experiment.
Figure 3: Monitoring enrichment of ssDNA aptamers during FACS-SELEX.
Figure 4: Determination of the relative amount of ssDNA library bound to Burkitt's lymphoma cells in the absence (w/o) and presence of increasing amounts of competitor (salmon sperm DNA).

Similar content being viewed by others

References

  1. Neri, D., Pini, A. & Nissim, A. Antibodies from phage display libraries as immunochemical reagents. Methods Mol. Biol. 80, 475–500 (1998).

    Article  CAS  Google Scholar 

  2. Marlind, J. et al. Antibody-mediated delivery of interleukin-2 to the stroma of breast cancer strongly enhances the potency of chemotherapy. Clin. Cancer Res. 14, 6515–6524 (2008).

    Article  CAS  Google Scholar 

  3. Schliemann, C. & Neri, D. Antibody-based targeting of the tumor vasculature. Biochim. Biophys. Acta. 1776, 175–192 (2007).

    CAS  PubMed  Google Scholar 

  4. Neri, D., Petrul, H. & Roncucci, G. Engineering recombinant antibodies for immunotherapy. Cell Biophys. 27, 47–61 (1995).

    Article  CAS  Google Scholar 

  5. Gafner, V., Trachsel, E. & Neri, D. An engineered antibody-interleukin-12 fusion protein with enhanced tumor vascular targeting properties. Int. J. Cancer 119, 2205–2212 (2006).

    Article  CAS  Google Scholar 

  6. Jäger, S. & Famulok, M. Generation and enzymatic amplification of high-density functionalized DNA double strands. Angew. Chem. Int. Ed. Engl. 43, 3337–3340 (2004).

    Article  Google Scholar 

  7. Jäger, S. et al. A versatile toolbox for variable DNA functionalization at high density. J. Am. Chem. Soc. 127, 15071–15082 (2005).

    Article  Google Scholar 

  8. Thum, O., Jäger, S. & Famulok, M. Functionalized DNA: a new replicable biopolymer. Angew. Chem. Int. Ed. Engl. 40, 3990–3993 (2001).

    Article  CAS  Google Scholar 

  9. Verma, S. & Eckstein, F. Modified oligonucleotides: synthesis and strategy for users. Annu. Rev. Biochem. 67, 99–134 (1998).

    Article  CAS  Google Scholar 

  10. Mayer, G. The chemical biology of aptamers. Angew. Chem. Int. Ed. Engl. 48, 2672–2689 (2009).

    Article  CAS  Google Scholar 

  11. Shamah, S.M., Healy, J.M. & Cload, S.T. Complex target SELEX. Acc. Chem. Res. 41, 130–138 (2008).

    Article  CAS  Google Scholar 

  12. Famulok, M., Hartig, J.S. & Mayer, G. Functional aptamers and aptazymes in biotechnology, diagnostics, and therapy. Chem. Rev. 107, 3715–3743 (2007).

    Article  CAS  Google Scholar 

  13. Famulok, M. & Verma, S. In vivo-applied functional RNAs as tools in proteomics and genomics research. Trends Biotechnol. 20, 462–466 (2002).

    Article  CAS  Google Scholar 

  14. Cerchia, L., Giangrande, P.H., McNamara, J.O. & de Franciscis, V. Cell-specific aptamers for targeted therapies. Methods Mol. Biol. 535, 59–78 (2009).

    Article  CAS  Google Scholar 

  15. Guo, K.T. et al. A new technique for the isolation and surface immobilization of mesenchymal stem cells from whole bone marrow using high-specific DNA aptamers. Stem Cells 24, 2220–2231 (2006).

    Article  CAS  Google Scholar 

  16. Pastor, F., Kolonias, D., Giangrande, P.H. & Gilboa, E. Induction of tumour immunity by targeted inhibition of nonsense-mediated mRNA decay. Nature 465, 227–230 (2010).

    Article  CAS  Google Scholar 

  17. Ellington, A.D. & Szostak, J.W. In vitro selection of RNA molecules that bind specific ligands. Nature 346, 818–822 (1990).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  19. Hermann, T. & Patel, D.J. Adaptive recognition by nucleic acid aptamers. Science 287, 820–825 (2000).

    Article  CAS  Google Scholar 

  20. Huang, D.B. et al. Crystal structure of NF-kappaB (p50)2 complexed to a high-affinity RNA aptamer. Proc. Natl Acad. Sci. USA 100, 9268–9273 (2003).

    Article  CAS  Google Scholar 

  21. Jaeger, J., Restle, T. & Steitz, T.A. The structure of HIV-1 reverse transcriptase complexed with an RNA pseudoknot inhibitor. EMBO J. 17, 4535–4542 (1998).

    Article  CAS  Google Scholar 

  22. Kettenberger, H. et al. Structure of an RNA polymerase II-RNA inhibitor complex elucidates transcription regulation by noncoding RNAs. Nat. Struct. Mol. Biol. 13, 44–48 (2006).

    Article  CAS  Google Scholar 

  23. Sassanfar, M. & Szostak, J.W. An RNA motif that binds ATP. Nature 364, 550–553 (1993).

    Article  CAS  Google Scholar 

  24. Burgstaller, P. & Famulok, M. Isolation of RNA aptamers for biological cofactors by in vitro selection. Angew. Chem. Int. Ed. 33, 1084–1087 (1994).

    Article  Google Scholar 

  25. Famulok, M. Molecular recognition of amino-acids by RNA-aptamers—an L-citrulline binding RNA motif and its evolution into an L-arginine binder. J. Am. Chem. Soc. 116, 1698–1706 (1994).

    Article  CAS  Google Scholar 

  26. Nastasijevic, B., Becker, N.A., Wurster, S.E. & Maher, L.J. III . Sequence-specific binding of DNA and RNA to immobilized nickel ions. Biochem. Biophys. Res. Commun. 366, 420–425 (2008).

    Article  CAS  Google Scholar 

  27. Proske, D., Hofliger, M., Soll, R.M., Beck-Sickinger, A.G. & Famulok, M. A Y2 receptor mimetic aptamer directed against neuropeptide Y. J. Biol. Chem. 277, 11416–11422 (2002).

    Article  CAS  Google Scholar 

  28. Rentmeister, A., Bill, A., Wahle, T., Walter, J. & Famulok, M. RNA aptamers selectively modulate protein recruitment to the cytoplasmic domain of beta-secretase BACE1 in vitro. RNA 12, 1650–1660 (2006).

    Article  CAS  Google Scholar 

  29. Zichi, D., Eaton, B., Singer, B. & Gold, L. Proteomics and diagnostics: let's get specific, again. Curr. Opin. Chem. Biol. 12, 78–85 (2008).

    Article  CAS  Google Scholar 

  30. Mayer, G., Raddatz, M.S., Grunwald, J.D. & Famulok, M. RNA ligands that distinguish metabolite-induced conformations in the TPP riboswitch. Angew. Chem. Int. Ed. Engl. 46, 557–560 (2007).

    Article  CAS  Google Scholar 

  31. Rentmeister, A., Mayer, G., Kuhn, N. & Famulok, M. Conformational changes in the expression domain of the Escherichia coli thiM riboswitch. Nucleic Acids Res. 35, 3713–3722 (2007).

    Article  CAS  Google Scholar 

  32. Rentmeister, A., Mayer, G., Kuhn, N. & Famulok, M. Secondary structures and functional requirements for thiM riboswitches from Desulfovibrio vulgaris, Erwinia carotovora and Rhodobacter spheroides. Biol. Chem. 389, 127–134 (2008).

    Article  CAS  Google Scholar 

  33. Lunse, C.E. et al. An aptamer targeting the apical-loop domain modulates pri-miRNA processing. Angew. Chem. Int. Ed. Engl. 49, 4674–4677 (2010).

    Article  Google Scholar 

  34. Mayer, G. & Hover, T. In vitro selection of ssDNA aptamers using biotinylated target proteins. Methods Mol. Biol. 535, 19–32 (2009).

    Article  CAS  Google Scholar 

  35. Hicke, B.J. et al. Tenascin-C aptamers are generated using tumor cells and purified protein. J. Biol. Chem. 276, 48644–48654 (2001).

    Article  CAS  Google Scholar 

  36. Li, N., Larson, T., Nguyen, H.H., Sokolov, K.V. & Ellington, A.D. Directed evolution of gold nanoparticle delivery to cells. Chem. Commun. (Camb.) 46, 392–394 (2010).

    Article  Google Scholar 

  37. Cerchia, L. et al. Neutralizing aptamers from whole-cell SELEX inhibit the RET receptor tyrosine kinase. PLoS Biol. 3, e123 (2005).

    Article  Google Scholar 

  38. Blank, M., Weinschenk, T., Priemer, M. & Schluesener, H. Systematic evolution of a DNA aptamer binding to rat brain tumor microvessels. selective targeting of endothelial regulatory protein pigpen. J. Biol. Chem. 276, 16464–16468 (2001).

    Article  CAS  Google Scholar 

  39. Sefah, K., Shangguan, D., Xiong, X., O'Donoghue, M.B. & Tan, W. Development of DNA aptamers using Cell-SELEX. Nat. Protoc. 5, 1169–1185 (2010).

    Article  CAS  Google Scholar 

  40. Raddatz, M.S. et al. Enrichment of cell-targeting and population-specific aptamers by fluorescence-activated cell sorting. Angew. Chem. Int. Ed. Engl. 47, 5190–5193 (2008).

    Article  CAS  Google Scholar 

  41. Li, N. et al. Technical and biological issues relevant to cell typing with aptamers. J. Proteome. Res. 8, 2438–2448 (2009).

    Article  CAS  Google Scholar 

  42. Lupold, S.E., Hicke, B.J., Lin, Y. & Coffey, D.S. Identification and characterization of nuclease-stabilized RNA molecules that bind human prostate cancer cells via the prostate-specific membrane antigen. Cancer Res. 62, 4029–4033 (2002).

    CAS  PubMed  Google Scholar 

  43. Proske, D. et al. Prion-protein-specific aptamer reduces PrPSc formation. Chembiochem 3, 717–725 (2002).

    Article  CAS  Google Scholar 

  44. Maecker, H.T. & Trotter, J. Flow cytometry controls, instrument setup, and the determination of positivity. Cytometry A 69, 1037–1042 (2006).

    Article  Google Scholar 

  45. Perfetto, S.P., Ambrozak, D., Nguyen, R., Chattopadhyay, P. & Roederer, M. Quality assurance for polychromatic flow cytometry. Nat. Protoc. 1, 1522–1530 (2006).

    Article  CAS  Google Scholar 

  46. Ahmed, M.S. & Mayer, G. Evolution of specific RNA motifs derived from pan-protein interacting precursors. Bioorg. Med. Chem. Lett. 20, 3793–3796 (2010).

    Article  CAS  Google Scholar 

  47. Mayer, G. et al. From selection to caged aptamers: identification of light-dependent ssDNA aptamers targeting cytohesin. Bioorg. Med. Chem. Lett. 19, 6561–6564 (2009).

    Article  CAS  Google Scholar 

  48. Mayer, G. et al. An RNA molecule that specifically inhibits G-protein-coupled receptor kinase 2 in vitro. RNA 14, 524–534 (2008).

    Article  CAS  Google Scholar 

  49. Paul, A., Avci-Adali, M., Ziemer, G. & Wendel, H. P. Streptavidin-coated magnetic beads for DNA strand separation implicate a multitude of problems during cell-SELEX. Oligonucleotides 19, 243–254 (2009).

    Article  CAS  Google Scholar 

  50. Mallikaratchy, P., Stahelin, R.V., Cao, Z., Cho, W. & Tan, W. Selection of DNA ligands for protein kinase C-delta. Chem. Commun. (Camb.) 2006, 3229–3231 (2006).

    Article  Google Scholar 

  51. Zueva, E., Rubio, L.I., Duconge, F. & Tavitian, B. Metastasis-focused cell-based SELEX generates aptamers inhibiting cell migration and invasion. Int. J. Cancer published online, doi:10.1002/ijc.25401 (19 April 2010).

Download references

Acknowledgements

This research was supported by research grants from the Deutsche Forschungsgemeinschaft Sonderforschungsbereich 704, the Graduiertenkolleg GRK 804 and the Fonds der Chemischen Industrie. We thank N. Krämer for excellent technical assistance and J. Müller for support during the preparation of peripheral blood mononuclear cells from whole blood.

Author information

Authors and Affiliations

  1. Department of Life and Medical Sciences, University of Bonn, Bonn, Germany

    Günter Mayer, Marie-Sophie L Ahmed & Michael Famulok

  2. Institute of Molecular Medicine and Experimental Immunology, University of Bonn, Bonn, Germany

    Andreas Dolf, Elmar Endl & Percy A Knolle

Authors
  1. Günter Mayer
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Marie-Sophie L Ahmed
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Andreas Dolf
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Elmar Endl
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Percy A Knolle
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Michael Famulok
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

G.M. and M.-S.L.A., along with M.F., performed and designed most of the included studies. E.E. and A.D. provided experimental input in setting up the FACS experiments and performed some of them to establish optimal conditions, assisted by M.-S.L.A. and G.M. P.A.K. provided conceptual input. M.F. and G.M. supervised the research project and assisted in the experimental design. All authors discussed the experimental results. M.F. and G.M. wrote the manuscript.

Corresponding authors

Correspondence to Günter Mayer or Michael Famulok.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Mayer, G., Ahmed, MS., Dolf, A. et al. Fluorescence-activated cell sorting for aptamer SELEX with cell mixtures. Nat Protoc 5, 1993–2004 (2010). https://doi.org/10.1038/nprot.2010.163

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2010.163

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing