Analytical and Bioanalytical Chemistry

, Volume 410, Issue 7, pp 1991–2000 | Cite as

G-quadruplex aptamer selection using capillary electrophoresis-LED-induced fluorescence and Illumina sequencing

  • Audrey Ric
  • Vincent Ecochard
  • Jason S. Iacovoni
  • Audrey Boutonnet
  • Frédéric Ginot
  • Varravaddheay Ong-Meang
  • Véréna Poinsot
  • Laurent Paquereau
  • François Couderc
Research Paper

Abstract

One of the major difficulties that arises when selecting aptamers containing a G-quadruplex is the correct amplification of the ssDNA sequence. Can aptamers containing a G-quadruplex be selected from a degenerate library using non-equilibrium capillary electrophoresis (CE) of equilibrium mixtures (NECEEM) along with high-throughput Illumina sequencing? In this article, we present some mismatches of the G-quadruplex T29 aptamer specific to thrombin, which was PCR amplified and sequenced by Illumina sequencing. Then, we show the proportionality between the number of sequenced molecules of T29 added to the library and the number of sequences obtained in Illumina sequencing, and we find that T29 sequences from this aptamer can be detected in a random library of ssDNA after the sample is fractionated by NECEEM, amplified by PCR, and sequenced. Treatment of the data by the counting of double-stranded DNA T29 sequences containing a maximum of two mismatches reveals a good correlation with the enrichment factor (fE). This factor is the ratio of the number of aptamer sequences found in the collected complex sample divided by the total number of sequencing reads (aptamer and non-aptamer) plus the quantity of T29 molecules (spiked into a DNA library) injected into CE.

Keywords

Aptamer Capillary electrophoresis Biomolecule Thrombin 

Notes

Acknowledgments

We thank La Région Occitanie (grant no. 13053107) and ANRT for their financial support.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

References

  1. 1.
    Berezovski M, Krylov SN. Nonequilibrium capillary electrophoresis of equilibrium mixtures—a single experiment reveals equilibrium and kinetic parameters of protein-DNA interactions. J Am Chem Soc. 2002;124:13674–5.CrossRefGoogle Scholar
  2. 2.
    Krylov SN, Berezovski M. Non-equilibrium capillary electrophoresis of equilibrium mixtures-appreciation of kinetics in capillary electrophoresis. Analyst. 2003;128:571–5.CrossRefGoogle Scholar
  3. 3.
    Ashley J, Ji K, Li SF. Selection of bovine catalase aptamers using non-SELEX. Electrophoresis. 2012;33:2783–9.CrossRefGoogle Scholar
  4. 4.
    Yufa R, Krylova SM, Bruce C, Bagg EA, Schofield CJ, Krylov SN. Emulsion PCR significantly improves nonequilibrium capillary electrophoresis of equilibrium mixtures-based aptamer selection: allowing for efficient and rapid selection of aptamer to unmodified ABH2 protein. Anal Chem. 2015;87:1411–9.CrossRefGoogle Scholar
  5. 5.
    Yu X, Yu Y. A mathematical analysis of the selective enrichment of NECEEM-based non-SELEX. Appl Biochem Biotechnol. 2014;173:2019–27.CrossRefGoogle Scholar
  6. 6.
    Gold L. The SELEX process: a surprising source of therapeutic and diagnostic compounds. Harvey Lect. 1995-1996;91:47–57.Google Scholar
  7. 7.
    Luo Z, Zhou H, Jiang H, Ou H, Li X, Zhang L. Development of a fraction collection approach in capillary electrophoresis SELEX for aptamer selection. Analyst. 2015;140:2664–70.CrossRefGoogle Scholar
  8. 8.
    Chambers VS, Marsico G, Boutell JM, Di Antonio M, Smith GP, Balasubramanian S. High-throughput sequencing of DNA G-quadruplex structures in the human genome. Nat Biotechnol. 2015;33:877–81.CrossRefGoogle Scholar
  9. 9.
    Kasahara Y, Irisawa Y, Fujita H, Yahara A, Ozaki H, Obika S, et al. Capillary electrophoresis-systematic evolution of ligands by exponential enrichment selection of base- and sugar-modified DNA aptamers: target binding dominated by 2′-O,4′-C-methylene-bridged/locked nucleic acid primer. Anal Chem. 2013;85:4961–7.CrossRefGoogle Scholar
  10. 10.
    Deng B, Lin Y, Wang C, Li F, Wang Z, Zhang H, et al. Aptamer binding assays for proteins: the thrombin example—a review. Anal Chim Acta. 2014;837:1–15.CrossRefGoogle Scholar
  11. 11.
    Tasset DM, Kubik MF, Steiner W. Oligonucleotide inhibitors of human thrombin that bind distinct epitopes. J Mol Biol. 1997;272:688–98.CrossRefGoogle Scholar
  12. 12.
    Wang J, Gu Y, Liu L, Wang C, Wang J, Ding S, et al. Novel application of fluorescence coupled capillary electrophoresis to resolve the interaction between the G-quadruplex aptamer and thrombin. J Sep Sci. 2017;  https://doi.org/10.1002/jssc.201700456.
  13. 13.
    Riley KR, Saito S, Gagliano J, Colyer CL. Facilitating aptamer selection and collection by capillary transient isotachophoresis with laser-induced fluorescence detection. J Chromatogr A. 2014;1368:183–9.CrossRefGoogle Scholar
  14. 14.
    Riley KR, Gagliano J, Xiao J, Libby K, Saito S, Yu G, et al. Combining capillary electrophoresis and next-generation sequencing for aptamer selection. Anal Bioanal Chem. 2015;407:1527–32.CrossRefGoogle Scholar
  15. 15.
    Huge BJ, Flaherty RJ, Dada OO, Dovichi NJ. Capillary electrophoresis coupled with automated fraction collection. Talanta. 2014;130:288–93.CrossRefGoogle Scholar
  16. 16.
    Ric A, Ong-Meang V, Poinsot V, Martins-Froment N, Chauvet F, Boutonnet A, et al. ssDNA degradation along capillary electrophoresis process using a Tris buffer. Electrophoresis. 2017;38:1624–31.CrossRefGoogle Scholar
  17. 17.
    Glenn TC. Field guide to next-generation DNA sequencers. Molec Ecol Res. 2011;11:759–69.CrossRefGoogle Scholar
  18. 18.
    Kanoatov M, Mehrabanfar S, Krylov SN. Systematic approach to optimization of experimental conditions in nonequilibrium capillary electrophoresis of equilibrium mixtures. Anal Chem. 2016;88:9300–8.CrossRefGoogle Scholar
  19. 19.
    Li Y, Guo L, Zhang F, Zhang Z, Tang J, Xie J. High-sensitive determination of human alpha-thrombin by its 29-mer aptamer in affinity probe capillary electrophoresis. Electrophoresis. 2008;29:2570–7.CrossRefGoogle Scholar
  20. 20.
    Bock LC, Griffin LC, Latham JA, Vermaas EH, Toole JJ. Selection of single-stranded DNA molecules that bind and inhibit human thrombin. Nature. 1992;355:564–6.CrossRefGoogle Scholar
  21. 21.
    Li HY, Deng QP, Zhang DW, Zhou YL, Zhang XX. Chemiluminescently labeled aptamers as the affinity probe for interaction analysis by capillary electrophoresis. Electrophoresis. 2010;31:2452–60.CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Audrey Ric
    • 1
    • 2
    • 3
  • Vincent Ecochard
    • 2
  • Jason S. Iacovoni
    • 4
  • Audrey Boutonnet
    • 3
  • Frédéric Ginot
    • 3
  • Varravaddheay Ong-Meang
    • 1
  • Véréna Poinsot
    • 1
  • Laurent Paquereau
    • 2
  • François Couderc
    • 1
  1. 1.Laboratoire des IMRCP, UMR 5623Université de Toulouse, Université Paul SabatierToulouseFrance
  2. 2.Institut de Pharmacologie et de Biologie Structurale, IPBSUniversité de Toulouse, CNRS, UPSToulouseFrance
  3. 3.Picometrics TechnologiesLabègeFrance
  4. 4.I2MC, UMR1048Toulouse Cedex 4France

Personalised recommendations