Advertisement

A Capture-SELEX Strategy for Multiplexed Selection of RNA Aptamers Against Small Molecules

  • Lasse H. Lauridsen
  • Holger B. Doessing
  • Katherine S. Long
  • Alex T. Nielsen
Part of the Methods in Molecular Biology book series (MIMB, volume 1671)

Abstract

In vitro selection of aptamers that recognize small organic molecules has proven difficult, in part due to the challenge of immobilizing small molecules on solid supports for SELEX (Systematic Evolution of Ligands by Exponential Enrichment). This study describes the implementation of RNA Capture-SELEX, a selection strategy that uses an RNA library to yield ligand-responsive RNA aptamers targeting small organic molecules in solution. To demonstrate the power of this method we selected several aptamers with specificity towards either the natural sweetener rebaudioside A or the food-coloring agent carminic acid. In addition, Bio-layer interferometry is used to screen clonal libraries of aptamer candidates and is used to interrogate aptamer affinity. The RNA-based Capture-SELEX strategy described here simplifies selection of RNA aptamers against small molecules by avoiding ligand immobilization, while also allowing selection against multiple candidate targets in a single experiment. This makes RNA Capture-SELEX particularly attractive for accelerated development of RNA aptamers targeting small metabolites for incorporation into synthetic riboswitches and for analytical biosensors.

Key words

SELEX RNA aptamer Small molecules Next-generation sequencing Bio-layer interferometry 

References

  1. 1.
    Tuerk C, Gold L (1990) Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 249:505–510CrossRefPubMedGoogle Scholar
  2. 2.
    Ellington AD, Szostak JW (1990) In vitro selection of RNA molecules that bind specific ligands. Nature 346:818–822CrossRefPubMedGoogle Scholar
  3. 3.
    Lynch SA, Desai SK, Sajja HK, Gallivan JP (2007) A high-throughput screen for synthetic riboswitches reveals mechanistic insights into their function. Chem Biol 14:173–184CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Lynch SA, Topp S, Gallivan JP (2009) High-throughput screens to discover synthetic riboswitches. Methods Mol Biol 540:321–333CrossRefPubMedGoogle Scholar
  5. 5.
    Link KH, Breaker RR (2009) Engineering ligand-responsive gene-control elements: lessons learned from natural riboswitches. Gene Ther 16:1189–1201CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Dietrich JA, McKee AE, Keasling JD (2010) High-throughput metabolic engineering: advances in small-molecule screening and selection. Annu Rev Biochem 79:563–590CrossRefPubMedGoogle Scholar
  7. 7.
    Yang J, Seo SW, Jang S et al (2013) Synthetic RNA devices to expedite the evolution of metabolite-producing microbes. Nat Commun 4:1413CrossRefPubMedGoogle Scholar
  8. 8.
    Nutiu R, Li Y (2005) In vitro selection of structure-switching signaling aptamers. Angew Chem Int Ed Engl 44:1061–1065CrossRefPubMedGoogle Scholar
  9. 9.
    Vallée-Bélisle A, Plaxco KW (2010) Structure-switching biosensors: inspired by nature. Curr Opin Struct Biol 20:518–526CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Suess B, Hanson S, Berens C et al (2003) Conditional gene expression by controlling translation with tetracycline-binding aptamers. Nucleic Acids Res 31:1853–1858CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Muranaka N, Abe K, Yokobayashi Y (2009) Mechanism-guided library design and dual genetic selection of synthetic OFF riboswitches. Chembiochem 10:2375–2381CrossRefPubMedGoogle Scholar
  12. 12.
    Topp S, Gallivan JP (2008) Random walks to synthetic riboswitches–a high-throughput selection based on cell motility. Chembiochem 9:210–213CrossRefPubMedGoogle Scholar
  13. 13.
    Stoltenburg R, Nikolaus N, Strehlitz B (2012) Capture-SELEX: selection of DNA aptamers for aminoglycoside antibiotics. J Anal Methods Chem 2012:415697CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Lauridsen LH, Sommer MOA, Nielsen AT (2015) Development of aptamers for in vivo and in vitro biosensor applications. Dissertation, Technical University of DenmarkGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  • Lasse H. Lauridsen
    • 1
  • Holger B. Doessing
    • 1
  • Katherine S. Long
    • 1
  • Alex T. Nielsen
    • 1
  1. 1.The Novo Nordisk Foundation Center for BiosustainabilityTechnical University of DenmarkKongens LyngbyDenmark

Personalised recommendations