Dual-Selection for Evolution of In Vivo Functional Aptazymes as Riboswitch Parts

  • Jonathan A. Goler
  • James M. Carothers
  • Jay D. Keasling
Part of the Methods in Molecular Biology book series (MIMB, volume 1111)


Both synthetic biology and metabolic engineering are aided by the development of genetic control parts. One class of riboswitch parts that has great potential for sensing and regulation of protein levels is aptamer-coupled ribozymes (aptazymes). These devices are comprised of an aptamer domain selected to bind a particular ligand, a ribozyme domain, and a communication module that regulates the ribozyme activity based on the state of the aptamer. We describe a broadly applicable method for coupling a novel, newly selected aptamer to a ribozyme to generate functional aptazymes via in vitro and in vivo selection. To illustrate this approach, we describe experimental procedures for selecting aptazymes assembled from aptamers that bind p-amino-phenylalanine and a hammerhead ribozyme. Because this method uses selection, it does not rely on sequence-specific design and thus should be generalizable for the generation of in vivo operational aptazymes that respond to any targeted molecules.

Key words

In vitro selection RNA aptamer Ribozyme Aptazyme Riboswitch Synthetic biology 


  1. 1.
    Vitreschak AG, Rodionov DA, Mironov AA, Gelfand MS (2004) Riboswitches: the oldest mechanism for the regulation of gene expression. Trends Genet 20:44–50PubMedCrossRefGoogle Scholar
  2. 2.
    Stormo GD, Ji Y (2001) Do mRNAs act as direct sensors of small molecules to control their expression? Proc Natl Acad Sci U S A 98:9465–9467PubMedCentralPubMedCrossRefGoogle Scholar
  3. 3.
    Patel DJ, Suri AK, Jiang F et al (1997) Structure, recognition and adaptive binding in RNA aptamer complexes. J Mol Biol 272:645–664PubMedCrossRefGoogle Scholar
  4. 4.
    Tuerk C (1997) Using the SELEX combinatorial chemistry process to find high affinity nucleic acid ligands to target molecules. Methods Mol Biol 67:219–230PubMedGoogle Scholar
  5. 5.
    Klug SJ, Hüttenhofer A, Famulok M (2000) In vitro selection of RNA aptamers that bind special elongation factor SelB, a protein with multiple RNA-binding sites, reveals one major interaction domain at the carboxyl terminus. RNA 5:1180–1190CrossRefGoogle Scholar
  6. 6.
    Lorsch JR, Szostak JW (1994) In vitro selection of RNA aptamers specific for cyanocobalamin. Biochemistry 33:973–982PubMedCrossRefGoogle Scholar
  7. 7.
    Winkler WC, Breaker RR (2003) Genetic control by metabolite-binding riboswitches. Chembiochem 4:1024–1032PubMedCrossRefGoogle Scholar
  8. 8.
    Nahvi A, Sudarsan N, Ebert MS et al (2002) Genetic control by a metabolite binding mRNA. Chem Biol 9:1043–1049PubMedCrossRefGoogle Scholar
  9. 9.
    Nahvi A, Barrick JE, Breaker RR (2004) Coenzyme B 12 riboswitches are widespread genetic control elements in prokaryotes. Nucleic Acids Res 32:143–150PubMedCentralPubMedCrossRefGoogle Scholar
  10. 10.
    Liu CC, Arkin AP (2010) The case for RNA. Science 330:1185–1186PubMedCrossRefGoogle Scholar
  11. 11.
    Hall B, Hesselberth JR, Ellington AD (2007) Computational selection of nucleic acid biosensors via a slip structure model. Biosens Bioelectron 22:1939–1947PubMedCrossRefGoogle Scholar
  12. 12.
    Lynch SA, Desai SK, Sajja HK, Gallivan JP (2007) A high-throughput screen for synthetic riboswitches reveals mechanistic insights into their function. Chem Biol 14:173–184PubMedCentralPubMedCrossRefGoogle Scholar
  13. 13.
    Suess B, Fink B, Berens C et al (2004) A theophylline responsive riboswitch based on helix slipping controls gene expression in vivo. Nucleic Acids Res 32:1610–1614PubMedCentralPubMedCrossRefGoogle Scholar
  14. 14.
    Soukup GA, Breaker RR (1999) Engineering precision RNA molecular switches. Proc Natl Acad Sci U S A 96:3584–3589PubMedCentralPubMedCrossRefGoogle Scholar
  15. 15.
    Carothers JM, Goler JA, Juminaga D, Keasling JD (2011) Model-driven engineering of RNA devices to quantitatively program gene expression. Science 334:1716–1719PubMedCrossRefGoogle Scholar
  16. 16.
    Carothers JM, Goler JA, Kapoor R, Lara LD, Keasling JD (2010) Selecting RNA aptamers for synthetic biology: investigating magnesium dependence and predicting binding affinity. Nucleic Acids Res 38:2736–2747PubMedCentralPubMedCrossRefGoogle Scholar
  17. 17.
    Thompson KM, Syrett HA, Knudsen SM, Ellington AD (2002) Group I aptazymes as genetic regulatory switches. BMC Biotechnol 2:21PubMedCentralPubMedCrossRefGoogle Scholar
  18. 18.
    Link KH, Guo L, Ames TD et al (2007) Engineering high-speed allosteric hammerhead ribozymes. Biol Chem 388:779–786PubMedCrossRefGoogle Scholar
  19. 19.
    Jenison RD, Gill SC, Pardi A, Polisky B (1994) High-resolution molecular discrimination by RNA. Science 263:1425–1429PubMedCrossRefGoogle Scholar
  20. 20.
    Dower WJ, Cwirla SE (1992) Guide to electroporation and electrofusion. Academic, San DiegoGoogle Scholar
  21. 21.
    Isambert H, Siggia E (2000) Modeling RNA folding paths with pseudo-knots: application to hepatitis delta virus ribozyme. Proc Natl Acad Sci U S A 97:6515–6520PubMedCentralPubMedCrossRefGoogle Scholar
  22. 22.
    Xayaphoummine A, Viasnoff V, Harlepp S, Isambert H (2007) Encoding folding paths of RNA switches. Nucleic Acids Res 35:614–622PubMedCentralPubMedCrossRefGoogle Scholar
  23. 23.
    Deana A, Celesnik H, Belasco JG (2008) The bacterial enzyme RppH triggers messenger RNA degradation by 5-prime pyrophosphate removal. Nature 451:355–358PubMedCrossRefGoogle Scholar
  24. 24.
    Bayer TS, Smolke CD (2005) Programmable ligand-controlled riboregulators of eukaryotic gene expression. Nat Biotechnol 23:337–343PubMedCrossRefGoogle Scholar
  25. 25.
    Wieland M, Hartig JS (2008) Improved aptazyme design and in vivo screening enable riboswitching in bacteria. Angew Chem Int Ed 47:2604–2607CrossRefGoogle Scholar
  26. 26.
    Weigand J, Sanchez M, Gunnesch EB et al (2008) Screening for engineered neomycin riboswitches that control translation initiation. RNA 14:89–97PubMedCentralPubMedCrossRefGoogle Scholar
  27. 27.
    Hillson NJ, Rosengarten RD, Keasling JD (2011) j5 DNA assembly design automation software. ACS Synth Biol 1:14–21PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Jonathan A. Goler
    • 1
    • 2
  • James M. Carothers
    • 1
    • 2
  • Jay D. Keasling
    • 1
    • 2
  1. 1.University of CaliforniaBerkeleyUSA
  2. 2.U.S. Department of Energy Joint BioEnergy InstituteEmeryvilleUSA

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