RNA Scaffolds pp 141-148 | Cite as

Aptazyme-Based Riboswitches and Logic Gates in Mammalian Cells

  • Yoko Nomura
  • Yohei YokobayashiEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1316)


This chapter describes a screening strategy to engineer synthetic riboswitches that can chemically regulate gene expression in mammalian cells. Riboswitch libraries are constructed by randomizing the key nucleotides that couple the molecular recognition function of an aptamer with the self-cleavage activity of a ribozyme. The allosteric ribozyme (aptazyme) candidates are cloned in the 3′ untranslated region (UTR) of a reporter gene mRNA. The plasmid-encoded riboswitch candidates are transfected into a mammalian cell line to screen for the desired riboswitch function. Furthermore, multiple aptazymes can be cloned into the 3′ UTR of a desired gene to obtain a logic gate response to multiple chemical signals. This screening strategy complements other methods to engineer robust mammalian riboswitches to control gene expression.

Key words

Riboswitch Aptamer Ribozyme Aptazyme Gene regulation RNA engineering 



We thank Linlin Zhou and Anh Miu for contributions to the development of the screening protocol. This work was supported by National Institutes of Health.


  1. 1.
    Wittmann A, Suess B (2012) Engineered riboswitches: expanding researchers’ toolbox with synthetic RNA regulators. FEBS Lett 586:2076–2083CrossRefPubMedGoogle Scholar
  2. 2.
    Chen YY, Jensen MC, Smolke CD (2010) Genetic control of mammalian T-cell proliferation with synthetic RNA regulatory systems. Proc Natl Acad Sci U S A 107:8531–8536CrossRefPubMedCentralPubMedGoogle Scholar
  3. 3.
    Nomura Y, Zhou L, Miu A et al (2013) Controlling mammalian gene expression by allosteric hepatitis delta virus ribozymes. ACS Synth Biol 2:684–689CrossRefPubMedCentralPubMedGoogle Scholar
  4. 4.
    Nomura Y, Kumar D, Yokobayashi Y (2012) Synthetic mammalian riboswitches based on guanine aptazyme. Chem Commun 48:7215–7217CrossRefGoogle Scholar
  5. 5.
    Liang JC, Smolke CD (2012) Rational design and tuning of ribozyme-based devices. Methods Mol Biol 848:439–454CrossRefPubMedGoogle Scholar
  6. 6.
    Piganeau N (2012) Selecting allosteric ribozymes. Methods Mol Biol 848:317–328CrossRefPubMedGoogle Scholar
  7. 7.
    Saragliadis A, Klauser B, Hartig JS (2012) In vivo screening of ligand-dependent hammerhead ribozymes. Methods Mol Biol 848:455–463CrossRefPubMedGoogle Scholar
  8. 8.
    Weigand JE, Wittmann A, Suess B (2012) RNA-based networks: using RNA aptamers and ribozymes as synthetic genetic devices. Methods Mol Biol 813:157–168CrossRefPubMedGoogle Scholar
  9. 9.
    Win MN, Smolke CD (2008) Higher-order cellular information processing with synthetic RNA devices. Science 322:456–460CrossRefPubMedCentralPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Department of Biomedical EngineeringUniversity of California, DavisDavisUSA

Personalised recommendations