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Programmable ligand-controlled riboregulators of eukaryotic gene expression

Nature Biotechnology volume 23, pages 337–343 (2005)Cite this article

Abstract

Recent studies have demonstrated the importance of noncoding RNA elements in regulating gene expression networks1,2. We describe the design of a class of small trans-acting RNAs that directly regulate gene expression in a ligand-dependent manner. These allosteric riboregulators, which we call antiswitches, are made fully tunable and modular by rational design. They offer flexible control strategies by adopting active or inactive forms in response to ligand binding, depending on their design. They can be tailor-made to regulate the expression of target transcripts in response to different cellular effectors. Coupled with in vitro selection technologies for generating nucleic acid ligand-binding species3,4, antiswitches present a platform for programming cellular behavior and genetic networks with respect to cellular state and environmental stimuli.

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Figure 1: Design and functional activity of an antiswitch regulator.
Figure 2: Tuning and expanding the switch response of an antiswitch regulator.
Figure 3: Redesign and characterization of an 'on' antiswitch regulator.
Figure 4: Simultaneous regulation of multiple genes through multiple antiswitch regulators.

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Acknowledgements

We thank S.V. Avery, A. Miyawaki and K. Weis for providing genes and plasmids used in assembling the constructs described in this work. This work was supported by startup funds provided by the California Institute of Technology.

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Authors and Affiliations

  1. Division of Chemistry and Chemical Engineering, 1200 East California Blvd., MC 210-41, California Institute of Technology, Pasadena, 91125, California, USA

    Travis S Bayer & Christina D Smolke

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Corresponding author

Correspondence to Christina D Smolke.

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The authors have a pending patent application whose value may be affected by the publication of this manuscript.

Supplementary information

Supplementary Fig. 1

Sequence and cleavage mechanism of ncRNA expression construct. (PPT 49 kb)

Supplementary Fig. 2

Structural probing of antiswitch s1 through nuclease mapping. (PPT 49 kb)

Supplementary Fig. 3

Sequences and predicted structures of antiswitches s5, s6, and s7 in the absence of ligand binding. (PPT 67 kb)

Supplementary Fig. 4

Sequence and structural switching of a tetracycline-responsive Venus (YFP) regulator, s9, and its target mRNA. (PPT 68 kb)

Supplementary Fig. 5

Set of plasmids used in the in vivo antiswitch characterization studies. (PPT 55 kb)

Supplementary Table 1

Relative RNA levels of target mRNA and antiswitch s1. (PPT 20 kb)

Supplementary Table 2

List of plasmids used in this study. (PPT 39 kb)

Supplementary Table 3

Sequences of antiswitch constructs, controls, and qRT-PCR primers used in this study. (PPT 33 kb)

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Bayer, T., Smolke, C. Programmable ligand-controlled riboregulators of eukaryotic gene expression. Nat Biotechnol 23, 337–343 (2005). https://doi.org/10.1038/nbt1069

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