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Allosteric selection of ribozymes that respond to the second messengers cGMP and cAMP

Nature Structural Biology volume 6, pages 1062–1071 (1999)Cite this article

Abstract

RNA transcripts containing the hammerhead ribozyme have been engineered to self-destruct in the presence of specific nucleoside 3′,5′-cyclic monophosphate compounds. These RNA molecular switches were created by a new combinatorial strategy termed 'allosteric selection,' which favors the emergence of ribozymes that rapidly self-cleave only when incubated with their corresponding effector compounds. Representative RNAs exhibit 5,000-fold activation upon cGMP or cAMP addition, display precise molecular recognition characteristics, and operate with catalytic rates that match those exhibited by unaltered ribozymes. These findings demonstrate that a vast number of ligand-responsive ribozymes with dynamic structural characteristics can be generated in a massively parallel fashion. Moreover, optimized allosteric ribozymes could serve as highly selective sensors of chemical agents or as unique genetic control elements for the programmed destruction of cellular RNAs.

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Figure 1: The tripartite design for allosteric ribozyme construction.
Figure 2: Allosteric selection scheme and the isolation of RNA molecular switches with new effector specificities.
Figure 3: Allosteric modulation of hammerhead ribozymes by cNMPs.
Figure 4: Molecular recognition of cAMP by cAMP-3 RNA.
Figure 5: Molecular recognition of cAMP by cAMP-1 RNA.
Figure 6: Selective molecular recognition by cNMP-dependent allosteric ribozymes.
Figure 7: Rapid effector-mediated activation of allosteric ribozymes.
Figure 8: Effector-binding affinities and the dynamic ranges for various allosteric ribozymes.

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Acknowledgements

We thank J. Shin for contributions to Figures 3 and 8, and we thank members of the Breaker laboratory for helpful discussions. We also thank T.E. Shrader at the Albert Einstein College of Medicine for the kind gift of the construct to express His-tagged T7 RNAP. Funding for this work was provided by grants from the National Institutes of Health (NIH), the Defense Advanced Research Projects Agency (DARPA) and the Yale Diabetes and Endocrinology Research Center (DERC). M.K. was supported by Sankyo Co. Ltd. of Japan and G.A.S. was supported by a Seessel postdoctoral fellowship from Yale University. R.R.B. is the recipient of a Hellman family fellowship and a fellowship from the David and Lucile Packard Foundation.

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  1. Department of Molecular, Cellular and Developmental Biology, Yale University, P.O. Box 208103, New Haven, 06520-8103, Connecticut, USA

    Makoto Koizumi, Garrett A. Soukup, Jo Nita Q. Kerr & Ronald R. Breaker

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  1. Makoto Koizumi
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Correspondence to Ronald R. Breaker.

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Koizumi, M., Soukup, G., Kerr, J. et al. Allosteric selection of ribozymes that respond to the second messengers cGMP and cAMP. Nat Struct Mol Biol 6, 1062–1071 (1999). https://doi.org/10.1038/14947

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