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Monitoring post-translational modification of proteins with allosteric ribozymes

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Abstract

An allosteric hammerhead ribozyme activated specifically by the unphosphorylated form of the protein kinase ERK2 was created through a rational design strategy that relies on molecular recognition of ERK2 to decrease the formation of an alternate, inactive ribozyme conformer. Neither closely related mitogen-activated protein kinases (MAPKs) nor the phosphorylated form of ERK2 induced ribozyme activity. The ribozyme quantitatively detected ERK2 added to mammalian cell lysates and also functioned quantitatively in a multiplexed solution-phase assay. This same strategy was used to construct a second ribozyme selectively activated by the phosphorylated (active) form of ERK2. This approach is generally applicable to the development of ribozymes capable of monitoring post-translational modification of specific proteins.

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Figure 1: A ribozyme activated by a protein kinase.
Figure 2: ERK2-concentration dependence of ERK-HH activation.
Figure 3: Specificity of ribozyme activation.
Figure 4: Detection of ERK2 in mammalian cell lysates.
Figure 5: A solution-phase assay.
Figure 6: A ribozyme responsive to phosphorylated ERK2.

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Acknowledgements

We thank Leonid Beigelman, James McSwiggen, Pamela Pavco, Nassim Usman, Shawn Zinnen, and others at Ribozyme Pharmaceuticals for helpful discussion, Olke Uhlenbeck (University of Colorado), Ronald Breaker (Yale University), and Scott Silverman (University of Illinois at Urbana-Champaign) for comments on the manuscript, and Terry Violette and Jeffery Hoehl for assistance in preparing graphics.

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Authors and Affiliations

  1. Ribozyme Pharmaceuticals, Inc., 2950 Wilderness Place, Boulder, 80301, CO

    Narendra K. Vaish, Fang Dong, Lori Andrews, Lawrence Blatt & Scott D. Seiwert

  2. Howard Hughes Medical Institute, University of Colorado at Boulder, Boulder, 80309-0215, CO

    Rebecca E. Schweppe & Natalie G. Ahn

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  1. Narendra K. Vaish
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  2. Fang Dong
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Correspondence to Narendra K. Vaish or Scott D. Seiwert.

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Vaish, N., Dong, F., Andrews, L. et al. Monitoring post-translational modification of proteins with allosteric ribozymes. Nat Biotechnol 20, 810–815 (2002). https://doi.org/10.1038/nbt719

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