Monitoring post-translational modification of proteins with allosteric ribozymes |
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Authors: | Vaish Narendra K Dong Fang Andrews Lori Schweppe Rebecca E Ahn Natalie G Blatt Lawrence Seiwert Scott D |
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Affiliation: | Ribozyme Pharmaceuticals, Inc., 2950 Wilderness Place, Boulder, CO 80301, USA. VaishN@rpi.com |
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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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