Monitoring post-translational modification of proteins with allosteric ribozymes.
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.
Ribozyme Pharmaceuticals, Inc., 2950 Wilderness Place, Boulder, CO 80301, USA. VaishN@rpi.com
Narendra K
NK
Vaish
Fang
F
Dong
Lori
L
Andrews
Rebecca E
RE
Schweppe
Natalie G
NG
Ahn
Lawrence
L
Blatt
Scott D
SD
Seiwert
D000494:Allosteric Regulation; D000818:Animals; D001483:Base Sequence; D002457:Cell Extracts; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D019950:Mitogen-Activated Protein Kinase 1 / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D016337:RNA, Catalytic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D013997:Time Factors
D002457:Cell Extracts; D011506:Proteins; D016337:RNA, Catalytic; C427204:hammerhead ribozyme; D019950:Mitogen-Activated Protein Kinase 1