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RNA aptamers with a nity for the Ras-binding domain (RBD) of Raf-1 were isolated from a degenerate pool by in vitroselection.These aptamers e ciently inhibited theRas interaction with the Raf-1 RBD, and also inhibited Ras-induced Raf-1 activation in a cell-free system. The RNA aptamer with the most potent inhibitory e ect speci®cally inhibited the RasáRaf-1 interaction and had no a nity for the RBD of the RGL protein, a homolog of the Ral GDP dissociation stimulator. | Eur. J. Biochem. 269 697-704 2002 FEBS 2002 Anti- Raf-l RNA aptamers that inhibit Ras-induced Raf-1 activation Michiko Kimoto1 2 Mikako Shirouzu2 3 Shin Mizutani4 Hiroshi Koide4 Yoshito Kaziro4 Ichiro Hirao5 and Shigeyuki Yokoyama1 2 3 5 1 Department of Biophysics and Biochemistry Graduate School of Science The University of Tokyo Bunkyo-ku Tokyo Japan 2Cellular Signaling Laboratory RIKEN Harima Institute Mikazuki-cho Sayo Hyogo Japan RIKEN Genomic Sciences Center Tsurumi Yokohama Japan 4Faculty of Bioscience and Biotechnology Tokyo Institute of Technology Yokohama Japan 5Yokoyama CytoLogic Project ERATO JST Wako-shi Saitama Japan RNA aptamers with affinity for the Ras-binding domain RBD of Raf-1 were isolated from a degenerate pool by in vitro selection. These aptamers efficiently inhibited the Ras interaction with the Raf-1 RBD and also inhibited Ras-induced Raf-1 activation in a cell-free system. The RNA aptamer with the most potent inhibitory effect specifically inhibited the Ras-Raf-1 interaction and had no affinity for the RBD of the RGL protein a homolog of the Ral GDP dissociation stimulator. Although the aptamer was capable of binding to the B-Raf RBD the RNA did not inhibit the interaction between Ras and the B-Raf RBD. Enzymatic and chemical probing experiments indicated that the aptamer was folded into a pseudoknot structure and some loop regions of the pseudoknot were located at the binding interface for the Raf-1 RBD. Keywords Raf-1 Ras RNA aptamer in vitro selection. The Raf-1 protein is a cytoplasmic serine threonine kinase that transmits cellular proliferative and developmental signals from the plasma membrane to the cytosol and the nucleus. Raf-1 phosphorylates and activates the MAPKK MEKs which in turn phosphorylate the MAPK ERKs 1 . Activated MAPK phosphorylates a number of proteins including protein kinases transcription factors and cyto-skeletal proteins and these proteins are thought to be critical for proliferation or differentiation. .
