mRNA display selection of a high-affinity, modification-specific phospho-IκBα-binding fibronectin

The complexity of the human proteome is greatly expanded by post-translational modifications. New tools capable of recognizing these modifications in a sequence-specific fashion provide a route to purify these modified proteins, to alter protein trafficking, and to visualize signal transduction in real time. Here, we have evolved novel, modification-specific ligands that target phosphorylated I kappa B alpha. To do this, we employed mRNA display-based in vitro selection using a 30-trillion-member protein library based on the fibronectin type III domain. The selection yielded one fibronectin molecule, 10C17C25, that binds a phospho-I kappa B alpha peptide with K-d = 18 nM and is over 1000-fold specific compared to the nonphosphorylated peptide. 10C17C25 specifically recognizes endogenous phosphorylated I kappa B alpha from mammalian cell extract and stabilizes phospho-I kappa B alpha in vivo. We also incorporated 10C17C25 into a FRET indicator that detects I kappa B kinase (IKK) activity in vitro, demonstrating the utility of selecting designed adaptors for kinase activity sensors.