Covalent tagging a DNA to its expressed protein in vitro

DESCRIPTION (provided by applicant): A novel method to covalently tag a DNA molecule to its own in vitro translated polypeptide is proposed. Based on this method, a convenient reagent kit can be developed that allows 1) easily generating a highly complex (up to 10exp15) DNA-tagged polypeptide library and 2) quickly identifying novel high affinity polypeptide ligands that recognize target molecules from this library using a simple PCR-based procedure. The kit will be a very useful tool to facilitate basic biomedical research in many areas that requires detection of disease markers. Furthermore, an automated ligand discovery process can also be developed based on this method. This automated process can provide a large collection of peptide ligands that detect cellular proteins for proteomic technologies such as protein array chips. These chips will be tremendously valuable to discover disease markers and drug targets. In this method, DNA that contains a binding motif and also encodes for a polypeptide is expressed in a coupled transcription /translation cell lysate. The unique feature of the expressed polypeptide is that it contains a capturing domain that binds to and forms a covalent bond with the binding motif on the DNA. The proposed study is to demonstrate that 1) the in vitro translated polypeptide can be tagged with its own encoding DNA via the joint of binding motif and capturing domain, and 2) a DNA that encodes for a target binding protein can be selectively enriched. The proposed study is intended to demonstrate the feasibility for developing the reagent kit for novel polypeptide ligand discovery using this method.