title: RasIns: Genetically Encoded Intrabodies of Activated Ras Proteins.
abstract: K- and H-Ras are the most commonly mutated genes in human tumors and are critical for conferring and maintaining the oncogenic phenotype in tumors with poor prognoses. Here, we design genetically encoded antibody-like ligands (intrabodies) that recognize active, GTP-bound K- and H-Ras. These ligands, which use the 10th domain of human fibronectin as their scaffold, are stable inside the cells and when fused with a fluorescent protein label, the constitutively active G12V mutant H-Ras. Primary selection of ligands against Ras with mRNA display resulted in an intrabody (termed RasIn1) that binds with a KDof 2.1μM to H-Ras(G12V) (GTP), excellent state selectivity, and remarkable specificity for K- and H-Ras. RasIn1 recognizes residues in the Switch I region of Ras, similar to Raf-RBD, and competes with Raf-RBD for binding. An affinity maturation selection based on RasIn1 resulted in RasIn2, which binds with a KDof 120nM and also retains excellent state selectivity. Both of these intrabodies colocalize with H-Ras, K-Ras, and G12V mutants inside the cells, providing new potential tools to monitor and modulate Ras-mediated signaling. Finally, RasIn1 and Rasin2 both display selectivity for the G12V mutants as compared with wild-type Ras providing a potential route for mutant selective recognition of Ras.
authors.affiliation: Department of Molecular and Computational Biology, University of Southern California, Los Angeles, CA 90089, USA.
authors.forename: Mehmet
authors.initials: M
authors.lastname: Cetin
authors.affiliation: Department of Chemistry, University of Southern California, Los Angeles, CA 90089, USA.
authors.forename: William E
authors.initials: WE
authors.lastname: Evenson
authors.affiliation: Department of Molecular and Computational Biology, University of Southern California, Los Angeles, CA 90089, USA.
authors.forename: Garrett G
authors.initials: GG
authors.lastname: Gross
authors.affiliation: Mork Family Department of Chemical Engineering and Materials Science, University of Southern California, Los Angeles, CA 90089, USA.
authors.forename: Farzad
authors.initials: F
authors.lastname: Jalali-Yazdi
authors.affiliation: Department of Chemistry, University of Southern California, Los Angeles, CA 90089, USA.
authors.forename: Daniel
authors.initials: D
authors.lastname: Krieger
authors.affiliation: Department of Molecular and Computational Biology, University of Southern California, Los Angeles, CA 90089, USA.
authors.forename: Don
authors.initials: D
authors.lastname: Arnold
authors.affiliation: Department of Chemistry, University of Southern California, Los Angeles, CA 90089, USA.
authors.forename: Terry T
authors.initials: TT
authors.lastname: Takahashi
authors.affiliation: Department of Molecular and Computational Biology, University of Southern California, Los Angeles, CA 90089, USA; Department of Chemistry, University of Southern California, Los Angeles, CA 90089, USA; Mork Family Department of Chemical Engineering and Materials Science, University of Southern California, Los Angeles, CA 90089, USA; USC Norris Comprehensive Cancer Center, Los Angeles, CA 90089, USA. Electronic address: richrob@usc.edu.
authors.forename: Richard W
authors.initials: RW
authors.lastname: Roberts
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000235:genetics* / Q000378:metabolism; D019556:COS Cells; D002522:Chlorocebus aethiops; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D009154:Mutation; D011487:Protein Conformation; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D018631:ras Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D000906:Antibodies; D012333:RNA, Messenger; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D018631:ras Proteins
keywords: E10FnIII; Ras; fibronectin; intrabody; mRNA display