title: Creating a TALE protein with unbiased 5'-T binding.
abstract: Transcription activator-like effectors (TALEs) are convenient tools for genome engineering at specific genomic sites. However, their use is constrained because most TALE binding sites are preceded by a highly conserved 5' terminal T nucleotide (5'-T). To remove the 5'-T constraint, we substituted tryptophan 232 in the repeat-1 loop region of the dHax3 N-terminal domain for other amino acids. Furthermore, we randomized four amino acid residues of the hairpin loop region of repeat-1. Although point mutation was insufficient to remove the 5'-T constraint, directed evolution from the randomized library yielded repeat-1 mutants with unbiased targeting sites for 5'-bases. Our result indicates that the repeat-1 loop region of dHax3 is important for 5'-base accommodation, and that molecular evolution of repeat-1 of TALEs is an efficient strategy to remove the 5'-T constraint and thus allow targeting of any DNA sequences.
authors.affiliation: Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan.
authors.forename: Shogo
authors.initials: S
authors.lastname: Tsuji
authors.forename: Shiroh
authors.initials: S
authors.lastname: Futaki
authors.forename: Miki
authors.initials: M
authors.lastname: Imanishi
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D019020:Directed Molecular Evolution; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D017354:Point Mutation / Q000235:genetics; D011485:Protein Binding; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics; D013936:Thymidine / Q000378:metabolism*; D015534:Trans-Activators / Q000737:chemistry / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
chemical_list: D004268:DNA-Binding Proteins; D015534:Trans-Activators; D013936:Thymidine
keywords: DNA binding; Directed evolution; Transcriptional activator-like effector (TALE)