Rational design of artificial zinc-finger proteins using a nondegenerate recognition code table.
We have developed a novel and simple method to rationally design artificial zinc-finger proteins (AZPs) targeting diverse DNA sequences using a nondegenerate recognition code table. The table was constructed based on known and potential DNA base-amino acid interactions. The table permits identification of an amino acid for each position (-1, 2, 3, and 6) of the alpha-helical region of the zinc-finger domain (position 1 is the starting amino acid in the alpha-helix) from overlapping 4-bp sequences in a given DNA target. Based on the table, we designed ten 3-finger AZPs, each of which targeted an arbitrarily chosen 10-bp DNA sequence, and characterized the binding properties. In vitro DNA-binding assays showed five of the AZPs tightly and specifically bound to their targets containing more than three guanine bases in the first 9-bp region. In addition, 6-finger AZPs, each of which was produced by combining two functional 3-finger AZPs, bound to their 19-bp targets with the dissociation constant of less than 3 pM. The in vivo functionality of the AZP was tested using Arabidopsis protoplasts. The AZP fused to a transcriptional activation domain efficiently activated expression of a reporter gene linked to a native promoter containing the AZP target site. Our simple AZP design method will provide a powerful approach to manipulation of endogenous gene expression by enabling rapid creation of numerous artificial DNA-binding proteins.
Sera
Takashi
T
Torrey Mesa Research Institute, 3115 Merryfield Row, Suite 100, San Diego, California 92121, USA. takashi.sera@syngenta.com
Uranga
Carla
C
D017360:Arabidopsis / Q000235:genetics* / Q000378:metabolism; D004247:DNA / Q000138:chemical synthesis* / Q000235:genetics; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics; D015195:Drug Design; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation / Q000502:physiology; D005813:Genes, Synthetic / Q000502:physiology; D011523:Protoplasts / Q000502:physiology; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology; D016335:Zinc Fingers / Q000235:genetics* / Q000502:physiology
D004268:DNA-Binding Proteins; D014157:Transcription Factors; D004247:DNA