Rational design of artificial zinc-finger proteins using a nondegenerate recognition code table

Biochemistry. 2002 Jun 4;41(22):7074-81. doi: 10.1021/bi020095c.

Abstract

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.

MeSH terms

  • Arabidopsis / genetics*
  • Arabidopsis / metabolism
  • DNA / chemical synthesis*
  • DNA / genetics
  • DNA-Binding Proteins / biosynthesis*
  • DNA-Binding Proteins / genetics
  • Drug Design
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Gene Expression Regulation / physiology
  • Genes, Synthetic / physiology
  • Protoplasts / physiology
  • Transcription Factors / biosynthesis
  • Transcription Factors / genetics
  • Transcription Factors / physiology
  • Zinc Fingers / genetics*
  • Zinc Fingers / physiology

Substances

  • DNA-Binding Proteins
  • Transcription Factors
  • DNA