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

Takashi Sera, Carla Uranga

Research output: Contribution to journalArticle

79 Citations (Scopus)

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 α-helical region of the zinc-finger domain (position 1 is the starting amino acid in the α-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.

Original languageEnglish
Pages (from-to)7074-7081
Number of pages8
JournalBiochemistry
Volume41
Issue number22
DOIs
Publication statusPublished - Jun 4 2002
Externally publishedYes

Fingerprint

Zinc Fingers
Zinc
Proteins
Fingers
DNA sequences
Amino Acids
DNA
Protoplasts
DNA-Binding Proteins
Guanine
Protein Transport
Reporter Genes
Arabidopsis
Gene expression
Transcriptional Activation
Assays
Genes
Chemical activation
Gene Expression

ASJC Scopus subject areas

  • Biochemistry

Cite this

Rational design of artificial zinc-finger proteins using a nondegenerate recognition code table. / Sera, Takashi; Uranga, Carla.

In: Biochemistry, Vol. 41, No. 22, 04.06.2002, p. 7074-7081.

Research output: Contribution to journalArticle

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