Toward controlling gene expression at will: Selection and design of zinc finger domains recognizing each of the 5′-GNN-3′ DNA target sequences

Abstract

We have taken a comprehensive approach to the generation of novel DNA binding zinc finger domains of defined specificity. Herein we describe the generation and characterization of a family of zinc finger domains developed for the recognition of each of the 16 possible 3-bp DNA binding sites having the sequence 5-GNN-3. Phage display libraries of zinc finger proteins were created and selected under conditions that favor enrichment of sequence-specific proteins. Zinc finger domains recognizing a number of sequences required refinement by site-directed mutagenesis that was guided by both phage selection data and structural information. In many cases, residues not expected to make basespecific contacts had effects on specificity. A number of these domains demonstrate exquisite specificity and discriminate between sequences that differ by a single base with >100-fold loss in affinity. We conclude that the three helical positions ؊1, 3, and 6 of a zinc finger domain are insufficient to allow for the fine specificity of the DNA binding domain to be predicted. These domains are functionally modular and may be recombined with one another to create polydactyl proteins capable of binding 18-bp sequences with subnanomolar affinity. The family of zinc finger domains described here is sufficient for the construction of 17 million novel proteins that bind the 5-(GNN) 6 -3 family of DNA sequences. These materials and methods should allow for the rapid construction of novel gene switches and provide the basis for a universal system for gene control.

Re-activation of a dormant tumor suppressor gene maspin by designed transcription factors

“…A BLAST-Search on the human genome has revealed that the three 18-bp targeted sites in the maspin promoter are unique (data not shown). Targets À126 and À97 were selected based on their close proximity to the transcription start site and because of the highly specific interactions predicted for the DNA triplets and the available ZF lexicons (Segal et al, 1999;Dreier et al, 2000Dreier et al, , 2002Dreier et al, , 2005. In addition to the proximal ATF sites, we have designed an ATF recognizing an 18-bp site comprising the hormone response element (HRE) sequence, which interacts with the hormone receptor both in breast (Khalkhali-Ellis et al, 2004) and prostate (Zhang et al, 1997) cells.…”

“…(b) a-Helical ZF sequences engineered to bind their corresponding target DNA triplets. These ZF sequences were chosen based on the available specific ZF lexicons (Segal et al, 1999;Dreier et al, 2000Dreier et al, , 2001Dreier et al, , 2005. The 6ZF ATFs were constructed by overlapping PCR; Supplementary methods).…”

Structure-based redesign of the dimerization interface reduces the toxicity of zinc-finger nucleases

“…A single zinc finger motif consists of 28 amino acids and includes an α-helix that contacts three bases in the major groove of DNA. Through modular assembly of such zinc finger modules, artificial DNA-binding domains can be created to bind to a wide 15 variety of DNA sequences [10][11][12][13][14] . A DNA-binding domain consisting of three zinc finger motifs has hence the potential to bind 9 bp of DNA.…”

Benzoate X receptor zinc-finger gene switches for drug-inducible regulation of transcription

“…7 ZFs are modular proteins that can be combined to create chains that recognize longer DNA sequences. [8][9][10] To date, ZF domains that recognized almost all of the GNN, ANN and CNN DNA triplets have been identified. [10][11][12][13][14] Approaches other than modular assembly also provide access to polydactyl ZF proteins.…”

“…[8][9][10] To date, ZF domains that recognized almost all of the GNN, ANN and CNN DNA triplets have been identified. [10][11][12][13][14] Approaches other than modular assembly also provide access to polydactyl ZF proteins. 15 Artificial transcription factors that use ZFs for DNA recognition typically use six ZFs to recognize 18 bases of DNA; this length of sequence is theoretically unique in the genome.…”