Design of polydactyl zinc-finger proteins for unique addressing within complex genomes

Abstract

Zinc-finger proteins of the Cys 2 -His 2 type represent a class of malleable DNA-binding proteins that may be selected to bind diverse sequences. Typically, zinc-finger proteins containing three zinc-finger domains, like the murine transcription factor Zif268 and the human transcription factor Sp1, bind nine contiguous base pairs. To create a class of proteins that would be generally applicable to target unique sites within complex genomes, we have utilized structurebased modeling to design a polypeptide linker that fuses two three-finger proteins. Two six-fingered proteins were created and demonstrated to bind 18 contiguous bp of DNA in a sequence-specific fashion. Expression of these proteins as fusions to activation or repression domains allows transcription to be specifically up-or down-modulated within human cells. Polydactyl zinc-finger proteins should be broadly applicable as genome-specific transcriptional switches in gene therapy strategies and the development of novel transgenic plants and animals.From the simplest of organisms to the most complex, transcriptional regulation is achieved primarily by proteins that bind nucleic acids. The advent of genomic sequencing and the availability of the complete sequences of several genomes provide new opportunities to study biology and to develop therapeutic strategies through specific modulation of the transcription of target genes. Of the DNA-binding motifs that have been manipulated by design or selection, the TFIIIA-related Cys 2 -His 2 zinc-finger proteins have demonstrated the greatest potential for manipulation into general and specific transcription factors (1, 2). Each Cys 2 -His 2 zinc-finger domain consists of approximately 30 amino acids and typically binds 3 base pairs of double-stranded DNA sequence (3, 4).Specific delivery of a DNA-binding protein to a single site within a genome as complex as that found in humans, 3.5 billion bp, requires an address of at least 16 bp. Statistically assuming random base distribution, a unique 16-bp sequence will occur only once in 4 16 or 4.3 billion nucleotides, roughly the same size of a human genome (3.5 ϫ 10 9 bp). An 18-bp address would be specific within 68 billion base pairs of sequence. The 18-bp address could be specified by a protein containing six zinc fingers if the periodicity of the protein domains could match that of the DNA over this extended sequence. An address of this length would be sufficient to uniquely specify any locus within all known genomes. Although natural proteins containing long polydactyl arrays of zinc-finger domains have been inferred from sequence, no zinc-finger proteins have been demonstrated to bind such a long, contiguous DNA sequence. Structural studies of the five-finger human glioblastoma (GLI) protein-DNA complex (5) and biochemical studies of the nine-finger protein TFIIIA (6, 7) have demonstrated that DNA binding in these polydactyl proteins is dominated by the interactions of a select few fingers. Sequence-specific binding of more than three contiguous zinc-fin...

The artificial zinc finger coding gene ‘Jazz’ binds the utrophin promoter and activates transcription

“…Notably, the complex Jazz/'J' target shows a relative Kd of 32 nm comparable to the value determined for several natural and synthetic zinc finger proteins. 5,7,19,20 These data indicate that the complex Jazz/'J' target is almost as stable as other natural and synthetic zinc finger proteins associated with their corresponding DNA targets. One of the crucial points in designing and selecting synthetic zinc finger peptides is the calibration of correct binding affinity/specificity.…”

“…[1][2][3][4][5][6][7] In the large class of proteins capable of binding DNA, zinc finger proteins have been preferred for the design of artificial transcription factors on the basis of structural plasticity and modularity. [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] As a result of the work of several research groups, a code that relates the amino-acid sequence of a single zincfinger domain to its associated DNA nucleotide triplet target has been established. This 'code' is described as 'syllabic', depending not only on invariant amino acid/base pair correspondence, but also on structural characteristics of every single zinc finger module and the context in which it is found.…”

Drug-inducible and simultaneous regulation of endogenous genes by single-chain nuclear receptor-based zinc-finger transcription factor gene switches

“…[3][4][5][6][7] Typically, designer TF ZF s are composed of up to six ZF domains; a six-finger ZF binds to 18 bp of DNA; this allows recognition of a unique sequence within the human genome. 8 When combined with repressor or activator domains, ZF proteins can be used to regulate transcription of genes. For instance, to regulate the ErbB-2/HER-2 gene, the E2C polydactyl ZF was designed to bind to a specific sequence within the promoter.…”

End effects in DNA recognition by zinc finger arrays

“…A related question is the extent to which extra fingers added to a protein such as Zif268 would increase affinity for a DNA site by extending the core of the complex. Interestingly, a recently constructed hexadactyl protein binds its DNA half-site with only a 10-fold lower K d relative to the full-length site (27). As zinc finger binding has been observed to unwind DNA (28), the benefit of extending the array of intermolecular contacts may be overcome by the energetic penalty of distorting the DNA structure.…”