[2] Modification of the endogenous DNA-binding zinc finger domain is the basis of the most advanced field in construction of gene-specific artificial transcription factors.
[4] Depending upon the requirements of the investigation, there are several techniques available to define a DNA-recognition domain that will confer the specificity of a ZFP-based transcription factor.
Three phage display strategies have been described, involving either parallel, sequential or bipartite selection of the constituent zinc fingers.
On this basis, existing predetermined domains should be usable with no additional design or selection, making it a rapid and accessible technique to any laboratory.
[5][6] This is not true in every case, such that this strategy is liable to suffer issues related to target-site overlap at a number of target sequences, as discussed later.
If necessary, it may be possible to surmount the problem of target site overlap by randomising the amino acid residues at the interface of two zinc fingers at which it occurs.
1 (B)), put forward by the Pabo group in 1997 embraces the cooperative binding between zinc fingers to produce DNA-binding domains of great affinity and specificity.
The domain with the best binding characteristics is selected and then included in another library in which the finger-one anchor is removed and another randomised finger is added to the opposite end.
The main drawback of this approach is the necessity to produce a separate library for each zinc finger, which exceeds the capacity of most laboratories.
In order to keep the library size within reasonable limits, this technique is restricted to randomisation of only the key residues involved in base recognition.
The oligonucleotide used may be synthesised to include a primary n‑hexyl amino group at its 5' end, later utilised to attach bovine serum albumin (BSA).
As an alternative to BSA, the hairpin target DNA may be biotinylated and later extracted using streptavidin-coated magnetic beads (streptavidin forms very strong bonds with biotin).
Subsequent rounds of panning involve increasing concentrations of specifically synthesised non-target oligonucleotides where all but the sequence of the target subsite remains the same, down to a single nucleotide difference.
Generating arrays of engineered Cys2His2 zinc fingers is the most developed method for creating proteins capable of targeting desired genomic DNA sequences.
[12] The structure of this protein bound to DNA was solved in 1991[13] and stimulated a great deal of research into engineered zinc finger arrays.
Arrays with 6 zinc finger motifs are particularly attractive because they bind a target site that is long enough to have a good chance of being unique in a mammalian genome.
A recent study demonstrated that a high proportion of 3-finger zinc finger arrays generated by modular assembly fail to bind their intended target with sufficient affinity in a bacterial two-hybrid assay and fail to function as zinc finger nucleases, but the success rate was somewhat higher when sites of the form GNNGNNGNN were targeted.
A promising new method to select novel 3-finger zinc finger arrays utilizes a bacterial two-hybrid system and has been dubbed "OPEN" by its creators.
Zinc finger nucleases have become useful reagents for manipulating genomes of many higher organisms including Drosophila melanogaster, Caenorhabditis elegans, tobacco, corn,[23] zebrafish,[38] various types of mammalian cells,[39] and rats.
[40] An ongoing clinical trial is evaluating Zinc finger nucleases that disrupt the CCR5 gene in CD4+ human T-cells as a potential treatment for HIV/AIDS.
Whilst purification is not necessary for multitarget ELISA, it is essential for measuring binding affinity by plasmon resonance and DNase footprints.
It can be performed using a Heparin-Sepharose FPLC column equilibrated with zinc buffer followed by confirmation of homogeneity by SDS PAGE gel densitometry[4] The same techniques are used to examine the binding properties of completed polydactyl ZFP chimera[42] The specificity of ZFPs selected by phage display, is tested using a multitarget enzyme-linked immunosorbent assay (ELISA).
[10] Alternatively, Kd can be calculated from a gel mobility shift assay in which the same purified protein is incubated with serial dilutions of gel-purified, 32P-end-labelled target oligonucleotide.