The affinity and specificity of these new proteins can also be improved by linking multiple fingers together or by designing proteins that bind as dimers and thus recognize an extended site on the DNA.These new proteins can further be modified by adding other domains for the activation or repression of transcription, for DNA cleavage, or silencing through methylation [ Measurements of the affinity and specificity of a synthetically designed zinc finger protein can help in evaluating both the potential utility for biological applications and the efficiency of the design/selection process.
Most existing tools for predicting DNA-binding specificity in zinc fingers are trained on data obtained from naturally occurring proteins, thereby skewing the predictions.However, a ZFP with the highest affinity for a particular DNA site need not bind that site with the highest degree of specificity.These factors have important implications on the design of ZFPs with favourable DNA binding specificities and thus, highlight the importance of incorporating affinity, specificity, and environmental requirements in the design process as a whole .Although there is no simple, general code for zinc finger protein–DNA recognition, selection strategies have been developed that allow these proteins to be designed to target almost any desired site on double-stranded DNA.The Cys2His2 zinc finger proteins, and more often, Zif-268, offers a stable and versatile framework for the design of such proteins .As highlighted above, certain factors influencing the DNA-ZFP binding specificity are unpredictable.
With binding not being solely dependent on the affinity, the possibility of the ZFP binding to an unwanted DNA site crops up.
Analysis of the predictions made by all three strategies indicate strong dependence of zinc finger binding specificity on the amino acid propensity and the position of a 3-bp DNA sub-site in the target DNA sequence.
Moreover, the binding affinity of the individual zinc fingers was found to increase in the order Finger 1 The field of targeted genome engineering is still incipient, and, there is a compelling need to develop tools which can meet the ever growing requirements of the field: designing DNA templates of our choice, construction and manipulation of DNA sequences, and tools for the implementation, testing and debugging of genome editing experiments.
In an attempt to elucidate the mechanism of zinc finger protein-DNA interactions, we evaluated and compared three approaches, differing in the amino acid mutations introduced in the Zif-268 parent template, and the mode of binding they try to mimic, i.e., modular and synergistic mode of binding.
Comparative evaluation of the three strategies reveals that the synergistic mode of binding appears to mimic the ideal mechanism of DNA-zinc finger protein binding.
Here, we review the available methods to study HP-PPI, with a focus on recent mass spectrometry based methods to decipher bacterial–human infectious diseases and examine their relevance in uncovering host cell rewiring by pathogens.