Gradually assemble new protein at desired binding site. Add and optimise one finger at a time as they proceed to target site. Use Zif268 structure.
3 selection steps are used. One for each finger of new protein
1) A finger that recognises 3'end of target site is selected by phage display. 2 wt Zif fingers are used as temporary anchors to position library of randomised fingers over target site. Use hybrid DNA site with Zif subsites fused to target site
2) Selected finger is retained as part of growing protein. After distal Zif finger is discarded, phage display selects a new finger that recognises central region of target site.
3) Remaining Zif finger is discarded. Phage display selects a 3rd finger. It recogninses 5' region of target site. Optimisation yields new zn finger protein.
New fingers are selected in relevant structural context. An intact binding site is present at every stage. Selections are performed in context of growing protein-DNA complex. Selects for fingers that function well together. To ensure that selection proteins will bind tightly to desired target sites, do all seelctions in presence of calf thymus competitor DNA. This counterselects against proteins that bind promiscuously or prefer alternative sites.
Modular design of articifial transcirpiton factors by Ansari and Mapp
Modular nature of eukaryotic TFs
Natural transcriptional regulators have DNA binding domain and regulatory domain (DBD and RD). DBD causes pseicificty in targeting RD to spec site in genome. REgulatory mediate effects on gene where theyt are delivered.
DBD are characterised by structure (HTH) but RDs are catgegorised by abundance of amino acids eg acid rich, gluatmin-erich, prioline rich etc. RDs are characterised by functional context. Some activator and repressor modules are categorised according to distance from promoter where they funciton.
Modular design of ATFs
DNA binding domains
Protein DBDs are attractive targets as they have high specificity and affinity for target sequences. No clear recognition code has been discoverd. Use genetic selection to generate protein DNa binding modules for spec sequence. Zn finger DBDs that target unique promoters have been isoalted and function in cultured cell lines.
Regulatory domain: activation
Activatin modules with multiple acidic and hydrophobic residues function robustly in all eukaryotes tested. Often used in ATAFs. Designed zinc fingers fused to acidic activating domain eg VP16, a potent viral coactivator upregulates genes.
Regulatory domains: repression
Fuse large segments of repressor proteins to DBD. eg KRAB repressor moduls us fused to designed zinc fingers, a modest repression of adjacent gene.
Inhibit by competition for DNA binding site of an endogenous TF. Polyamides which targeted TF binding sites inhibited expression of HIV genes in cell culture.
Linking DNA binding and regulatory domains
For active activation or repression DBD and RD must be linked together to function as an ATF. Tether with peptide linekrs.
Traffic and delivery
Obstacle. Trafficking of polyamide ATFDs into cell nucleir depends on cell type/ TFO-bsed regulators are devliered by electroporation or cationic liposomes. Unsusatinable and limits tissues it can be targeted. Attaching NLS is effective.