12.3 Design of DNA‐Binding roteins and Target Sites 249Taken together, the described results indicate that (i) zinc fingers retained
DNA‐binding activity when fused to different proteins and (ii) two orthogonal
zinc finger domains can simultaneously bind their target sequences in a DNA
scaffold and bring their fused protein domains into close proximity as evidenced
by the YFP reassembly.
12.3.2 TAL‐DNA Binding Domains
The recent discovery of the code underlying the nucleotide sequence recognition
by TAL effectors allows the design of protein domains that can bind to almost
any nucleotide sequence [20] (Chapter 13). Similar to the zinc finger proteins,
the TAL protein domains also seem to be ideal DNA‐binding proteins for use in
DNA scaffold applications. The typical TAL recognition site of 15–20 nucleo
tides is more than sufficient to provide the specificity required to build DNA
scaffolds, even when taking into account any cross‐interaction with similar
(a)
(b) (c)Zif PBSnYFPcYFPZif PBSnYFPcYFPFluorescence No fluorescenceSPR analysisResponse020406080100120β-GalactozidaseactivityDNA
TargetControl1% arabinose020406080100120β-GalactozidaseactivityArabinose − +1% arabinose 1% arabinose
b lacZaraCzfAAa lacZaraCzfAAAβ-Galactosidase0% arabinose
a lacZaraCzfAβ-GalactosidaseAFigure 12.5 Targeting DNA in vitro and in vivo with zinc finger domains. (a) The binding
affinity of zinc finger domains (e.g., Zif268) to their specific nucleotide target sequence was
determined using surface plasmon resonance (SPR). With increasing concentrations of
purified zinc finger protein, the response signal increases, indicating protein binding.
(b) The zinc finger domain (PBSII) was fused to the N‐terminal half (PBSII‐nYFP), and Zif268
was fused to the C‐terminal half (cYFP‐Zif268) of the yellow fluorescent protein (YFP).
Purified PBSII‐nYFP and cYFP‐Zif268 protein chimeras were mixed, either with DNA scaffolds,
containing PBSII, or Zif268 target sites separated by 2‐bp spacer, or DNA scaffolds with
random nucleotide sequences. Fluorescence was then measured [11]. (c) The binding of the
DNA‐binding domain (e.g., Zif268) in vivo was tested with the inhibition of β‐galactosidase
expression. The expression of the tested zinc finger was under the control of an arabinose‐
inducible promoter. The lacZ gene was controlled by the PSYN promoter, which contained
either the zinc finger target site or random DNA target site (CTCTATCAATGATAGAG).
β‐Galactosidase activity is measured in the presence of 1% or absence (0%) of arabinose and
normalized to the galactosidase levels of the unrepressed state. The β‐galactosidase activity is
detected when the DNA‐binding protein (e.g., zinc finger A) is not expressed (no arabinose).
Arabinose induces the expression of zinc finger A, which binds to the DNA‐target site “a”
upstream of the β‐galactosidase gene, and represses the expression of β‐galactosidase.
If the DNA‐target site “b” is not recognized by the DNA‐binding protein, the expression of
β‐galactosidase is not affected.