GCT - GCG - 3 ′ over the nonpermuted site. The results indicated that observa-
tions on the specifi city of zinc fi ngers can be extended to generalized zinc - fi nger
structures and realized the use of zinc fi ngers for the design of site - specifi c
DNA - binding proteins.^36
The Pabo group has described a method for selecting DNA - binding pro-
teins that recognize desired DNA sequences.^37 The research began with amino
acid sequences and secondary structure of the three Zif268 zinc fi ngers, Zif1,
Zif2, and Zif3. These are shown schematically in Figure 2.23A as adapted from
reference 37. Figure 2.23B shows how the residues in the α - helical regions of
fi ngers Zif1, Zif2, and Zif3 interact with 3 base - pair subsites in the bound
DNA. The bold numbers above the helical residue positions denote the residue
within the helix, with − 1 denoting a residue outside the N - terminal end of the
helix. Note that many contacts are from arginine (R) residues to guanines on
the same (primary) DNA strand. Other contacts from position 2 of the α -
helical portion often contact with bases on the complementary strand at posi-
tions not within the 3 - base - pair subsite. In this research the amino acid residues
inα - helical portions of Zif268 ′ s Zif1, Zif2, and Zif3 zinc fi ngers known to make
contact with DNA were randomized into a library. The proteins of the library
then were subjected to multiple cycles of selection and amplifi cation by genetic
engineering techniques calledphage display. Phage display has proven to be
a powerful enabling technology in genomics and drug development. It allows
the directed evolution of proteins engineered for specifi c properties and selec-
tivity and provides an approach for the engineering of human antibodies, as
well as protein ligands, and for such diverse applications as arrays, separations,
and drug development. The use of phage display in screening for novel high -
affi nity ligands and their receptors has been useful in functional genomics and
proteomics. Display methods promise to have benefi t in the development of
therapeutics targeting many different disorders, including cancer, AIDS, auto-
immune disorders, and other diseases. In the Pabo group ’ s work the phage
display - selected proteins were successively substituted for Zif1, Zif2, and Zif3
in binding to different DNA strands such as the so - called TATA box sequence
3 ′ - GAAAATATCGG - 5 ′ shown in Figure 2.23C. The protein sequences were
deemed successful if they would bind tightly to the selected DNA sequences —
that is, bind with nanomolar dissociation constants and discriminate at greater
than 20,000 - fold in binding to nonspecifi c DNA sequences. The authors believe
that the protocol described could be adapted to fi nding zinc fi ngers capable
of binding to many different DNA - and RNA - binding domains and also that
their sequential selection strategy could be applicable to the designing of zinc -
fi nger proteins to be used in gene therapy. Reference 34b discusses some
recent developments in this research area.
Various other research groups have been working since the early 1990s
toward rule formation for zinc - fi nger proteins and their interactions with
DNA. Researchers believe that there are key amino acid positions on the
zinc - fi nger protein that interact with base or phosphate positions on DNA in
similar ways for different zinc - fi nger – DNA systems. These positions may form
ZINC-FINGER PROTEINS 69