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Design of novel sequence-specific DNA-binding proteins
David J Segal and Carlos F Barbas III*
The design and selection of DNA-binding proteins or individual
domains capable of novel sequence recognition continues to
make great strides. Recent studies have also highlighted the
role of the non-DNA-contacting portions of the protein and the
optimal assembly of the domains. For the first time, it appears
that it is possible to produce proteins capable of targeting any
gene with an 18 base pair recognition domain. A variety of
applications are being explored, such as targeted
transcriptional regulation, recombination and viral integration.
These proteins will probably find diverse applications in gene
therapy, functional genomics, and agriculture.
Addresses
The Skaggs Institute for Chemical Biology and the Department of
Molecular Biology, The Scripps Research Institute, BCC-515, North
Torrey Pines Road, La Jolla, CA 92037, USA
*e-mail: carlos@scripps.edu
Current Opinion in Chemical Biology 2000, 4:34–39
1367-5931/00/$ — see front matter © 2000 Elsevier Science Ltd.
All rights reserved.
Abbreviations
DBD
DNA-binding domain
ER
estrogen receptor
PR
progesterone receptor
Introduction
The design of proteins with the capacity to recognize
extended nucleotide sequences with high affinity and
specificity has been a goal of two decades of research; a
goal that has remained elusive until the past two years.
Ideally, proteins could be designed to specifically regulate the transcription of a single gene in a genome as
complex as our own four billion base pair genome. These
proteins would allow scientists to reach into genomes
and to modulate the transcription of a gene of interest, to
assign its function or to change the phenotype of
the organism.
Specific recognition of double-stranded DNA by proteins is most typically accomplished using an α-helix as
the key recognition element. An α-helix provides a rigid
scaffold from which amino-acid sidechains are presented
to the accessible edges of the bases in the major grove of
DNA. This presentation is modulated by ‘framework’
structural elements of the protein. For proteins in which
the α-helix is the key recognition element, the task of
creating DNA-binding proteins with novel sequence
specificity consists of modifying the DNA-contacting
residues in the α-helix, then modifying the framework to
optimize the display of the α-helix. In this review, we
discuss the recent successful application of these principles to create novel DNA-binding proteins, with
particular emphasis on the advances achieved using
Cys2-His2 zinc finger proteins.
Helical domains
For proteins that do not contact DNA with an organized
α-helix, success in generating novel sequence-specific proteins has been extremely limited. For example, the
base-specific hydrogen bonds between the restriction
endonuclease EcoRV and its recognition base pairs are
made through a highly cooperative set of interactions by
five residues in a surface loop of the enzyme (Figure 1a;
[1]). Through application of DNA shuffling, mutagenesis
and screening, Pingoud and co-workers [2••] were able to
extend recognition to include the base pairs immediately
flanking the EcoRV site. Although this is an impressive
study, it remains to be seen if more can be expected from
these types of complex recognition motifs.
A decade ago, the dimeric bZIP proteins were proposed as
candidates for the development of novel DNA-binding
proteins [3]. Each bZIP monomer contacts DNA with an
α-helix that is oriented and constrained by being part of
longer α-helix, which makes up the leucine zipper dimerization interface (Figure 1b; [4]). In vitro selection methods
produced mutants of the bZIP protein C/EBP that could
recognize sequences that differed in two of its five half-site
nucleotides [5]. Interestingly, some of the mutations
occurred in the hinge region between the α-helix of the
DNA-binding domain (DBD) and the leucine zipper
framework, suggesting that reorientation of the α-helix
may have been required.
Theoretically, DBDs of bZIP heterodimers, such as JunFos, could be modified to recognize novel 10-nucleotide,
non-palindromic sites. Alternatively, tandem monomers of
the bZIP DBDs could potentially be used to recognize
extended sequences, but these typically bind poorly to
DNA. In an elegant design approach, the affinity of a bZIP
monomer DBD, that of GCN4, was improved 270-fold by
grafting the DNA-contacting residues from the α-helix of
GCN4 onto the stable and compact scaffold of the avian
pancreatic polypeptide [6••]. This miniature 42-aminoacid protein, consisting of a single amphipathic α-helix
that is stabilized through hydrophobic interactions with a
type II polyproline helix, was able to bind a GCN4 half site
with an affinity and specificity greater than that of the
parental α-helix. This study again illustrates that binding
can be improved by optimizing not only the DNA-contacting residues but also the display and stability of the
α-helix. It remains unclear if these domains can be effectively linked as tandem multimers to recognize extended
sites. Chemical linkage of two mini-proteins provided a
‘dimer’ with increased affinity but lower specificity [6••].
Nuclear receptors, such as the estrogen receptor (ER)
and progesterone receptor (PR), also bind as dimers to
their DNA targets. Each monomer inserts an α-helix of a
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Design of novel sequence-specific DNA-binding proteins Segal and Barbas
35
Figure 1
DBDs that have been used to create proteins
with novel sequence specificity. (a) EcoRV
[1]. (b) bZIP of GCN4 [4]. (c) Cys4 zinc
fingers of glucocorticoid receptor [8].
(d) Helix-turn-helix domains of Myb [10].
(e) Cys2–His2 zinc fingers of Zif268 [13].
Double stranded DNA is shown in orange and
brown, DBD domains are shown in red, blue
or green ribbon representation, and zinc ions
are shown as yellow spheres.
(b)
(a)
(d)
(c)
(e)
Current Opinion in Chemical Biology
Cys4-type zinc finger into the major grove, and for some
receptors the DBD contains a strong dimerization interface (Figure 1c; [7,8]). Using in vivo survival-based
selection of randomized libraries, Shapiro and co-workers
[9••] found PR-DBD mutants that lost their ability to recognize the PR response element (PRE) but could bind
the ER response element (ERE), which differs in two of
the six half-site nucleotides. The mutants bound the
ERE with a 15-fold higher affinity than wild-type ERDBD, albeit with a specificity considerably broader than
wild-type ER-DBD.
From domains to proteins
Tandemly repeated elements, although having the potential
for extended, non-palindromic recognition, introduce another level of complexity: inter-domain cooperativity. One
domain may make protein–protein contacts with the next
domain, affect the binding geometry of adjacent domains, or
it may contact the nucleotides of another domain’s binding
site. These interactions that allow for concerted recognition
may be considered as evolutionary levers for optimizing the
binding of a particular protein; however, for those seeking to
design novel binding proteins these interactions present
additional challenges. For example, Myb domains
(Figure 1d) consist of two or three tandem repeats (designated R1, R2 and R3) of a helix-turn-helix motif, similar to
the motif found in the λ repressor and homeodomian proteins [10]. Myb DBDs with novel specificity have been
generated by combining the R2 and R3 of different species
[11]; however, the combination of two tandem R3 repeats
severely reduced binding affinity [12]. The authors concluded that cooperative interactions between the R2 and R3
domains were required for high affinity binding.
By any measure, the greatest success in producing proteins
with novel binding specificity has been achieved with the
classic Cys2–His2 zinc-finger domains (Figure 1e). These
zinc fingers are compact domains containing a single
amphipathic α-helix stabilized by two β-strands and zinc
ligation [13]. Like the Myb domains, zinc-finger proteins
contain multiple tandem repeats and display varying
degrees of inter-domain cooperativity. Fortunately, a subset
of zinc-finger proteins, including the murine transcription
factor Zif268 and the human protein Sp1, display only minimal — though still troublesome — cooperativity. In these
proteins, each finger recognizes a three nucleotide site with
relative independence, which has allowed several groups to
produce zinc fingers with novel specificities using rational
or combinatorial methods (reviewed in [14••]).
Early attempts to produce zinc fingers with novel
sequence recognition gave hope to the idea that there
might be a simple 1:1 amino acid to base recognition code
that could be used to build fingers that could specifically
recognize any three nucleotide sequence [15,16].
Unfortunately, this simplistic code is poorly predictive of
the actual specificity of these domains [17,18•]. In one
report of a code-based three-finger protein, only five of the
nine nucleotides appear to be correctly specified [17].
There are at least three reasons why a simple code is insufficient for comprehensive recognition. The first is
technical. Phage display has proven to be a powerful tool
for revealing the repertoire of zinc-finger–DNA interactions. This procedure involves the display of a large
(typically 107–109 member) library of randomized proteins
on the surface of a filamentous bacteriophage. Because
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Interaction, assembly and processing
Table 1
Affinities of sequentially-selected and stitched three-finger
proteins.
Protein
Target sequence
Published
KD (nM)‡
Normalized
KD (nM)§
Stitched study
Zif268*
B3*
C5*
E2C-HS2(SP1)*
GCG
GGA
GGA
GCC
TGG
GGG
GGC
GCA
GCG
GAC
GGG
GTG
10
4
30
25
10
4
30
25
Sequential study
Zif268†
TATAZF†
p53ZF†
NREZF†
GCG
GCT
GGG
AAG
GGG
ATA
ACA
GGT
GCG
AAA
TGT
TCA
0.010
0.12
0.11
0.038
10
120
110#
38
*Data from [23••]. †Data from [22]. ‡Standard deviation < 60%.
§Published values normalized to Zif268 = 10 nM. #p53
ZF was later
reported to bind with an affinity higher than NREZF [18•].
each phage contains the gene for the protein it displays,
the sequence of proteins having the desired properties can
be identified through repeated cycles of in vitro selection
and amplification of the phage library (known as ‘panning’). There is, however, a common misconception that
any sequence that is strongly selected from panning must
be the optimal sequence. The flaw in that logic is that the
researcher is not always aware of what the actual selection
pressure is during an experiment. Our approach [14••] has
been to optimize the output of phage display using sitedirected mutagenesis. This study revealed that some
residues were selected during panning because they
increased the affinity of the interaction at the cost of specificity. The end product of this study was a collection of 16
well-characterized domains recognizing each of the possible GNN binding sites. A number of these domains
demonstrated exquisite specificity and discriminated
between sequences that differ by one in nine bases with
> 100-fold loss in affinity.
Sequential selection versus stitchery
The second reason for the problems with simple coded interactions relates to cooperativity. A central theme in recent
zinc-finger research has been to refocus on the question: just
how independent are the zinc fingers of Zif268? An early
clue was the difficulty that every laboratory encountered in
selecting fingers that could recognize sequences of the type
ANN or CNN, instead of GNN — the sequence type recognized by the natural protein. With Zif268 as their scaffold
protein, a consensus in the field emerged that aspartate in
position 2 of one α-helix contacted the binding site of the
finger next to it, forcing recognition at that neighboring site
to be GNN or TNN [19,20•]. The significance of this contact was underappreciated in the original Zif268 crystal
structure [13] but is now widely acknowledged and constitutes what is known as the target site overlap problem in zinc
fingers [14••]. Limitations imposed by this contact can be
addressed by randomization of residues at the interface of
two α-helices including position 2 [20•,21], or by replacement of the offending domain with a finger that does not
contain aspartate in position 2 and is not expected to have
any cooperative effects.
Concerns regarding cooperativity can also be addressed by
the selection methodology if sequential selections are
applied [22]. In this approach (Figure 2a) finger 1 of a
three-finger protein is combinatorially selected in the context of two ‘anchor’ fingers. Subsequently, the terminal
anchor finger is removed, finger 1 becomes finger 2, and a
new finger 1 is selected to bind the next three nucleotides
in the DNA sequence. After one additional round of
exchanges, a new three-finger protein is created in which
all of the fingers have been selected in the ‘context’ of the
finger next to it. This is in contrast to direct selection of
defined fingers from a single library that are then ‘stitched’
together (or at least the contact residues) to make a new
three-finger protein (Figure 2b). The advantage of the
stitchery method is that once fingers of high affinity and
specificity are created, they can be rapidly reassembled to
recognize any desired sequence. However, this aim
requires that each finger be completely modular and independent. On the other hand, sequential selection requires
that three sequential libraries be constructed and selected
for each three-finger protein, making zinc-finger technology inaccessible to most researchers.
Is sequential selection necessary? Several stitched threefinger proteins have been constructed from an optimized
set of GNN-binding fingers [14••,23••]. In these fingers,
cooperativity effects caused by aspartate in position 2 are
minimized as all of the target sites are of the GNN type.
The published specificity of sequentially selected proteins
appears to be no better than that of the stitched proteins
[18•,23••]. The affinity of the sequentially selected proteins was reported to be in the sub-nanomolar range [22].
Regrettably, however, the issue of affinity has become
somewhat distorted in the zinc-finger field. As one example, the affinity of Zif268 for its operator ‘improved’
600-fold by modification of the binding measurements
[13,22]. Although the use of non-standard binding conditions when measuring affinities is legitimate, the values
obtained can not be directly compared to affinities measured in a more conventional way. A more accurate
comparison can be obtained by normalizing the published
affinities to a common value for Zif268 as this measurement is also reported. As shown in Table 1, the
sequentially selected proteins bind their target with a
lower affinity than the stitched proteins. On the basis of
this comparison the advantages of sequential selection are
not clear. The comparison is not entirely fair, however, as
the sequentially selected proteins contain fingers that recognize ANN and TNN sites, which may be inherently
more difficult for zinc fingers to bind with high affinity and
specificity. Studies in live cells suggest that specificity is a
problem as the TATA protein was not effective in reporter
gene studies [24].
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Design of novel sequence-specific DNA-binding proteins Segal and Barbas
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Figure 2
Sequential and parallel selection methods.
Boxes represent zinc finger domains. Letters
beneath the boxes represent DNA sequences.
See text for details.
(a) Sequential selection
(b)
Anchor
Anchor
Library
GCG
TGG
ABC
DEF
Anchor
ABC
Library
TGG
ABC
DEF
GCG
ABC
GCG
TGG
ABC
ABC
DEF
DEF
DEF
GHI
Parallel selection and stitchery
Anchor
Library
Anchor
GCG
ABC
GCG
Anchor
Library
Anchor
GHI
GCG
DEF
Library
Anchor
Library
GHI
GCG
GHI
GHI
ABC
DEF
GCG
Anchor
GCG
GHI
Current Opinion in Chemical Biology
Framework effects
A third reason for the problems with simple coded interactions is that a code fails to consider the influence of the
linker region, the β-strands, and the carboxy-terminal end
of the α-helix (collectively referred to as the framework).
Framework effects on specificity and affinity are only
beginning to be appreciated and will undoubtedly represent a new area of research in this field. Structures of
Zif268 mutants binding their designed targets clearly show
a repositioning of the α-helix relative to the DNA, suggesting that the α-helix might require reorientation for the
optimal presentation of its contacting residues to variant
DNA sites [25••]. Variation in helical presentation is also
clearly evident in recent structural studies of three-finger
and six-finger proteins derived from TFIIIA [26,27]. In our
own studies [23••], a 50-fold increase in affinity was
observed when the same six α-helices were displayed on
different frameworks. Ryan and Darby [28] reported an
eightfold loss in affinity and a 50-fold loss in specificity
after modifying only the linker regions of TFIIIA.
Appropriate display of the α-helix becomes particularly
important as the number of tandem domains is increased
to six, the number required to recognize a unique
sequence in the human genome [29]. No natural zinc finger proteins have been found that bind specifically to 18
contiguous nucleotides. Additionally, the DNA duplex is
unwound slightly upon binding, causing concern that the
contact residues in a long protein may come out of register
with the nucleotides, resulting in a loss in specificity and
affinity. Our studies [23••,29] suggest that the affinity of a
six-finger protein is highly dependant on the framework.
In another example, the use of an 11-amino-acid linker
peptide produced a six-finger protein with an affinity
> 6000-fold greater than that of its three-finger components [30•]. Framework mutations have tended to be
context-dependent in the zinc finger area and application
of this 11-amino-acid linker to other proteins has not
resulted in other examples with exceptional affinity.
Additional studies that correlate structure with quantitative affinity and specificity data will be required to further
reveal how framework effects influence the ability of zincfinger proteins to both recognize specific sequences and
exclude highly related ones.
Despite these complexities, a polydactyl protein specifically recognizing an 18-nucleotide sequence in the
5′-untranslated region of the human erbB-2 gene has been
constructed. This protein bound the target sequence with
0.5 nM affinity. When expressed as a fusion protein with
repressor or activator domains, the transcription factor was
able to specifically regulate the erbB-2 promoter [23••].
Extension of this work to the study of the regulation of the
endogenous erbB-2 gene has resulted in the first demonstration of the modulation of endogenous genes with
designed transcription factors (CF Barbas III, unpublished
data; see Note added in proof). These proteins were shown
to be effective in cells derived from three mammalian
species that conserve the erbB-2 binding site. Amazingly,
the erbB-3 gene is not regulated despite a 15 of 18
nucleotide match to the erbB-2 target site at the same relative position. Furthermore, construction of an
erbB-3-targetting transcription factor enables specific regulation of the gene as well. Thus it appears that
genome-specific regulation is now in reach.
Conclusions
With the demonstration that polydactyl zinc-finger proteins can be readily assembled to recognize 18 base-pair
sequences of the 5′-(GNN)6-3′ type, and that these proteins can specifically regulate an endogenous gene, much
additional study will be needed to extend the type of
sequences that can be addressed by this approach. Some
sequence motifs may require the use of other DNA-binding proteins or combinations of domains of different types.
This might be accomplished through the selection of novel
dimerizing regions that serve to link varied proteins noncovalently [31••]. Now that we have begun to be able place
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Interaction, assembly and processing
proteins at specific addresses within complex genomes,
what activities might these proteins direct? Given the
modular nature of proteins that act on DNA, a vast array of
tools that facilitate the manipulation of genomes is possible. Appending activation or repression domains onto a
DBD allows for transcriptional modulation [23••,29], while
endonuclease [32,33•], methylase [34], topoisomerase [35],
and HIV-1 integrase [36] domains have allowed for some
degree of site-directed action on DNA. Although many of
these novel functions are still too inefficient for practical
application, they are certain to have an impact on basic and
applied biology in this new era of the genome.
Note added in proof
The paper referred to in the text as (CF Barbas III, unpublished data) has now been accepted for publication [37].
References and recommended reading
Papers of particular interest, published within the annual period of review,
have been highlighted as:
• of special interest
•• of outstanding interest
1.
Winkler FK, Banner DW, Oefner C, Tsernoglou D, Brown RS,
Heathman SP, Bryan RK, Martin PD, Petratos K, Wilson KS: The
crystal structure of EcoRV endonuclease and of its complexes
with cognate and non-cognate DNA fragments. EMBO J 1993,
12:1781-1795.
2.
••
Lanio T, Jeltsch A, Pingoud A: Towards the design of rare cutting
restriction endonucleases: using directed evolution to generate
variants of EcoRV differing in their substrate specificity by two
orders of magnitude. J Mol Biol 1998, 283:59-69.
This paper describes one of the very few modifications of DNA recognition
in an enzyme using a non-α-helical recognition motif. DNA shuffling was
used to evolve the specificity of the enzyme to a longer sequence than its
native target. Extension of the sequence specificity of restriction enzymes to
longer sequences promises many practical applications in cloning and
recombination.
3.
4.
5.
O’Neil KT, Hoess RH, DeGrado WF: Design of DNA-binding
peptides based on the leucine zipper motif. Science 1990,
249:774-778.
Ellenberger TE, Brandl CJ, Struhl K, Harrison SC: The GCN4 basic
region leucine zipper binds DNA as a dimer of uninterrupted
α-helices: crystal structure of the protein–DNA complex. Cell
1992, 71:1223-1237.
Sera T, Schultz PG: In vivo selection of basic region-leucine zipper
proteins with altered DNA-binding specificities. Proc Natl Acad Sci
USA 1996, 93:2920-2925.
6.
••
Zondlo NJ, Schepartz A: Highly specific DNA recognition by a
designed miniature protein. J Am Chem Soc 1999, 121:69386939.
This study demonstrates minimalist design at its best. Simple grafting of the
recognition residues of GCN4 onto the well-studied avian pancreatic
polypeptide and a little fine-tuning provided a small 42-residue protein with
exquisite affinity and specificity for the target DNA, much greater than the
native protein.
7.
8.
9.
••
Schwabe JW, Chapman L, Finch JT, Rhodes D: The crystal structure
of the estrogen receptor DNA-binding domain bound to DNA: how
receptors discriminate between their response elements. Cell
1993, 75:567-578.
Luisi BF, Xu WX, Otwinski Z, Freedman LP, Yamamoto KR, Sigler PB:
Crystallographic analysis of the interaction of the glucocorticoid
receptor with DNA. Nature 1991, 352:497-505.
Chusacultanachai S, Glenn KA, Rodriguez AO, Read EK, Gardner JF,
Katzenellenbogen BS, Shapiro DJ: Analysis of estrogen response
element binding by genetically selected steroid receptor DNA
binding domain mutants exhibiting altered specificity and
enhanced affinity. J Biol Chem 1999, 274:23591-23598.
This study is the most extensive analysis of the elements of recognition for
Cys4-type zinc-finger binding motifs. Novel randomization and in vivo selec-
tion methods are described. The authors are poised to discover if these Cys4
zinc fingers are capable of the versatility of their Cys2–His2 cousins.
10. Ogata K, Morikawa S, Nakamura H, Sekikawa A, Inoue T, Kanai H,
Sarai A, Ishii S, Nishimura Y: Solution structure of a specific DNA
complex of the Myb DNA-binding domain with cooperative
recognition helices. Cell 1994, 79:639-648.
11. Williams CE, Grotewold E: Differences between plant and animal
Myb domains are fundamental for DNA binding activity, and
chimeric Myb domains have novel DNA binding specificities.
J Biol Chem 1997, 272:563-571.
12. Oda M, Furukawa K, Sarai A, Nakamura H: Construction of an
artificial tandem protein of the c-Myb DNA-binding domain and
analysis of its DNA binding specificity. Biochem Biophys Res
Commun 1999, 262:94-97.
13. Pavletich NP, Pabo CO: Zinc finger-DNA recognition: crystal
structure of a Zif268-DNA complex at 2.1 Å. Science 1991,
252:809-817.
14. Segal DJ, Dreier B, Beerli RR, Barbas CF III: Toward controlling
•• gene expression at will: selection and design of zinc finger
domains recognizing each of the 5′′-GNN-3′′ DNA target
sequences. Proc Natl Acad Sci USA 1999, 96:2758-2763.
The domains reported in this paper were optimized using a combination of
phage display and rational mutagenesis. Domains are disclosed that recognize any GNN target sequence (guanine followed by any two bases. Many
of these domains exhibit exquisite specificities not previously observed for
zinc-finger proteins. Together with [23••], this paper describes a system by
which any laboratory can build a zinc finger protein capable, perhaps, of
uniquely targeting any gene in a complex genome.
15. Choo Y, Klug A: Selection of DNA binding sites for zinc fingers
using rationally randomized DNA reveals coded interactions. Proc
Natl Acad Sci USA 1994, 91:11168-11172.
16. Desjarlais JR, Berg JM: Use of a zinc-finger consensus sequence
framework and specificity rules to design specific DNA binding
proteins. Proc Natl Acad Sci USA 1993, 90:2256-2260.
17.
Corbi N, Libri V, Fanciulli M, Passananti C: Binding properties of the
artificial zinc fingers coding gene Sint1. Biochem Biophys Res
Commun 1998, 253:686-692.
18. Wolfe SA, Greisman HA, Ramm EI, Pabo CO: Analysis of zinc
•
fingers optimized via phage display: evaluating the utility of a
recognition code. J Mol Biol 1999, 285:1917-1934.
This paper reports the specificity of the proteins constructed by sequential
selection (see [22]). The authors also argue that many of the interactions
they observed were not predicted by a recognition code.
19. Isalan M, Choo Y, Klug A: Synergy between adjacent zinc fingers in
sequence-specific DNA recognition. Proc Natl Acad Sci USA 1997,
94:5617-5621.
20. Isalan M, Klug A, Choo Y: Comprehensive DNA recognition through
•
concerted interactions from adjacent zinc fingers. Biochemistry
1998, 37:12026-12033.
By randomizing residues at the interface of two domains, the authors
showed that they could select residues that would allow the recognition of
sequences starting with any base. It is still not clear if these results can be
generalized, however, and the authors have yet to show that they can apply
their methods to make multi-finger proteins of high affinity and specificity or
that purified rather than phage-bound proteins actually bind the target
sequence.
21. Jamieson AC, Wang H, Kim S-H: A zinc finger directory for highaffinity DNA recognition. Proc Natl Acad Sci USA 1996, 93:1283412839.
22. Greisman HA, Pabo CO: A general strategy for selecting highaffinity zinc finger proteins for diverse DNA target sites. Science
1997, 275:657-661.
23. Beerli RR, Segal DJ, Dreier B, Barbas CF III: Toward controlling
•• gene expression at will: specific regulation of the erbB-2/HER-2
promoter by using polydactyl zinc finger proteins constructed
from modular building blocks. Proc Natl Acad Sci USA 1998,
95:14628-14633.
This paper presents the assembly, affinity, specificity, and biological activity
of six-domain polydactyl zinc-finger proteins using the domains described in
[14••]. Transcription factors recognizing an 18 base pair sequence are
assembled and demonstrated to specifically control the erbB-2 promoter in
human cells, the first time that transcription factors have been fashioned to
regulate a specific promoter. Together, these papers provide the necessary
tools by which any laboratory can build a zinc finger protein capable, perhaps, of uniquely targeting any gene in the human genome.
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Design of novel sequence-specific DNA-binding proteins Segal and Barbas
24. Kim JS, Pabo CO: Transcriptional repression by zinc finger
peptides. Exploring the potential for applications in gene therapy.
J Biol Chem 1997, 272:29795-29800.
25. Elrod-Erickson M, Benson TE, Pabo CO: High-resolution structures
•• of variant Zif268–DNA complexes: implications for understanding
zinc finger–DNA recognition. Structure 1998, 6:451-464.
This paper presents the structures of phage display selected mutants. The
structures show interactions in cognate and non-cognate sites. The paper
also describes the repositioning of the α-helix relative to the DNA. This is an
excellent and necessary descriptive work, although the paucity of quantitative affinity and specificity data for these proteins allows for few conclusions
regarding specific interactions that provide for binding one sequence while
excluding others.
26. Nolte RT, Conlin RM, Harrison SC, Brown RS: Differing roles for
zinc fingers in DNA recognition: structure of a six-finger
transcription factor IIIA complex. Proc Natl Acad Sci USA 1998,
95:2938-2943.
27.
Wuttke DS, Foster MP, Case DA, Gottesfeld JM, Wright PE: Solution
structure of the first three zinc fingers of TFIIIA bound to the
cognate DNA sequence: determinants of affinity and sequence
specificity. J Mol Biol 1997, 273:183-206.
28. Ryan RF, Darby MK: The role of zinc finger linkers in p43 and
TFIIIA binding to 5S rRNA and DNA. Nucleic Acids Res 1998,
26:703-709.
29. Liu Q, Segal DJ, Ghiara JB, Barbas CF III: Design of polydactyl zincfinger proteins for unique addressing within complex genomes.
Proc Natl Acad Sci USA 1997, 94:5525-5530.
30. Kim JS, Pabo CO: Getting a handhold on DNA: design of poly-zinc
•
finger proteins with femtomolar dissociation constants. Proc Natl
Acad Sci USA 1998, 95:2812-2817.
This work suggests that modifications to the framework of a zinc-finger protein can lead to dramatic improvements in binding. The authors state that by
using a noncanonical linker between zinc fingers 3 and 4, a > 6000-fold
increase in affinity can be obtained by a six-finger protein over that of its constituent three-fingers proteins. It remains to be seen if specificity is sacrificed, or if this effect can be reproduced with other proteins. To date, further
application of this approach has failed. More importantly, it is not clear that
39
the effect has anything to do with the noncanonical linker, as it appears that
the canonical linker was never tested. Kinetic measurements are used to
derive dissociation constants. As performed, these measurements can lead
to very large errors. Therefore, the use of the term ‘femtomolar’ in the title and
abstract of this paper is provocatively misleading. Normalizing the reported
affinities to a more traditional value for Zif268 produces dissociation constants in the picomolar range. Such affinities are still impressive, but are
achievable with canonical linkers.
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