Published online 23 September 2008Nucleic Acids Research, 2009, Vol. 37, Database issueD279–D283
Zinc Finger Database (ZiFDB): a repository for
information on C2H2 zinc fingers and engineered
Fengli Fu1,2, Jeffry D. Sander1,2, Morgan Maeder3, Stacey Thibodeau-Beganny3,
J. Keith Joung3,4, Drena Dobbs1,2, Leslie Miller2,5and Daniel F. Voytas6,*
1Department of Genetics, Development & Cell Biology, 1043 Roy J. Carver Co-Laboratory,2Interdepartmental
Graduate Program in Bioinformatics & Computational Biology, 2114 Molecular Biology Building, Iowa State
University, Ames, Iowa 50011,3Molecular Pathology Unit, Center for Cancer Research and Center for
Computational and Integrative Biology, Massachusetts General Hospital, 149 13th Street, Charlestown, MA 02129,
4Department of Pathology, Harvard Medical School, Boston, MA 02115,5Department of Computer Science, 227
Atanasoff Hall, Iowa State University, Ames, IA 50011 and6Department of Genetics, Cell Biology & Development
and Center for Genome Engineering, 321 Church Street SE, University of Minnesota, MN 55455, USA
Received August 15, 2008; Accepted September 6, 2008
Zinc fingers are the most abundant DNA-binding
motifs encoded by eukaryotic genomes and one of
it is possible to engineer zinc-finger arrays (ZFAs)
that recognize extended DNA sequences by linking
together individual zinc fingers. Engineered zinc-
finger proteins have proven to be valuable tools for
gene regulation and genome modification because
they target specific sites in a genome. Here we
describe ZiFDB (Zinc Finger Database; http://bindr.
gdcb.iastate.edu/ZiFDB), a web-accessible resource
that compiles information on individual zinc fingers
and engineered ZFAs. To enhance its utility, ZiFDB is
linked to the output from ZiFiT—a software package
that assists biologists in finding sites within target
genes for engineering zinc-finger proteins. For
many molecular biologists, ZiFDB will be particularly
valuable for determining if a given ZFA (or portion
whether or not it has the requisite DNA-binding
activity for their experiments. ZiFDB will also be a
valuable resource for those scientists interested
in better understanding how zinc-finger proteins
recognize target DNA.
amino acids in length and conforms to the pattern
(F/Y)-X-C-X2–5-C-X3-(F/Y)-X5-c-X2-H-X3–5-H, where X
is an amino acid and c is a hydrophobic residue (1). The
amino acids fold into a bba structure, which is stabilized by
a zinc ion coordinated by the two conserved cysteine and
histidine residues. In binding DNA, each finger typically
recognizes three adjacent nucleotides: amino acids at
positions ?1, +3 and +6 (relative to the beginning of
the a-helix) contact nucleotides at the 30-end, middle and
50-end of the target nucleotide triplet. Zinc fingers can func-
tion as monomers, making it possible to link zinc fingers
together in extended arrays that recognize unique DNA
sequences. Such artificial or engineered zinc-finger arrays
(ZFAs) are often fused to effector domains (e.g. transcrip-
tional activation domains or nucleases), and engineered
valuable as reagents for gene regulation and genome
A simple approach for engineering ZFAs is known as
‘modular assembly’, wherein individual fingers are joined
together to create an array that recognizes a novel target
sequence. To facilitate modular assembly, efforts have
been made to obtain zinc fingers that recognize all 64
possible triplets. Approaches to create such a finger
archive have included parallel selection from random
libraries (3–7), rational design (8,9) and characterization
of naturally occurring zinc-finger proteins (10). Zinc fin-
gers have thus far been identified that recognize all 16
GNN triplets as well as most CNN, ANN and some
TNN triplets. Modular assembly assumes that individual
zinc fingers are not influenced by neighboring fingers in an
array. However, systematic testing of a large number of
ZFAs constructed with available zinc-finger modules
resulted in success rates of 56%, 20%, 4% and 0% for
*To whom correspondence should be addressed. Tel: +1 515 294 1963; Fax: +1 515 294 7155; Email: email@example.com
? 2008 The Author(s)
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9-bp target sites composed of three, two, one or no GNN
triplets, respectively (11). It is clear from this work that
zinc fingers are not always modular and that a better
understanding of context-dependent effects is needed to
increase the reliability of modular assembly.
To minimize the context dependence associated with
modular design, sequential selection (12), bipartite selec-
tion (13) and bacterial-based selection methods have been
described (14,15). These methods typically involve screen-
ing large libraries of ZFA variants. The few arrays in the
library that recognize a specific target with high affinity
and specificity are identified using phage display or a bac-
terial two-hybrid system in which target binding activates
expression of a reporter gene. An advantage of these selec-
tion-based strategies is that context-dependent effects are
addressed through the process of selection. Selection-
based approaches, however, require considerable mole-
cular biological expertise and are time consuming.
Recently, a selection platform was described, called
OPEN (Oligomerized Pool ENgineering), that overcomes
shortcomings of traditional selection-based methods in
that it is simpler and faster; it is also much more effective
than modular assembly (11,16). However, even the sim-
plified OPEN strategy remains more labor-intensive and
difficult to perform than modular assembly.
Because many scientists are interested in generating
zinc-finger proteins for gene regulation and genome
modification, we implemented a Zinc Finger Database
(ZiFDB)—an archive of information about zinc fingers
and engineered ZFAs. The database is one of several, inte-
grated resources provided by the Zinc Finger Consortium
(http://www.zincfingers.org), a group of academic labora-
tories dedicated to improving methods for engineering
ZFAs and developing new applications for their use.
The Consortium has previously created a repository of
zinc-finger encoding plasmids which simplify and facilitate
modular assembly of ZFAs (17). Consortium labs also
developed andvalidated theOPENplatform forZFA engi-
neering (11,16). Further, Consortium-supported software,
called ZiFiT (Zinc Finger Targeter), allows users to
scan DNA sequences to identify potential sites for which
ZFAs might be engineered by either modular assembly
or OPEN (18). A particularly important feature of
ZiFDB is its linkage to ZiFiT, which allows users to readily
other investigators that recognize a target sequence of
ZiFDB currently contains 716 zinc fingers, each of which
is assigned a unique numerical designator. The database
was initially populated with fingers generated and charac-
terized by four different research groups:
(1) Sangamo BioSciences: Sangamo Biosciences designed
zinc fingers that recognize GNN triplets at each of
the three positions in a three-finger array (7). It is
assumed that these fingers function optimally in the
position for which they were designed. Sangamo has
also generated several zinc fingers that recognize
non-GNN triplets, and these are also included in
the database (17,19,20).
(2) Barbas laboratory: The Barbas laboratory at the
number of zinc fingers that recognize diverse nucleo-
tide triplets. The fingers are presumed to be modu-
lar and function at any position within a ZFA
(3) Toolgen: Toolgen Inc. assembled a collection of zinc
fingers derived from naturally occurring zinc-finger
proteins encoded in the human genome (10).
Massachusetts General Hospital used the OPEN
platform to generate a large number of ZFAs (16).
All of these ZFAs are three finger domains that
recognize a 9-bp target. Unique zinc fingers compris-
ing ZFAs generated by OPEN are also included in
In some cases, investigators have used the DNA-recogni-
tion helices from the above fingers within the context of a
different zinc-finger backbone. In ZiFDB, such fingers are
given a different numerical designator, since the backbone
may affect zinc-finger function.
The following information is provided for each zinc
(1) Triplet target: The sequence is provided for the cog-
nate 3-bp DNA target of each finger.
(2) Recognition helix: The sequence is provided for the
seven amino acids that constitute the recognition
helix: positions ?1 to +6 relative to the start of
the a-helix that contacts DNA in the finger.
(3) Position within a three-finger array: As indicated
above, some fingers were designed to function opti-
mally at certain positions within a three-finger array.
The fingers described by Sangamo BioSciences, for
example, were selected at different positions within
a three-finger protein, and the recognition helices for
the same triplet target at different positions in a three
finger protein usually differ (7). These differences are
likely due to position and context-dependent effects.
In addition, the fingers generated by OPEN were
selected to function in a specific position within a
three finger protein (16). The database records position
information for the fingers described by Sangamo
BioSciences and generated by OPEN. By convention,
finger 1 (F1) specifies the 30-triplet in a 9-bp target,
F2 specifies the middle triplet and F3 specifies the
(4) Source: The name of the group that generated the
zinc finger is provided. The groups at present include
theBarbas and Joung
BioSciences (SGMO) and ToolGen.
(5) Amino acid sequence: The amino acid sequence of
the entire finger is provided.
(6) Article: A link to the relevant citation is provided so
the user can get more detailed information about a
Nucleic Acids Research, 2009, Vol. 37, Databaseissue
(7) Experiment: A link directs the user to information
describing the method used to generate the finger,
relevant functional data and other observations
made by the experimenter.
At present, ZiFDB houses 652 three-finger arrays col-
lected from the published literature. Although engineered
ZFAs of four or more fingers have been reported, the
Consortium has developed reagents are only designed
for constructing three-finger arrays. Larger arrays, there-
fore, are currently not supported by ZiFDB. Each three-
finger array in the database has a unique numerical
designator, and the following information is provided:
whichthe Zinc Finger
(1) Identification numbers (IDs) of the component
fingers: The IDs correspond to the zinc-finger desig-
nator and are hyperlinks that allow a user to look up
detailed information about individual fingers in an
(2) Binding subsites: These sequences correspond to the
3-bp nucleotide target of the corresponding compo-
(3) Recognition helix: For each finger in an array, the
sequence is provided of the seven amino acids that
make up the recognition helix.
(4) Article: A link to the relevant citation is provided so
the user can get more detailed information about
(5) Experiment: A link is provided to information
describing the method used to generate the arrays
and relevant functional data or other observations
made by the experimenter.
To retrieve information about a particular zinc finger, any
combination of three search criteria can be used: triplet
target, finger position and/or finger source. By providing
partial information, a list of all fingers matching the input
is returned (Figure 1). By clicking the link in the article or
experiment column, detailed information about related
citations or experiments is displayed.
When searching for information about an array, the
user provides the nucleotide triplets (subsites) recognized
by each finger in the array. The database returns not
only the arrays recognizing the three input subsites, but
also arrays matching any two of three triplet subsites
(Figure 2). This partial information may inform the design
of novel ZFAs by providing the user with a two finger
protein that could be modified and tested for function.
For arrays that match only two of the input subsites, the
matching subsites and their recognition helices are colored
Figure 1. Sample output from the finger search page when GGG is provided as the target and the position and source are left blank.
Nucleic Acids Research, 2009,Vol. 37,Database issue D281
in red for easy identification. The output also provides
the amino acid sequence of the array, IDs for each finger
in the array and IDs for related articles or experiments.
By clicking the relevant ID, the user is directed to infor-
mation on specific fingers, articles or experiments.
Interface withpre-existing ZiFiT software
To enhance its utility, ZiFDB is interfaced directly with
ZiFiT—a web accessible software package that assists bio-
logists with zinc-finger protein design (http://bindr.gdcb.
iastate.edu/ZiFiT/) (16,18). ZiFiT provides users with a
list of target sites within their DNA sequence of interest
as well as the corresponding Consortium-developed
reagents available for engineering the ZFAs. Target site
hyperlinks within the ZiFiT output directly query ZiFDB
to determine if any previously constructed arrays exist
that bind to completely or partially matched target
sequences. In addition, ZiFiT users can query ZiFDB
for finger information for a specific triplet subsite by click-
ing on the triplet. Thus, ZiFiT and ZiFDB work synergis-
tically to aid in ZFA design.
ZiFDB stores zinc-finger information as a set of objects
defined by Java classes. The atomic information is con-
tained in two main classes (Zinc Finger and Zinc Finger
Array). Both classes reference additional Article and
Experiment classes. The Article class includes the title,
journal, volume, page, year and authors for the article
being represented. The Experiment class includes informa-
tion on the submitter, assay, results and other comments
provided by the author or submitter. The authors of arti-
cles and experiments are organized using an Author class.
Each finger or array object can be associated with multiple
Articles or Experiments. The objects representing the Java
classes are mapped to a MySQL version 4.0.15 database
Apache Tomcat version 5.5.15 is used as the servlet
engine for the web site. The web application uses Struts
1.2 to implement the Model-View-Controller (MVC) pat-
tern. Java Server Pages (JSP) are used to implement the
user’s view. The web application was developed using
ZiFDB is one of an increasing repertoire of tools for zinc-
finger protein engineering provided by the Zinc Finger
Consortium. ZiFDB houses information on individual
zinc fingers, including the 141 zinc-finger modules cur-
Consortium. Importantly, the database also provides
users with information on ZFAs and their target
sequences, and we believe this feature will be particularly
a clone librarybythe
Figure 2. Sample output returned when searching for arrays with the binding site 50-GAAGGCGGC-30.
Nucleic Acids Research, 2009, Vol. 37, Databaseissue
valuable for the rapidly growing number of molecular biol- Download full-text
ogists interested in generating ZFAs for modification or
regulation of their genome locus of interest. Further, ana-
lysis of the data housed in ZiFDB will also likely provide
new insight into how zinc-finger proteins recognize their
DNA targets, which, in turn, may lead to additional
improvements in ZFA engineering.
Future versions of ZiFDB will allow users to directly input
information on novel zinc fingers or ZFAs. To ensure data
quality, a shadow database will be created that is asso-
ciated with the persistent database. Users will be permitted
to submit data to the shadow database where it will be
held pending approval by the curator and subsequent
loading into the persistent database. This feature will
ensure that the database capitalizes upon new information
generated by the scientific community, including unpub-
lished ZFAs, and thereby should expand the utility of the
The National Science Foundation (0501678 and 0209818
to D.F.V.); National Institutes of Health (GM069906 and
GM078369 to J.K.J.). Funding for open access charge:
National Science Foundation 0501678.
Conflict of interest statement. None declared.
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