Zinc Finger Tools: custom DNA-binding domains for transcription factors and nucleases

Abstract

Individual zinc finger (ZF) domains that recognize DNA triplets with high specificity and affinity can be used to create designer transcription factors and nucleases that are specific for nearly any site in the genome. These domains can be treated as modular units and assembled to create a polydactyl protein that recognizes extended DNA sequences. Deter-mination of valid target sites and the subsequent design of ZF proteins (ZFPs) is error-prone and not trivial, however. As a result, the use of ZFPs have been restricted primarily to those labs with the appropriate expertise. To address these limitations, we have created a user-friendly utility called Zinc Finger Tools (ZF Tools) that can be accessed at the URL http://www.zincfingertools.org. User-supplied DNA sequences can be searched for target sites appropriate for either gene regulation or nuclease targeting. Using a database of experimentally characterized zinc finger domains, the amino acid sequence for a ZFP expected to bind to any chosen target site can be generated. A reverse engineering utility is provided to predict the binding site for a ZFP of known sequence.

Figure 1

Home page and the search tool. The interface for the tool for searching a DNA sequence to find potential target sites is shown. The other ZF Tools appear below this on the website and not visible here. On the left are hyperlinks to various information pages. Parameters (described in the text) for searching for either contiguous or separated target sites are shown.

Figure 2

Results of a separated target site search. A search was performed on a DNA sequence 188 bp long for two 9 bp target sites separated by a random 6 bp core. The coverage map highlights those bases that are members of one or more target sites (black bases are in no target sites, red bases are on the top strand, and green bases on the bottom strand). The lower half of the figure displays target sites and non-targeted core sequence (underlined). Each triplet is hyperlinked to the appropriate multitarget ELISA specificity assay. Triplets that do not meet TSO requirements are colored (red) in the list of target sites. Next to each target site are icons either for obtaining the amino acid sequence of a ZFP predicted to bind to the target, or for parsing the target sequence into subsites.

Figure 3

Generation of the amino acid sequence for a ZFP. (A) The 18 bp target sequence shown was input. Triplets that do not meet TSO requirements are colored (red). Each DNA triplet is shown with the corresponding amino acid sequence of its recognition helix. Clicking on a hyperlinked triplet (blue) opens a window displaying results of a multitarget ELISA assay for the relevant ZF domain. A hyperlink is provided to the NCBI BLAST tool so that the human or mouse genomes can be searched for the target sequence. At the bottom of the screen is the complete amino acid sequence of the ZFP predicted to bind the target. This sequence can be copied to other applications for further manipulation. (B) Order of assembly for fixed, backbone, helix and linker amino acid sequences to generate the sequence of a ZFP. The default amino acid sequence is displayed below each box. The linker is in parenthesis because it is present only between domains and would not be included for a single ZF, as shown here. Sequences between the N-term and C-term fixed sequences repeat with each ZF.