Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Review Article
  • Published:

Drug discovery with engineered zinc-finger proteins

Nature Reviews Drug Discovery volume 2, pages 361–368 (2003)Cite this article

Key Points

  • C2H2 zinc fingers are the most common DNA-binding motif found in the human genome.

  • The Zif268–DNA crystal structure shows how zinc fingers interact with DNA. The fingers act as modular units (each contacting three to four base pairs of DNA) and the structure reveals which residues should be changed to alter the specificity.

  • Researchers have engineered zinc finger proteins (ZFPs) to bind a diverse set of DNA sequences, and thereby target specific locations in the human genome, such as the promoters of therapeutically relevant genes. Exquisite specificity can be obtained with proteins that have six fingers.

  • ZFP transcription factors (ZFP TFs) are made by combining the ZFPs with domains that either activate or repress genes. ZFP TFs are used in drug discovery to regulate genes for target validation, high-throughput screening and human therapeutics.

  • ZFP-mediated regulation of endogenous genes could make it possible to use genes in a drug discovery application that would otherwise require securing intellectual property rights for a corresponding complementary DNA sequence.

  • Recently, ZFP TFs have been used to promote angiogenesis in a mouse ear model, and are now undergoing further preclinical testing.

  • Combining ZFPs with novel functional domains makes it possible to target DNA for chromatin and DNA modification, DNA cleavage and for targeted integration of exogenous DNA.

Abstract

Zinc-finger proteins (ZFPs) that recognize novel DNA sequences are the basis of a powerful technology platform with many uses in drug discovery and therapeutics. These proteins have been used as the DNA-binding domains of novel transcription factors (ZFP TFs), which are useful for validating genes as drug targets and for engineering cell lines for small-molecule screening and protein production. Recently, they have also been used as a basis for novel human therapeutics. Most of our advances in the design and application of these ZFP TFs rely on our ability to engineer ZFPs that bind short stretches of DNA (typically 9–18 base pairs) located within the promoters of target genes. Here, we summarize the methods used to design these DNA-binding domains, explain how they are incorporated into novel transcription factors (and other useful molecules) and describe some key applications in drug discovery.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Modular interactions between zinc fingers and DNA.
Figure 2: Incorporation of zinc-finger modules into multi-finger arrays.
Figure 3: Combining zinc-finger proteins with different functional domains.
Figure 4: The zinc-finger protein transcription factor assembly process.
Figure 5: Zinc-finger protein transcription factor-mediated regulation of the Vegfa gene in a mouse ear model.

Similar content being viewed by others

References

  1. Tupler, R., Perini, G. & Green, M. R. Expressing the human genome. Nature 409, 832–833 (2001).

    Article  CAS  PubMed  Google Scholar 

  2. Miller, J., McLachlan, A. D. & Klug, A. Repetitive zinc-binding domains in the protein transcription factor IIIA from Xenopus oocytes. EMBO J. 4, 1609–1614 (1985).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Berg, J. M. Proposed structure for the zinc-binding domains from transcription factor IIIA and related proteins. Proc. Natl Acad. Sci. USA 85, 99–102 (1988).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Lee, M. S. et al. Three-dimensional solution structure of a single zinc finger DNA-binding domain. Science 245, 635–637 (1989).

    Article  CAS  PubMed  Google Scholar 

  5. Pavletich, N. P. & Pabo, C. O. Zinc finger-DNA recognition: crystal structure of a Zif268–DNA complex at 2. 1 A. Science 252, 809–817 (1991). The first crystal structure to give a detailed view of how ZFPs recognize DNA. This paper laid the foundation for the subsequent experiments in zinc finger protein engineering.

    Article  CAS  PubMed  Google Scholar 

  6. Houbaviy, H. B. et al. Cocrystal structure of YY1 bound to the adeno-associated virus P5 initiator. Proc. Natl Acad. Sci. USA 93, 13577–13582 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Nolte, R. T. et al. Differing roles for zinc fingers in DNA recognition: structure of a six-finger transcription factor IIIA complex. Proc. Natl Acad. Sci. USA 95, 2938–2943 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Fairall, L. et al. The crystal structure of a two zinc-finger peptide reveals an extension to the rules for zinc-finger/DNA recognition. Nature 366, 483–487 (1993).

    Article  CAS  PubMed  Google Scholar 

  9. Rebar, E. J. & Pabo, C. O. Zinc finger phage: affinity selection of fingers with new DNA-binding specificities. Science 263, 671–673 (1994).

    Article  CAS  PubMed  Google Scholar 

  10. Jamieson, A. C., Kim, S. H. & Wells, J. A. In vitro selection of zinc fingers with altered DNA-binding specificity. Biochemistry 33, 5689–5695 (1994).

    Article  CAS  PubMed  Google Scholar 

  11. Choo, Y. & Klug, A. Toward a code for the interactions of zinc fingers with DNA: selection of randomized fingers displayed on phage. Proc. Natl Acad. Sci. USA 91, 11163–11167 (1994). One of several early phage display papers showing how ZFPs can be engineered to recognize different DNA target sequences.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Wolfe, S. A., Nekludova, L. & Pabo, C. O. DNA recognition by Cys2His2 zinc finger proteins. Annu. Rev. Biophys. Biomol. Struct. 29, 183–212 (2000).

    Article  CAS  PubMed  Google Scholar 

  13. Smith, G. P. Surface presentation of protein epitopes using bacteriophage expression systems. Curr. Opin. Biotechnol. 2, 668–673 (1991).

    Article  CAS  PubMed  Google Scholar 

  14. Rebar, E. J., Greisman, H. A. & Pabo, C. O. Phage display methods for selecting zinc finger proteins with novel DNA-binding specificities. Methods Enzymol. 267, 129–149 (1996).

    Article  CAS  PubMed  Google Scholar 

  15. Isalan, M., Choo, Y. & Klug, A. Synergy between adjacent zinc fingers in sequence-specific DNA recognition. Proc. Natl Acad. Sci. USA 94, 5617–5621 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Isalan, M., Klug, A. & Choo, Y. Comprehensive DNA recognition through concerted interactions from adjacent zinc fingers. Biochemistry 37, 12026–12033 (1998).

    Article  CAS  PubMed  Google Scholar 

  17. Segal, D. J. et al. 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 96, 2758–2763 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Dreier, B. et al. Development of zinc finger domains for recognition of the 5'-ANN-3' family of DNA sequences and their use in the construction of artificial transcription factors. J. Biol. Chem. 276, 29466–29478 (2001).

    Article  CAS  PubMed  Google Scholar 

  19. Beerli, R. R. & Barbas, C. F. 3rd. Engineering polydactyl zinc-finger transcription factors. Nature Biotechnol. 20, 135–141 (2002).

    Article  CAS  Google Scholar 

  20. Pabo, C. O., Peisach, E. & Grant, R. A. Design and selection of novel Cys2His2 zinc finger proteins. Annu. Rev. Biochem. 70, 313–340 (2001). A comprehensive review of zinc finger DNA recognition that will allow the reader to find detailed biochemical information as needed.

    Article  CAS  PubMed  Google Scholar 

  21. Choo, Y. & Klug, A. Selection of DNA binding sites for zinc fingers using rationally randomized DNA reveals coded interactions. Proc. Natl Acad. Sci. USA 91, 11168–11172 (1994).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Jamieson, A. C., Wang, H. & Kim, S. H. A zinc finger directory for high-affinity DNA recognition. Proc. Natl Acad. Sci. USA 93, 12834–12839 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Liu, Q. et al. Validated zinc finger protein designs for all 16 GNN DNA triplet targets. J. Biol. Chem. 277, 3850–3856 (2002).

    Article  CAS  PubMed  Google Scholar 

  24. Liu, P. Q. et al. Regulation of an endogenous locus using a panel of designed zinc finger proteins targeted to accessible chromatin regions. Activation of vascular endothelial growth factor A. J. Biol. Chem. 276, 11323–11334 (2001).

    Article  CAS  PubMed  Google Scholar 

  25. Zhang, L. et al. Synthetic zinc finger transcription factor action at an endogenous chromosomal site. Activation of the human erythropoietin gene. J. Biol. Chem. 275, 33850–33860 (2000).

    Article  CAS  PubMed  Google Scholar 

  26. Beerli, R. R., Dreier, B. & Barbas, C. F. 3rd. Positive and negative regulation of endogenous genes by designed transcription factors. Proc. Natl Acad. Sci. USA 97, 1495–1500 (2000). A convincing account of how ZFPs can be engineered to regulate endogenous genes.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Greisman, H. A. & Pabo, C. O. A general strategy for selecting high-affinity zinc finger proteins for diverse DNA target sites. Science 275, 657–661 (1997).

    Article  CAS  PubMed  Google Scholar 

  28. Isalan, M., Klug, A. & Choo, Y. A rapid, generally applicable method to engineer zinc fingers illustrated by targeting the HIV-1 promoter. Nature Biotechnol. 19, 656–660 (2001). This paper shows how, using phage display, a comprehensive archive of zinc fingers can be made for DNA recognition.

    Article  CAS  Google Scholar 

  29. Bartsevich, V. V. & Juliano, R. L. Regulation of the MDR1 gene by transcriptional repressors selected using peptide combinatorial libraries. Mol. Pharmacol. 58, 1–10 (2000).

    Article  CAS  PubMed  Google Scholar 

  30. Joung, J. K., Ramm, E. I. & Pabo, C. O. A bacterial two-hybrid selection system for studying protein–DNA and protein–protein interactions. Proc. Natl Acad. Sci. USA 97, 7382–7387 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Blancafort, P., Magnenat, L. & Barbas, C. F. Scanning the human genome with combinatorial transcription factor libraries. Nature Biotechnol. 21, 269–274 (2003).

    Article  CAS  Google Scholar 

  32. Liu, Q. et al. Design of polydactyl zinc-finger proteins for unique addressing within complex genomes. Proc. Natl Acad. Sci. USA 94, 5525–5530 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Kim, J. S. & Pabo, C. O. Getting a handhold on DNA: design of poly-zinc finger proteins with femtomolar dissociation constants. Proc. Natl Acad. Sci. USA 95, 2812–2817 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Nekludova, L. & Pabo, C. O. Distinctive DNA conformation with enlarged major groove is found in Zn- finger–DNA and other protein–DNA complexes. Proc. Natl Acad. Sci. USA 91, 6948–6952 (1994).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Moore, M., Klug, A. & Choo, Y. Improved DNA binding specificity from polyzinc finger peptides by using strings of two-finger units. Proc. Natl Acad. Sci. USA 98, 1437–1441 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Pomerantz, J. L., Wolfe, S. A. & Pabo, C. O. Structure-based design of a dimeric zinc finger protein. Biochemistry 37, 965–970 (1998).

    Article  CAS  PubMed  Google Scholar 

  37. Wolfe, S. A., Ramm, E. I. & Pabo, C. O. Combining structure-based design with phage display to create new Cys(2)His(2) zinc finger dimers. Structure Fold. Des. 8, 739–750 (2000).

    Article  CAS  PubMed  Google Scholar 

  38. Beerli, R. R. et al. Chemically regulated zinc finger transcription factors. J. Biol. Chem. 275, 32617–32627 (2000).

    Article  CAS  PubMed  Google Scholar 

  39. Gross, D. S. & Garrard, W. T. Nuclease hypersensitive sites in chromatin. Annu. Rev. Biochem. 57, 159–197 (1988).

    Article  CAS  PubMed  Google Scholar 

  40. Bushman, F. D. & Miller, M. D. Tethering human immunodeficiency virus type 1 preintegration complexes to target DNA promotes integration at nearby sites. J. Virol. 71, 458–464 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Xu, G. L. & Bestor, T. H. Cytosine methylation targetted to pre-determined sequences. Nature Genet. 17, 376–378 (1997).

    Article  CAS  PubMed  Google Scholar 

  42. Kim, Y. G., Cha, J. & Chandrasegaran, S. Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain. Proc. Natl Acad. Sci. USA 93, 1156–1160 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Kalderon, D. et al. A short amino acid sequence able to specify nuclear location. Cell 39, 499–509 (1984).

    Article  CAS  PubMed  Google Scholar 

  44. Sadowski, I. et al. GAL4-VP16 is an unusually potent transcriptional activator. Nature 335, 563–564 (1988).

    Article  CAS  PubMed  Google Scholar 

  45. Ruben, S. M. et al. Isolation of a rel-related human cDNA that potentially encodes the 65- kD subunit of NF-κB. Science 251, 1490–1493 (1991).

    Article  CAS  PubMed  Google Scholar 

  46. Thiesen, H. J. et al. Conserved KRAB protein domain identified upstream from the zinc finger region of Kox 8. Nucleic Acids Res. 19, 3996 (1991).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Guan, X. et al. Heritable endogenous gene regulation in plants with designed polydactyl zinc finger transcription factors. Proc. Natl Acad. Sci. USA 99, 13296–13301 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Ren, D. et al. PPARγ knockdown by engineered transcription factors: exogenous PPARγ2 but not PPARγ1 reactivates adipogenesis. Genes Dev. 16, 27–32 (2002). A 'target validation' paper showing how ZFPs can be used to elucidate the role of a receptor isoform in fat cell differentiation.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Falke, D. et al. Design of artificial transcription factors to selectively regulate the pro-apoptotic bax gene. Nucleic Acids Res. 31, E10–20 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Hopkins, A. L. & Groom, C. R. The druggable genome. Nature Rev. Drug Discov. 1, 727–730 (2002).

    Article  CAS  Google Scholar 

  51. Aranda, A. & Pascual, A. Nuclear hormone receptors and gene expression. Physiol. Rev. 81, 1269–1304 (2001).

    Article  CAS  PubMed  Google Scholar 

  52. Reik, A., Gregory, P. D. & Urnov, F. D. Biotechnologies and therapeutics: chromatin as a target. Curr. Opin. Genet. Dev. 12, 233–242 (2002).

    Article  CAS  PubMed  Google Scholar 

  53. Snowden, A. W. et al. Gene-specific targeting of h3k9 methylation is sufficient for initiating repression in vivo. Curr. Biol. 12, 2159–2166 (2002).

    Article  CAS  PubMed  Google Scholar 

  54. Minucci, S. et al. Histone deacetylases: a common molecular target for differentiation treatment of acute myeloid leukemias? Oncogene 20, 3110–3115 (2001).

    Article  CAS  PubMed  Google Scholar 

  55. Rebar, E. J. et al. Induction of angiogenesis in a mouse model using engineered transcription factors. Nature Med. 8, 1427–1432 (2002). A state-of-the-art paper showing how ZFPs can be used to stimulate the growth of blood vessels in a mouse ear.

    Article  CAS  PubMed  Google Scholar 

  56. Grunstein, J. et al. Isoforms of vascular endothelial growth factor act in a coordinate fashion to recruit and expand tumor vasculature. Mol. Cell. Biol. 20, 7282–7291 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Modrek, B. & Lee, C. A genomic view of alternative splicing. Nature Genet. 30, 13–19 (2002).

    Article  CAS  PubMed  Google Scholar 

  58. Rowen, L. et al. Analysis of the human neurexin genes: alternative splicing and the generation of protein diversity. Genomics 79, 587–597 (2002).

    Article  CAS  PubMed  Google Scholar 

  59. Young, J. C., Hoogenraad, N. J. & Hartl, F. U. Molecular chaperones hsp90 and hsp70 deliver preproteins to the mitochondrial import receptor tom70. Cell 112, 41–50 (2003).

    Article  CAS  PubMed  Google Scholar 

  60. Pollock, R. et al. Regulation of endogenous gene expression with a small-molecule dimerizer. Nature Biotechnol. 20, 729–733 (2002).

    Article  CAS  Google Scholar 

  61. Johnson, L. et al. Selectively replicating adenoviruses targeting deregulated E2F activity are potent, systemic antitumor agents. Cancer Cell 1, 325–337 (2002).

    Article  CAS  PubMed  Google Scholar 

  62. Dranoff, G. GM-CSF-based cancer vaccines. Immunol. Rev. 188, 147–154 (2002).

    Article  CAS  PubMed  Google Scholar 

  63. Reynolds, L. et al. Repression of the HIV-1 5' LTR promoter and inhibition of HIV-1 replication by using engineered zinc-finger transcription factors. Proc. Natl Acad. Sci. USA 100, 1615–1620 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Papworth, M. et al. Inhibition of herpes simplex virus 1 gene expression by designer zinc-finger transcription factors. Proc. Natl Acad. Sci. USA 100, 1621–1626 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Chandrasegaran, S. & Smith, J. Chimeric restriction enzymes: what is next? Biol. Chem. 380, 841–848 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Bibikova, M. et al. Stimulation of homologous recombination through targeted cleavage by chimeric nucleases. Mol. Cell. Biol. 21, 289–297 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Bibikova, M. et al. Targeted chromosomal cleavage and mutagenesis in Drosophila using zinc-finger nucleases. Genetics 161, 1169–1175 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  68. Porteus, M. H. & Baltimore, D. Chimeric nucleases stimulate gene targeting in human cells. Science (in the press).

  69. Hacein-Bey-Abina, S. et al. A serious adverse event after successful gene therapy for X-linked severe combined immunodeficiency. N. Engl. J. Med. 348, 255–256 (2003).

    Article  PubMed  Google Scholar 

  70. Robertson, K. D. & Wolffe, A. P. DNA methylation in health and disease. Nature Rev. Genet. 1, 11–19 (2000).

    Article  CAS  PubMed  Google Scholar 

  71. McNamara, A. R. et al. Characterisation of site-biased DNA methyltransferases: specificity, affinity and subsite relationships. Nucleic Acids Res. 30, 3818–3830 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Jouvenot, Y. et al. Targeted regulation of imprinted genes by synthetic zinc-finger transcription factors. Gene Ther. (in the press).

  73. Kim, J. H. et al. Dopamine neurons derived from embryonic stem cells function in an animal model of Parkinson's disease. Nature 418, 50–56 (2002).

    Article  CAS  PubMed  Google Scholar 

  74. Stix, G. Legal circumvention. Sci. Am. 287, 36 (2002).

    PubMed  Google Scholar 

Download references

Acknowledgements

The authors would like to express their indebtedness to all those who contributed to the critical reading of this manuscript, including Dr P. Gregory, D. E. Wolffe, Dr E. Rebar, Dr A. McNamara, Dr A. Reik, Dr F. Urnov, Dr M. Holmes and T. J. Cradick. The authors also apologize to those researchers whose many contributions were not included owing to space limitations.

Author information

Authors and Affiliations

  1. Sangamo Biosciences, Inc., Point Richmond Technical Center, 501 Canal Boulevard, Suite A100, Richmond, 98404, CA, USA

    Andrew C. Jamieson, Jeffrey C. Miller & Carl O. Pabo

Authors
  1. Andrew C. Jamieson
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jeffrey C. Miller
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Carl O. Pabo
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Carl O. Pabo.

Related links

Related links

DATABASES

LocusLink

ABCB1

BAX

CCKBR

EPO

ERBB2

ERBB3

NR4A2

PPARγ

RB1

VEGF

Online Mendelian Inheritance in Man

Beckwith–Wiedemann syndrome

Parkinson's disease

FURTHER INFORMATION

Encyclopedia of Life Sciences

Aaron Klug

Protein–nucleic acid interaction: major groove recognition determinants

Protein–DNA interactions

Glossary

NUCLEOSOME

The basic structural subunit of chromatin, which consists of ∼200 base pairs of DNA and an octamer of histones (a family of small, highly conserved basic proteins).

CHROMATIN

The compact form that DNA is organized into in eukaryotic cells, which contains genomic DNA, histones and non-histone proteins.

HISTONE ACETYLTRANSFERASES AND DEACETYLASES

Enzymes that modify histones by adding and removing acetyl groups, a chemical modification that can affect chromatin structure.

ANGIOGENESIS

Growth of new blood vessels.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Jamieson, A., Miller, J. & Pabo, C. Drug discovery with engineered zinc-finger proteins. Nat Rev Drug Discov 2, 361–368 (2003). https://doi.org/10.1038/nrd1087

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1038/nrd1087

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing