A rapid, generally applicable method to engineer zinc fingers illustrated by targeting the HIV-1 promoter - PubMed (original) (raw)

A rapid, generally applicable method to engineer zinc fingers illustrated by targeting the HIV-1 promoter

M Isalan et al. Nat Biotechnol. 2001 Jul.

Abstract

DNA-binding domains with predetermined sequence specificity are engineered by selection of zinc finger modules using phage display, allowing the construction of customized transcription factors. Despite remarkable progress in this field, the available protein-engineering methods are deficient in many respects, thus hampering the applicability of the technique. Here we present a rapid and convenient method that can be used to design zinc finger proteins against a variety of DNA-binding sites. This is based on a pair of pre-made zinc finger phage-display libraries, which are used in parallel to select two DNA-binding domains each of which recognizes given 5 base pair sequences, and whose products are recombined to produce a single protein that recognizes a composite (9 base pair) site of predefined sequence. Engineering using this system can be completed in less than two weeks and yields proteins that bind sequence-specifically to DNA with Kd values in the nanomolar range. To illustrate the technique, we have selected seven different proteins to bind various regions of the human immunodeficiency virus 1 (HIV-1) promoter.

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Figures

Figure 1

Overview of the protein engineering strategy. In step 1, two pre-made zinc finger phage-display libraries (Lib12 and Lib23) contain randomized DNA-binding amino acid positions in fingers 1 and 2 (black) or fingers 2 and 3 (gray), respectively. Selections of “one-and-a-half” fingers from each master library are carried out in parallel using DNA sequences in which five nucleotides have been fixed to a sequence of interest. In step 2, zinc finger genes are amplified from the recovered phage using PCR, and sets of “one-and-a-half” fingers are paired to yield recombinant three-finger DNA-binding domains. In step 3, the recombinant DNA-binding domains are cloned back into phage and subjected to additional rounds of selection, or immediately validated for binding to a composite 9 base pair DNA of predefined sequence.

Figure 2

Composition of the “bipartite” library. (A) DNA recognition by the two zinc finger master libraries, Lib12 and Lib23. The libraries are based on the three-finger DNA-binding domain of Zif268, and the putative binding scheme is based on the crystal structure of the wild-type domain in complex with DNA (refs 6,32). The DNA-binding positions of each zinc finger are numbered, and randomized residues in the two libraries are circled. Broken arrows denote possible DNA contacts from Lib12 to bases H’IJKLM and from Lib23 to bases MNOPQ. Solid arrows show DNA contacts from those regions of the two libraries that carry the wild-type Zif268 amino acid sequence, as observed in the crystal structure. The wild-type portion of each library target site (white boxes) determines the register of the zinc finger-DNA interactions, such that the selected portions of the two libraries can be recombined to recognize the composite site H’IJKLMNOPQ. (B) Amino acid composition of the randomized DNA-binding positions on the α-helix of each zinc finger. A subset of the 20 amino acids was included in each DNA-binding position. Note that positions 4 and 5 of F2 (LS) are specified by the codons CTG AGC, which contain the recognition site of the restriction enzyme _Dde_I (underlined), used as a breakpoint to recombine the products of the two libraries.

Figure 3

Matrix specificity assay for seven zinc finger DNA-binding domains designed to bind sequences in the HIV-1 promoter. The seven constructs and their respective binding sites are labeled A-G. Binding of zinc fingers to 0.4 pmol DNA per 50 μl well is plotted vertically from phage ELISA absorbance readings (A450-A650). Each clone was tested using all seven DNA sequences, but strong binding was only observed to those sequences against which they had been designed.

Figure 4

Gel shift assays to determine the apparent equilibrium dissociation constants (_K_d values) of three engineered zinc finger peptides. Each purified peptide (HIV A, D, and F) was tested for binding to its target DNA site. Two-fold serial dilutions of each peptide resulted in binding reactions ranging from 80 nM peptide (lane 11), down to <80 pM peptide (lane 1). Lane 0 was a control without added protein. The letter “f” denotes free DNA and “b” denotes protein-bound complex. Approximate _K_d values for the three clones are shown above the corresponding figures. The binding behavior of the DNA-binding domain of wild-type Zif268 and its optimal DNA site is shown for comparison.

Figure 5

Matrix sequence-discrimination assay for three-zinc finger peptides generated using three regions of the HIV-1 LTR. 0, 5, and 20 nM of each purified peptide (HIV A, D, and F) were tested against each DNA-binding site (DNA: A, D and F). The letter “f” denotes free DNA and “b” denotes protein-bound complex. DNA binding by each protein was only observed to the sequences against which the zinc fingers had been designed.

Figure 6

Construction of a gene cassette coding for the zinc finger phage-display libraries used in this study. (A) The scheme used to generate selective randomization throughout the α-helix of a zinc finger. A set of complementary oligonucleotides is used to construct a series of “minicassettes” that can be annealed and ligated together to construct the randomized portion of the gene. Note that several similar minicassettes, coding for other fingers, need to be constructed to achieve the full randomization scheme outlined in Figure 2B. After ligation of all the minicassettes, the full-length construct is recovered by PCR using primers that contain _Sfi_I/_Not_I restriction enzyme sites for cloning into phage vector. (B) Examples of the oligonucleotides used to achieve the selective randomization of a single zinc finger (Finger1, Lib12). Key to randomized nucleotides: m = A/C; r = A/G; w = A/T; s = G/C; y = T/C; k = T/G; b = C/G/T; n = A/C/G/T; v = A/C/G; h = A/C/T; d = A/G/T.

References

1. Choo Y, Sanchez-Garcia I, Klug A. In vivo repression by a site-specific DNA-binding protein designed against an oncogenic sequence. Nature. 1994;372:642–645. - PubMed
1. Greisman HA, Pabo CO. A general strategy for selecting high-affinity zinc finger proteins for diverse DNA target sites. Science. 1997;275:657–661. - PubMed
1. Klug A, Rhodes D. ‘Zinc fingers’: a novel protein motif for nucleic acid recognition. Trends Biochem. Sci. 1987;12:464–469.
1. Choo Y, Klug A. Designing DNA-binding proteins on the surface of filamentous phage. Curr. Opin. Biotechnol. 1995;6:431–436. - PubMed
1. Miller J, McLachlan AD, Klug A. Repetitive zinc-binding domains in the protein transcription factor IIIA from Xenopus oocytes. EMBO J. 1985;4:1609–1614. - PMC - PubMed

A rapid, generally applicable method to engineer zinc fingers illustrated by targeting the HIV-1 promoter - PubMed (original) (raw)