Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-01-29
2002-06-25
Celsa, Bennett (Department: 1627)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S004000, C435S005000, C435S006120, C435S069100, C435S091500, C435S091500, C435S091500, C435S091500, C435S091500, C436S501000
Reexamination Certificate
active
06410248
ABSTRACT:
STATEMENT AS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT
Not applicable.
BACKGROUND OF THE INVENTION
Design of DNA binding proteins that will recognize desired target sites on double-stranded DNA presents a challenging situation. A number of DNA-binding motifs have yielded variants with altered specificities, and zinc finger proteins related to TFIIIA (Miller et al.,
EMBO J
. 4:1609 (1985)) and Zif268 (Christy et al.,
Proc. Natl. Acad. Sci. U.S.A
. 85:7857 (1988)) appear to provide a versatile framework for design. Modeling, sequence comparisons, and phage display have been used to alter the specificity of an individual zinc finger within a multifinger protein (Nardelli et al.,
Nucleic Acids Res
. 20:4137 (1992); Thukral et al.,
Mol. Cell. Biol
. 12:2784 (1992); Desjarlais et al.,
Proteins
12:101 (1992) Desjarlais et al.,
Proteins
13:272 (1992);
Proc. Natl. Acad. Sci. U.S.A
. 89:7345 (1992);
Proc. Natl. Acad. Sci. U.S.A
. 91:11099 (1994); Rebar et al.,
Science
263:671 (1994); Choo et al.,
Proc. Natl. Acad. Sci. U.S.A
. 91:11163 (1994); Choo et al.,
Proc. Natl. Acad. Sci. U.S.A
. 91:11168; Jamieson et al.,
Biochemistry
33:5689 (1994); Wu et al.,
Proc Natl. Acad. Sci. U.S.A
. 92:344 (1995); Taylor et al.,
Biochemistry
34:3222 (1995); Cheng et al.,
J. Mol. Biol
. 251:1 (1995)), and fingers also have been “mixed and matched” to construct new DNA binding proteins (Desjarlais et al.,
Proc. Natl. Acad. Sci. U.S.A
. 90:2256 (1993); Choo et al.,
Nature
372:642 (1994)).
These design and selection studies have assumed that each finger (with a corresponding, 3 base pair (bp) subsite) can be treated as an independent unit (FIG.
1
B). This assumption has provided a useful starting point for design studies, but crystallographic studies of zinc finger-DNA complexes reveal many examples of contacts that couple neighboring fingers and subsites (Pavletich et al.,
Science
252:809 (1991); Fairall et al.,
Nature
366:483 (1993); Paveltich et al.,
Science
261:1701 (1993); Elrod-Erickson et al.,
Structure
4:1171 (1996)). Context-dependent interactions are therefore important for zinc finger-DNA recognition (Nardelli et al.,
Nucleic Acids Res
. 20:4137 (1992); Thukral et al.,
Mol. Cell. Biol
. 12:2784 (1992); Desjarlais et al.,
Proteins
12:101 (1992); Desjarlais et al.,
Proteins
13:272 (1992);
Proc. Natl. Acad. Sci. U.S.A
. 89:7345 (1992);
Proc. Natl. Acad. Sci. U.S.A
. 91:11099 (1994); Taylor et al.,
Biochemistry
34:3222 (1995); Cheng et al.,
J. Mol. Biol
. 251:1 (1995); Desjarlais et al.,
Proc. Natl. Acad. Sci. U.S.A
. 90:2256 (1993)).
“Mix and match” design strategies have, so far, been limited to binding sites in which the primary strand (
FIG. 1B
) contains at least one guanine within each 3 bp subsite (Nardelli et al.,
Nucleic Acids Res
. 20:4137 (1992); Thukral et al.,
Mol. Cell. Biol
. 12:2784 (1992); Desjarlais et al.,
Proteins
12:101 (1992); Desjarlais et al.,
Proteins
13:272 (1992);
Proc. Natl. Acad. Sci. U.S.A
. 89:7345 (1992);
Proc. Natl. Acad. Sci. U.S.A
. 91:11099 (1994); Desjarlais et al.,
Proc. Natl. Acad. Sci. U.S.A
. 90:2256 (1993); Choo et al.,
Nature
372:642 (1994)). The affinities of designed zinc finger proteins also have varied widely, and some K
d
s have been in the micromolar range (Desjarlais et al.,
Proc. Natl. Acad. Sci. U.S.A
. 90:2256 (1993); Choo et al,
Nature
372:642 (1994)). Subtle, context-dependent interactions may have a critical cumulative effect when optimizing, multifinger proteins: A modest (10-fold) increase in affinity for each finger may yield a substantial (1000-fold) increase in affinity for a three-finger protein. However, existing strategies have not taken these context-dependent interactions into account when designing multi-finger zinc finger proteins that bind to a target site.
SUMMARY OF THE INVENTION
The present invention therefore provides a selection strategy for a making multi-finger zinc finger proteins that takes into account context-dependent interactions of zinc fingers and target subsites. This strategy thus provides a means for making zinc finger proteins that bind to a specific target site.
In one aspect, the present invention provides a method of making a zinc finger protein that binds to a target site, the method comprising the steps of: (i) providing a target site comprising first, second, and third subsites; (ii) identifying a first finger of the zinc finger protein by: (a) providing a nucleic acid library encoding variants of a zinc finger protein comprising a randomized first finger, and constant fingers that bind to known subsites; and (b) selecting a first zinc finger protein that binds to a target site comprising the first subsite and the known subsites, the first zinc finger protein comprising a selected variant first finger and the constant fingers; (iii) identifying a second finger of the zinc finger protein by: (a) providing a nucleic acid library encoding variants of a zinc finger protein comprising the selected variant first finger, a randomized second finger, and a constant finger that binds to a known subsite; and (b) selecting a second zinc finger protein that binds to a target site comprising the first and second subsites and the known subsite, the second zinc finger protein comprising the selected variant first finger, a selected variant second finger, and the constant finger; and (iv) identifying a third finger of a zinc finger protein by: (a) providing a nucleic acid library encoding variants of a zinc finger protein comprising the selected variant first finger, the selected variant second finger, and a randomized third finger; and (b) selecting a third zinc finger protein that binds to the target site comprising the first, second, and third subsites, the third zinc finger protein comprising the selected variant first finger, the selected variant second finger, and a selected variant third finger, thereby making a zinc finger protein that binds to the target site.
In one embodiment, the first, second, and third fingers are randomized at positions −1, 1, 2, 3, 5, and 6. In another embodiment, the first, second, and third fingers are randomized using degenerate oligonucleotides. In another embodiment, the constant finger is from Zif268, Tramtrack, GLI, or TFIIIA. In another embodiment, a dissociation constant of the zinc finger protein is less than about 0.1 nM. In another embodiment, the steps of selecting the first, second, or third zinc finger protein comprises selecting a pool of variants of the first, second or third zinc finger protein. In one embodiment, the constant fingers are on the N-terminal side of the randomized first finger or the selected variant first finger.
In one embodiment, the nucleic acid library is a phagemid display vector library or a phage display vector library. In another embodiment, the vector phagemid comprises a C-terminal subsequence of the M13 gene III protein.
In one embodiment, the method comprises identifying additional fingers and the target site comprises more than three subsites. In another embodiment, three additional fingers are identified to make a six fingered protein, and the target site comprises six subsites.
In one embodiment, the step of selecting the first, second or third zinc finger protein comprises using a biotinylated target site. In another embodiment, the target site is a TATA box, a p53 binding site or a nuclear receptor element.
In one embodiment, the zinc finger protein is fused to a heterologous DNA binding domain. In another embodiment, the zinc finger protein is fused to a heterologous modular domain involved in protein-protein recognition.
In another embodiment, the method further comprises identifying a heterologous DNA binding domain fused to a zinc finger protein, wherein the heterologous DNA binding domain binds to a secondary target, further comprising the steps of: (a) providing a nucleic acid library encoding variants of a fusion zinc finger protein comprising a randomized heterologous DNA binding domain, and further comprising the first, middle, and last select
Greisman Harvey A.
Pabo Carl O.
Brennan Sean M.
Celsa Bennett
Massachusetts Institute of Technology
Robins & Pasternak LLP
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