Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-11-20
2004-09-21
Borin, Michael (Department: 1631)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C702S019000
Reexamination Certificate
active
06794136
ABSTRACT:
BACKGROUND
Sequence-specific binding of proteins to DNA, RNA, protein and other molecules is involved in a number of cellular processes such as, for example, transcription, replication, chromatin structure, recombination, DNA repair, RNA processing and translation. The binding specificity of cellular binding proteins that participate in protein-DNA, protein-RNA and protein-protein interactions contributes to development, differentiation and homeostasis. Alterations in specific protein interactions can be involved in various types of pathologies such as, for example, cancer, cardiovascular disease and infection.
Increased understanding of the nature and mechanism of protein binding specificity has encouraged the hope that specificity of a binding protein could be altered in a predictable fashion, or that a binding protein of predetermined specificity could be constructed de novo. See, for example, Blackburn (2000)
Curr. Opin. Struct. Biol
. 10:399-400; Segal et al. (2000)
Curr. Opin. Chem. Biol
. 4:34-39. To date, the greatest progress in both of these areas has been obtained with a class of binding proteins known as zinc finger proteins.
Zinc finger proteins (ZFPs) are proteins that can bind to DNA in a sequence-specific manner. Zinc fingers were first identified in the transcription factor TFIIIA from the oocytes of the African clawed toad,
Xenopus laevis
. An exemplary motif characterizing one class of these proteins (C
2
H
2
class) is -Cys-(X)
2-4
-Cys-(X)
12
-His-(X)
3-5
-His (SEQ ID NO: 1), where X is any amino acid. A single zinc finger domain is about 30 amino acids in length, and several structural studies have demonstrated that it contains a beta turn (containing the two invariant cysteine residues) and an alpha helix (containing the two invariant histidine residues), which are held in a particular conformation through coordination of a zinc atom by the two cysteines and the two histidines. To date, over 10,000 zinc finger sequences have been identified in several thousand known or putative transcription factors. Zinc finger domains are involved not only in DNA recognition, but also in RNA binding and in protein-protein binding. Current estimates are that this class of molecules will constitute about 2% of all human genes.
The x-ray crystal structure of Zif268, a three-finger domain from a murine transcription factor, has been solved in complex with a cognate DNA sequence. Pavletich et al. (1991)
Science
252:809-817. The structure suggests that each finger interacts independently with a 3-nucleotide DNA subsite, with side-chains at positions −1, +2, +3 and +6 (with respect to the start of the &agr;-helix) making contacts with bases in a DNA triplet subsite. The amino terminus of Zif268 is situated at the 3′ end of the DNA strand with which it makes most contacts. Some zinc fingers can bind to a fourth base in a target segment. If the strand with which a zinc finger protein makes most contacts is designated the target strand, some zinc finger proteins bind to a three base triplet in the target strand and a fourth base on the non-target strand. The fourth base is complementary to the base immediately 3′ of the three base subsite. See Wolfe et al. (2000)
Annu. Rev. Biophys. Biomol. Struct
. 3:183-212 for a recent review on DNA recognition by zinc finger proteins.
The structure of the Zif268-DNA complex also suggested that the DNA sequence specificity of a zinc finger protein could be altered by making amino acid substitutions at the four positions (−1, +2, +3 and +6) involved in DNA base recognition. Phage display experiments using zinc finger combinatorial libraries to test this observation were published in a series of papers in 1994. Rebar et al. (1994)
Science
263:671-673; Jamieson et al. (1994)
Biochemistry
33:5689-5695; Choo et al. (1994)
Proc. Natl. Acad. Sci. USA
91:11163-11167 (1994). Combinatorial libraries were constructed with randomized amino acid residues in either the first or middle finger of Zif268, and members of the library able to bind to an altered Zif268 binding site (in which the appropriate DNA sub-site was replaced by an altered DNA triplet) were selected. The amino acid sequences of the selected fingers were correlated with the nucleotide sequences of the new binding sites for which they had been selected. In additional experiments, correlations were observed between the nature of mutations introduced into a recognition helix and resulting alterations in binding specificity. The results of these experiments have led to a number of proposed substitution rules for design of ZFPs with altered binding specificity. Most of these substitution rules concern amino acids occupying positions −1, +2, +3 and +6 in the recognition helix of a zinc finger protein, which have been reported to be the principal determinants of binding specificity. Some of these rules are supported by site-directed mutagenesis of the three-finger domain of the transcription factor, Sp-1. Desjarlais et al. (1992a)
Proc. Natl. Acad. Sci. USA
89:7345-7349; Desjarlais et al. (1992b)
Proteins: Structure, Function and Genetics
12:101-104; Desjarlais et al. (1993)
Proc. Natl. Acad. Sci. USA
90:2256-2260.
Two general classes of design rules for zinc finger proteins have been proposed. The first relates one or more amino acids at a particular position in the recognition helix with a nucleotide at a particular position in the target subsite. For example, if the 5′-most nucleotide in a three-nucleotide target subsite is G, certain design rules specify that the amino acid at position +6 of the recognition helix is arginine, and optionally position +2 of an adjacent carboxy-terminal finger is aspartic acid. The second class of design rules relates the sequence of an entire recognition helix with the sequence of a three- or four-nucleotide target subsite. These and related design rules have been elaborated in, for example, U.S. Pat. No. 6,140,081; PCT WO98/53057; PCT WO98/53058; PCT WO98/53059; PCT WO98/53060; PCT WO00/23464; Choo et al. (2000)
Curr. Opin. Struct. Biol
. 10:411-416; Segal et al. (2000)
Curr. Opin. Chem. Biol
. 4:34-39; and references cited in these publications.
In addition, two strategies for identifying a zinc finger which binds to a specific triplet subsite have emerged. In the first strategy, the sequence of a portion (generally a single finger but, in some cases, one-and-a-half fingers) of a multi-finger protein is randomized (generally at positions −1, +2, +3 and +6 of the recognition helix), and members of the randomized population able to bind to a particular subsite are selected. The second strategy relies on de novo synthesis of a zinc finger specific for a particular subsite, using existing design rules as set forth supra. See, for example, Choo et al. (1997)
Curr. Opin. Struct. Biol
. 7:117-125; Greisman et al. (1997)
Science
275:657-661.
In attempting to construct a ZFP of predetermined specificity able potentially to discriminate a target sequence in a eucaryotic genome, it is necessary to join individual zinc fingers into a multi-finger protein. However, because of overlap in the recognition of adjacent subsites in a target sequence by adjacent zinc fingers in a ZFP, cooperativity and synergistic interactions between adjacent fingers, currently existing design and selection methods have been limited largely to zinc fingers which recognize G-rich target subsites; in particular triplets of the form GNN and, to a lesser extent, TNN. Although certain selection methods not limited to GNN triplets have been devised, they involve construction of multiple libraries; hence they are more difficult to practice and the degree of possible randomization is limited.
Another deficiency of current design rules is that they do not provide zinc finger sequences able to recognize every one of the 64 possible triplet subsites. Moreover, even for those subsites that are covered, the design rules are degenerate, in that they often specify more than on
Eisenberg Stephen P.
Jamieson Andrew
Liu Qiang
Rebar Edward
Borin Michael
Sangamo Biosciences, Inc.
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