Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-05-08
2004-05-11
Brusca, John S. (Department: 1631)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S069100, C435S091410, C435S091510
Reexamination Certificate
active
06733970
ABSTRACT:
The present application relates to a method for screening zinc finger polypeptides for a desired binding ability. In particular, the invention relates to a polysome display technique which permits the isolation of binding polypeptides without resorting to phage display techniques.
Protein-nucleic acid recognition is a commonplace phenomenon which is central to a large number of biomolecular control mechanisms which regulate the functioning of eukaryotic and procaryotic cells. For instance, protein-DNA interactions form the basis of the regulation of gene expression and are thus one of the subjects most widely studied by molecular biologists.
A wealth of biochemical and structural information explains the details of protein-DNA recognition in numerous instances, to the extent that general principles of recognition have emerged. Many DNA-binding proteins contain independently folded domains for the recognition of DNA, and these domains in turn belong to a large number of structural families, such as the leucine zipper, the “helix-turn-helix” and zinc finger families.
Most sequence-specific DNA-binding proteins bind to the DNA double helix by inserting an &agr;-helix into the major groove (Pabo & Sauer 1992 Annu. Rev. Biochem. 61, 1053-1095; Harrison 1991 Nature (London) 353, 715-719; and Klug 1993 Gene 135, 83-92). Sequence specificity results from the geometrical and chemical complementarity between the amino acid side chains of the &agr;-helix and the accessible groups exposed on the edges of base-pairs. In addition to this direct reading of the DNA sequence. interactions with the DNA backbone stabilise the complex and are sensitive to the conformation of the nucleic acid, which in turn depends on the base sequence (Dickerson & Drew 1981 J. Mol. Biol. 149, 761-786. Crystal structures of protein-DNA complexes have shown that proteins can be idiosyncratic in their mode of DNA recognition, at least partly because they may use alternative geometries to present their sensory &agr;-helices to DNA, allowing a variety of different base contacts to be made by a single amino acid and vice versa (Matthews 1988 Nature (London) 335, 294-295).
Protein engineering experiments have shown that it is possible to alter rationally the DNA-binding characteristics of individual zinc fingers when one or more of the &agr;-helical positions is varied in a number of proteins (Nardelli et al., 1991 Nature (London) 349, 175-178; Nardelli et al. 1992 Nucleic Acids Res. 20, 4137-4144; and Desjarlais & Berg 1992a Proteins 13, 272). It has already been possible to propose some principles relating amino acids on the &agr;-helix to corresponding bases in the bound DNA sequence (Desjarlais & Berg 1992b Proc. Natl. Acad. Sci. USA 89, 7345-7349). However in this approach the altered positions on the &agr;-helix are prejudged, making it possible to overlook the role of positions which are not currently considered important; and secondly, owing to the importance of context, concomitant alterations are sometimes required to affect specificity (Desjarlais & Berg 1992b), so that a significant correlation between an amino acid and base may be misconstrued.
More sophisticated principles describing the relationship between the sequence of the zinc finger and the nucleic acid target have been described, for example in WO 96/06166 (Medical Research Council).
To investigate binding of mutant Zf proteins, Thiesen and Bach (1991 FEBS 283, 23-26) mutated Zf fingers and studied their binding to randomised oligonucleotides, using electrophoretic mobility shift assays. Subsequent use of phage display technology has permitted the expression of random libraries of Zf mutant proteins on the surface of bacteriophage. The three Zf domains of Zif268, with 4 positions within finger one randomised, have been displayed on the surface of filamentous phage by Rebar and Pabo (1994 Science 263, 671-673). The library was then subjected to rounds of affinity selection by binding to target DNA oligonucleotide sequences in order to obtain Zf proteins with new binding specificities. Randomised mutagenesis (at the same postions as those selected by Rebar & Pabo) of finger 1 of Zif 268 with phage display has also been used by Jamieson et al., (1994 Biochemistry 33, 5689-5695) to create novel binding specificity and affinity.
In summary, it is known that Zf protein motifs are widespread in DNA binding proteins and that binding is via three key amino acids, each one contacting a single base pair in the target DNA sequence. Motifs are modular and may be linked together to form a set of fingers which recognise a contiguous DNA sequence (e.g. a three fingered protein will recognise a 9 mer etc). The key residues involved in DNA binding have been identified through sequence data and from structural information. Directed and random mutagenesis has confirmed the role of these amino acids in determining specificity and affinity. Phage display has been used to screen for new binding specificities of random mutants of fingers. Therefore, the combination of a set of rules with a selection process appears to provide the most promising avenue for the development of zinc finger proteins.
SUMMARY OF THE INVENTION
According to a first aspect of, the present invention, there is provided a method for producing a zinc finger nucleic acid binding protein comprising preparing a zinc finger protein according design rules, varying the protein at one or more positions, and selecting variants which bind to a target nucleic acid sequence by polysome display.
According to a second aspect of the present invention, there is provided a method for producing a zinc finger nucleic acid binding protein comprising an at least partially varied sequence and selecting variants thereof which bind to a target DNA strand, comprising the steps of:
(i) preparing a nucleic acid binding protein of the Cys2-His2 zinc finger class capable of binding to a nucleic acid triplet in a target nucleic acid sequence, wherein binding to each base of the triplet by an cc-helical zinc finger nucleic acid binding motif in the protein is determined as follows:
a) if the 5′ base in the triplet is G, then position +6 in the &agr;-helix is Arg; or position +6 is Ser or Thr and position ++2 is Asp;
b) if the 5′ base in the triplet is A, then position +6 in the &agr;-helix is Gln and ++2 is not Asp;
c) if the 5′ base in the triplet is T, then position +6 in the &agr;-helix is Ser or Thr and position ++2 is Asp;
d) if the 5′ base in the triplet is C, then position +6 in the &agr;-helix may be any amino acid, provided that position ++2 in the &agr;-helix is not Asp;
e) if the central base in the triplet is G, then position +3 in the &agr;-helix is His,
f) if the central base in the triplet is A, then position +3 in the &agr;-helix is Asn;
g) if the central base in the triplet is T, then position +3 in the &agr;-helix is Ala. Ser, or Val; provided that if it is Ala, then one of the residues at −1 or +6 is a small residue;
h) if the central base in the triplet is C, then position +3 in the &agr;-helix is Ser, Asp, Glu, Leu, Thr or Val;
i) if the 3′ base in the triplet is G, then position −1 in the &agr;-helix is Arg;
j) if the 3′ base in the triplet is A, then position −1 in the &agr;-helix is Gln;
k) if the 3′ base in the triplet is T, then position −1 in the &agr;-helix is Asn or Gln;
l) if the 3′ base in the triplet is C, then position −1 in the &agr;-helix is Asp;
(ii) varying the resultant polypeptide at at least one position; and
(iii) selecting the variants which bind to a target nucleic acid sequence by polysome display.
SEQ ID NOs: are assigned as follows:
SEQ ID NO:1
Nucleotide sequence encoding the zinc finger protein;
SEQ ID NO:2
Amino acid sequence of the zinc finger protein;
SEQ ID NO:3
Sequence of the zinc finger framework as described page 6 lines 21-24 of the specification
XXCXXXXXCX XXXXXXXXXX XXXHXXXXXX H
SEQ ID NO:4
Alternate Sequen
Choo Yen
Moore Michael
Brennan Sean M.
Brusca John S.
Gendaq Limited
Robins & Pasternak LLP
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