Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-02-29
2004-06-08
Low, Christopher S. F. (Department: 1653)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007100, C435S091100, C536S023100, C530S350000
Reexamination Certificate
active
06746838
ABSTRACT:
The present invention relates to nucleic acid binding proteins. In particular, the invention relates to a method for designing a protein which is capable of binding to any predefined nucleic acid sequence.
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 prokaryotic 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.
Despite the great variety of structural domains, the specificity of the interactions observed to date between protein and DNA most often derives from the complementarity of the surfaces of a protein &agr;-helix and the major groove of DNA [Klug, (1993) Gene 135:83-92]. In light of the recurring physical interaction of &agr;-helix and major groove, the tantalising possibility arises that the contacts between particular amino acids and DNA bases could be described by a simple set of rules; in effect a stereochemical recognition code which relates protein primary structure to binding-site sequence preference.
It is clear, however, that no code will be found which can describe DNA recognition by all DNA-binding proteins. The structures of numerous complexes show significant differences in the way that the recognition &agr;-helices of DNA-binding proteins from different structural families interact with the major groove of DNA, thus precluding similarities in patterns of recognition. The majority of known DNA-binding motifs are not particularly versatile. and any codes which might emerge would likely describe binding to a very few related DNA sequences.
Even within each family of DNA-binding proteins. moreover, it has hitherto appeared that the deciphering of a code would be elusive. Due to the complexity of the protein-DNA interaction. there does not appear to be a simple “alphabetic” equivalence between the primary structures of protein and nucleic acid which specifies a direct amino acid to base relationship.
International patent application WO 96/06166 addresses this issue and provides a “syllabic” code which explains protein-DNA interactions for zinc finger nucleic acid binding proteins. A syllabic code is a code which relies on more than one feature of the binding protein to specify binding to a particular base, the features being combinable in the forms of “syllables”, or complex instructions, to define each specific contact.
However, this code is incomplete, providing no specific instructions permitting the specific selection of nucleotides other than G in the 5′ position of each triplet. The method relies on randomisation and subsequent selection in order to generate nucleic acid binding proteins for other specificities. Even with the aid of partial randomisation and selection, however, neither the method reported in WO 96/06166 nor any other methods of the prior art have succeeded in isolating a zinc finger polypeptide based on the first finger of Zif268 capable of binding triplets wherein the 5′ base is other than G or T. This is a serious shortfall in any ability to design zinc finger proteins.
Moreover, this document relies upon the notion that zinc fingers bind to a nucleic acid triplet or multiples thereof, as does all of the prior art. We have now determined that zinc finger binding sites are determined by overlapping 4 bp subsites, and that sequence-specificity at the boundary between subsites arises from synergy between adjacent fingers. This has important implications for the design and selection of zinc fingers with novel DNA binding specificities.
SUMMARY OF THE INVENTION
The present invention provides a more complete code which permits the selection of any nucleic acid sequence as the target sequence. and the design of a specific nucleic acid-binding protein which will bind thereto. Moreover, the invention provides a method by which a zinc finger protein specific for any given nucleic acid sequence may be designed and optimised. The present invention therefore concerns a recognition code which has been elucidated for the interactions of classical zinc fingers with nucleic acid. in this case a pattern of rules is provided which covers binding to all nucleic acid sequences.
The code set forth in the present invention takes account of synergistic interactions between adjacent zinc fingers. thereby allowing the selection of any desired binding site.
According to a first aspect of the present invention. therefore, we provide a method for preparing a nucleic acid binding protein of the Cys2-His2 zinc finger class capable of binding to a nucleic acid quadruplet in a target nucleic acid sequence, wherein binding to base 4 of the quadruplet by an &agr;-helical zinc finger nucleic acid binding motif in the protein is determined as follows:
a) if base 4 in the quadruplet is A. then position +6 in the &agr;-helix is Glu. Asn or Val;
b) if base 4 in the quadruplet is C, then position +6 in the &agr;-helix is Ser, Thr, Val, Ala, Glu or Asn.
Preferably, binding to base 4 of the quadruplet by an &agr;-helical zinc finger nucleic acid binding motif in the protein is additionally determined as follows:
c) if base 4 in the quadruplet is G, then position +6 in the &agr;-helix is Arg or Lys;
d) if base 4 in the quadruplet is T. then position +6 in the &agr;-helix is Set, Thr, Val or Lys.
The quadruplets specified in the present invention are overlapping, such that, when read 3′ to 5′ on the -strand of the nucleic acid, base 4 of the first quadruplet is base 1 of the second, and so on. Accordingly, in the present application, the bases of each quadruplet are referred by number, from 1 to 4, 1 being the 3′ base and 4 being the 5′ base. Base 4 is equivalent to the 5′ base of a classical zinc finger binding triplet.
All of the nucleic acid-binding residue positions of zinc fingers, as referred to herein, are numbered from the first residue in the &agr;-helix of the finger, ranging from +1 to +9. “−1” refers to the residue in the framework structure immediately preceding the &agr;-helix in a Cys2-His2 zinc finger polypeptide.
Residues referred to as “++2” are residues present in an adjacent (C-terminal) finger. They reflect the synergistic cooperation between position +2 on base 1 (on the + strand) and position +6 of the preceding (N-terminal) finger on base 4 of the preceding (3′) quadruplet, which is the same base due to the overlap. Where there is no C-terminal adjacent finger, “++” interactions do not operate.
Cys2-His2 zinc finger binding proteins, as is well known in the art, bind to target nucleic acid sequences via &agr;-helical zinc metal atom coordinated binding motifs known as zinc fingers. Each zinc finger in a zinc finger nucleic acid binding protein is responsible for determining binding to a nucleic acid quadruplet in a nucleic acid binding sequence. Preferably, there are 2 or more zinc fingers, for example 2, 3, 4, 5 or 6 zinc fingers, in each binding protein. Advantageously, there are 3 zinc fingers in each zinc finger binding protein.
The method of the present invention allows the production of what are essentially artificial nucleic acid binding proteins. In these proteins, artificial analogues of amino acids may be used, to impart the proteins with desired properties or for other reasons. Thus, the term “amino acid”, particularly in the context where “any amino acid” is referred to, means any sort of natural or artificial amino acid or amino acid analo
Choo Yen
Isalan Mark
Klug Aaron
Gendaq Limited
Low Christopher S. F.
Robinson Hope A.
Townsend and Townsend / and Crew LLP
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