Nucleic acid binding proteins
AssigneeGendaq Limited
InventorsYen Choo, Aaron Klug, Mark Isalan
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Abstract
The invention provides a method for designing 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 alpha-helical zinc finger nucleic acid binding motif in the protein is determined as follows: if base 4 in the quadruplet is A, then position +6 in the alpha-helix is Glu, Asn or Val; if base 4 in the quadruplet is C, then position +6 in the alpha-helix is Ser, Thr, Val, Ala, Glu or Asn; if base 4 of the quadruplet is G, the position +6 in the alpha helix is Arg or Lys; if position 4 in the quadruplet is T, then position +6 in the alpha helix is Ser, Thr, Val or Lys.
Claims12
A method for designing a nucleic acid binding protein that binds to a target nucleotide sequence, wherein the binding protein comprises a plurality of zinc fingers of the Cys2-His2 class, wherein the second His in Cys2-His2 is His or Cys, and wherein each zinc finger has an alpha helix and amino acids numbered −1, 1, 2, 3, 4, 5, 6, 7, 8, and 9 with position 1 being the first amino acid of the alpha helix, further wherein adjacent zinc fingers bind synergistically to overlapping quadruplet target subsites, wherein the method comprises:
- i) selecting a quadruplet within the target nucleotide sequence;
- ii) designing the binding protein such that binding of a zinc finger to the quadruplet is obtained by choosing the sequence of particular residues of the zinc finger depending on the nucleotide sequence of the quadruplet, as follows:
- a) if base 4 in the quadruplet is A, then position +6 in the α-helix is Glu, Asn or Val;
- b) if base 4 in the quadruplet is C, then position +6 in the α-helix is Ser, Thr, Val, Ala, Glu or Asn.
A method for designing a nucleic acid binding protein that binds to a target nucleotide sequence, wherein the binding protein comprises a plurality of zinc fingers of the Cys2-His2 class, wherein the second His in Cys2-His2 is His or Cys, and wherein each zinc finger has an alpha helix and amino acids are numbered −1, 1, 2, 3, 4, 5, 6, 7, 8, and 9 with position 1 being the first amino acid of the alpha helix, and adjacent zinc fingers bind synergistically to overlapping quadruplet target subsites, wherein the method comprises:
- i) selecting a quadruplet within the target nucleotide sequence;
- ii) designing the binding protein such that binding of a zinc finger to the quadruplet is obtained by choosing the sequence of particular residues of the zinc finger depending on the nucleotide sequence of the quadruplet, as follows:
- a) if base 4 in the quadruplet is G, then position +6 in the α-helix is Arg or Lys;
- b) if base 4 in the quadruplet is A, then position +6 in the α-helix is Glu, Asn or Val;
- c) if base 4 in the quadruplet is T, then position +6 in the α-helix is Ser, Thr, Val or Lys;
- d) if base 4 in the quadruplet is C, then position +6 in the α-helix is Ser, Thr, Val, Ala, Glu or Asn;
- e) if base 3 in the quadruplet is G, then position +3 in the α-helix is His;
- f) if base 3 in the quadruplet is A, then position +3 in the α-helix is Asn;
- g) if base 3 in the quadruplet is T, then position +3 in the α-helix is Ala, Ser or Val;
- h) if base 3 in the quadruplet is C, then position +3 in the α-helix is Ser, Asp, Glu, Leu, Thr or Val;
- i) if base 2 in the quadruplet is G, then position −1 in the α-helix is Arg;
- j) if base 2 in the quadruplet is A, then position −1 in the α-helix is Gln;
- k) if base 2 in the quadruplet is T, then position −1 in the α-helix is His or Thr;
- l) if base 2 in the quadruplet is C, then position −1 in the α-helix is Asp or His;
- m) if base 1 in the quadruplet is G, then position +2 is Glu;
- n) if base 1 in the quadruplet is A, then position +2 Arg or Gln;
- o) if base 1 in the quadruplet is C, then position +2 is Asn, Gln, Arg, His or Lys;
- (p) if base 1 in the quadruplet is T, then position +2 is Ser or Thr.
Dependencies are inferred from the claim text.