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US 6,205,404 B1Granted
DNA-binding proteins of the zinc-finger class
OwnerGeorge S. Michaels, Raik-Hiio Mikelsaar +1inventors
InventorsGeorge S. Michaels, Raik-Hiio Mikelsaar, Richard J. Feldmann
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Abstract
The invention is directed to the design of DNA-binding proteins (DBP's) with capabilities of binding to any predetermined target double-stranded DNA sequence. Disclosed are the rules for design of the proteins; an algorithm for screening for the optimal DBP's; a computer system employing the rules and the algorithm; general formulae encompassing the proteins; and methods of use of the proteins.
Claims14
A method for designing a DNA-binding protein (DBP), with multiple zinc-finger (ZF) domains connected by linker sequences, that binds selectively to a target DNA sequence within a given gene, each of said ZF domains having the formula
- A1XCX2-4CA2A3XFXZ3XXZ2LXZ1HX3-5H (SEQ ID NO:
- 3)
- and each of said linkers having the formula
- A4A5X0-2EA6 P (SEQ ID NO 4),
- wherein
- (i) X is any amino acid;
- (ii) X2-4 is a peptide from 2 to 4 amino acids in length;
- (iii) X3-5 is a peptide from 3 to 5 amino acids in length;
- (iv) X0-2 is a peptide from 0 to 2 amino acids in length;
- (iv) A1 is selected from the group consisting of phenylalanine and tyrosine;
- (v) A2 is selected from the group consisting of glycine and aspartic acid;
- (vi) A3 is selected from the group consisting of lysine and arginine;
- (vii) A4 is selected from the group consisting of threonine and serine;
- (viii) A5 is selected from the group consisting of glycine and glutamic acid;
- (ix) A6 is selected from the group consisting of lysine and arginine;
- (x) C is cysteine;
- (xi) F is phenylalanine;
- (xii) L is leucine;
- (xiii) H is histidine;
- (xiv) E is glutamic acid;
- (xv) P is proline;
- and (xvi) Z1, Z2 and Z3 are the base-contacting amino acids, which method comprises an algorithm comprising the steps of:
- (a) setting a genome to be screened;
- (b) selecting the target DNA sequence in the genome for binding;
- (c) setting the number of ZF domains to nd;
- (d) dividing the target DNA sequence into nucleotide blocks wherein each block contains nz nucleotides using a first routine where nz is determined using the following relationship:
- nz=3nd;
- (e) assigning base-contacting amino acids at Z1, Z2 and Z3 to each ZF domain, according to the A Rules and/or B Rules set forth in Tables 1-3 of the specification, of a DBP which binds to the first nucleotide block from step (d) as numbered from the first 5′ nucleotide of the target gene sequence to generate a block-specific DBP and calculating the binding energy, Binding Energyblock, of each ZF domain of each such block-specific DBP as the product of the binding energies, Binding Energydomain, of all ZF domains of the DBP, each determined using the formula:
- Binding Energydomain=(5×the number of hydrogen bonds)+(2×the number of H2O contacts)+(the number of hydrophobic contacts);
- (f) subdividing the DBP from step (d) into blocks using a second routine to generate a subdivided DBP having three ZF domains;
- (g) screening the subdivided DBP from step (f) against the genome using a third routine to determine the number of binding sites in the genome for each subdivided DBP in the genome and assigning a binding energy for each such site using the following formula:
- Binding Energysite n=(5×the number of hydrogen bonds)+(2×the number of H2O contacts)+(the number of hydrophobic contacts);
- (h) calculating a ratio of binding energy, Rb, using a fourth routine for each nucleotide block-specific DBP from step (e) using the following formula:
- Rb=Binding Energyblock/the sum of all Binding Energysite n's for all subdivided DBP's from step (g);
- (i) repeating steps (f) through (h) for each subdivided DBP wherein nd≧4;
- (j) repeating steps (d) through (i) for each nucleotide block in the target DNA sequence containing nz nucleotides;
- (k) rank-ordering Rb numerical values obtained from step (h);
- and
- (l) selecting a DBP with an acceptable Rb value.
36 more elements
A computer system for designing a DNA-binding protein (DBP), with multiple zinc-finger (ZF) domains connected by linker sequences, that binds selectively to a target DNA sequence within a given gene, each of said ZF domains having the formula
- A1XCX2-4CA2A3XFXZ3XXZ2LXZ1HX3-5H (SEQ ID NO 3)
- and each of said linkers having the formula
- A4A5X0-2EA6 P (SEQ ID NO 4),
- wherein
- (i) X is any amino acid;
- (ii) X2-4 is a peptide from 2 to 4 amino acids in length;
- (iii) X3-5 is a peptide from 3 to 5 amino acids in length;
- (iv) X0-2 is a peptide from 0 to 2 amino acids in length;
- (iv) A1 is selected from the group consisting of phenylalanine and tyrosine;
- (v) A2 is selected from the group consisting of glycine and aspartic acid;
- (vi) A3 is selected from the group consisting of lysine and arginine;
- (vii) A4 is selected from the group consisting of threonine and serine;
- (viii) A5 is selected from the group consisting of glycine and glutamic acid;
- (ix) A6 is selected from the group consisting of lysine and arginine;
- (x) C is cysteine;
- (xi) F is phenylalanine;
- (xii) L is leucine;
- (xiii) H is histidine;
- (xiv) E is glutamic acid;
- (xv) P is proline;
- and (xvi) Z1, Z2 and Z3 are the base-contacting amino acids, which computer system comprises a computer readable memory which executes a method for designing the DBP, wherein the method comprises the steps of:
- (a) setting a genome to be screened;
- (b) selecting the target DNA sequence in the genome for binding;
- (c) setting the number of ZF domains to nd;
- (d) dividing the target DNA sequence into nucleotide blocks wherein each block contains nz nucleotides using a first routine where nz is determined using the following relationship:
- nz=3nd;
- (e) assigning base-contacting amino acids at Z1, Z2 and Z3 to each ZF domain, according to the A Rules and/or B Rules set forth in Tables 1-3 of the specification, of a DBP which binds to the first nucleotide block from step (d) as numbered from the first 5′ nucleotide of the target gene sequence to generate a block-specific DBP and calculating the binding energy, Binding Energyblock, of each ZF domain of each such block-specific DBP as the product of the binding energies, Binding Energydomain, of all ZF domains of the DBP, using the formula:
- Binding Energydomain=(5×the number of hydrogen bonds)+(2×the number of H2O contacts)+(the number of hydrophobic contacts);
- (f) subdividing the DBP from step (d) into blocks using a second routine to generate a subdivided DBP having three ZF domains;
- (g) screening the subdivided DBP from step (f) against the genome using a third routine to determine the number of binding sites in the genome for each subdivided DBP in the genome and assigning a binding energy for each such site using the following formula:
- Binding Energysite n=(5×the number of hydrogen bonds)+(2×the number of H2O contacts)+(the number of hydrophobic contacts);
- (h) calculating a ratio of binding energy, Rb, using a fourth routine for each nucleotide block-specific DBP from step (e) using the following formula:
- Rb=Binding Energyblock/the sum of all Binding Energysite n's for all subdivided DBP's from step (g);
- (i) repeating steps (f) through (h) for each subdivided DBP wherein nd≧4;
- (j) repeating steps (d) through (i) for each nucleotide block in the target DNA sequence containing nz nucleotides;
- (k) rank-ordering Rb numerical values obtained from step (h);
- and
- (l) selecting a DBP with an acceptable Rb value.
35 more elements
Dependencies are inferred from the claim text.