CA2290886A1 - Nucleic acid binding proteins - Google Patents
Nucleic acid binding proteins Download PDFInfo
- Publication number
- CA2290886A1 CA2290886A1 CA002290886A CA2290886A CA2290886A1 CA 2290886 A1 CA2290886 A1 CA 2290886A1 CA 002290886 A CA002290886 A CA 002290886A CA 2290886 A CA2290886 A CA 2290886A CA 2290886 A1 CA2290886 A1 CA 2290886A1
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- Prior art keywords
- nucleic acid
- quadruplet
- base
- helix
- alpha
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Classifications
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- C—CHEMISTRY; METALLURGY
- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B40/00—Libraries per se, e.g. arrays, mixtures
- C40B40/02—Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
- C07K1/047—Simultaneous synthesis of different peptide species; Peptide libraries
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4702—Regulators; Modulating activity
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1034—Isolating an individual clone by screening libraries
- C12N15/1037—Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
Abstract
The invention provides 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 .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 Gln and position ++2 is not Asp; and if base 4 in the quadruplet is C, then position +6 in the .alpha.-helix may be any residue, as long as position ++2 in the .alpha.-helix is not Asp.
Claims (29)
1. A method for preparing a nucleic acid binding protein of the Cyst-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 a-helical zinc finder 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 .alpha.-helix is Gln and position + +2 is not Asp;
b) if base 4 in the quadruplet is C, then position +6 in the a-helix may be any residue, as long as position + +2 in the a-helix is not Asp.
a) if base 4 in the quadruplet is A, then position +6 in the .alpha.-helix is Gln and position + +2 is not Asp;
b) if base 4 in the quadruplet is C, then position +6 in the a-helix may be any residue, as long as position + +2 in the a-helix is not Asp.
2. A method according to claim 1, wherein binding to base 4 of the quadruplet by an .alpha.-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 .alpha.-helix is Arg; or position +6 is Ser or Thr and position + +2 is Asp;
d) if base 4 in the quadruplet is T, then position +6 in the .alpha.-helix is Ser or Thr and position + +2 is Asp.
c) if base 4 in the quadruplet is G, then position +6 in the .alpha.-helix is Arg; or position +6 is Ser or Thr and position + +2 is Asp;
d) if base 4 in the quadruplet is T, then position +6 in the .alpha.-helix is Ser or Thr and position + +2 is Asp.
3. A method for preparing a nucleic acid binding protein of the Cys2-His2 zinc finder class capable of binding to a nucleic acid quadruplet in a target nucleic acid sequence, wherein binding to each base of the quadruplet by an a-helical zinc finder nucleic acid binding motif in the protein is determined as follows:
a) if base 4 in the quadruplet is G, then position +6 in the .alpha.-helix is Arg; or position +6 is Ser or Thr and position ++2 is Asp;
b) if base 4 in the quadruplet is A, then position +6 in the .alpha.-helix is Gln and position + +2 is not Asp;
c) if base 4 in the quadruplet is T, then position +6 in the .alpha.-helix is Ser or Thr and position + +2 is Asp;
d) if base 4 in the quadruplet is C, then position +6 in the .alpha.-helix may be any amino acid, provided that position + +2 in the .alpha.-helix is not Asp;
e) if base 3 in the quadruplet is G, then position +3 in the .alpha.-helix is His:
f) if base 3 in the quadruplet is A, then position +3 in the .alpha.-helix is Asn;
g) if base 3 in the quadruplet is T, then position +3 in the .alpha.-helix is Ala, Ser or Val;
provided that if it is Ala, then the residues at -1 or +6 are small residues;
h) if base 3 in the quadruplet is C, then position +3 in the .alpha.-helix is Ser. Asp, Glu, Leu, Thr or Val;
i) if base 2 in the quadruplet is G, then position -1 in the .alpha.-helix is Ark:
j) if base 2 in the quadruplet is A, then position -1 in the .alpha.-helix is Gln;
k) if base 2 in the quadruplet is T, then position -1 in the .alpha.-helix is Asn or Gln;
l) if base 2 in the quadruplet is C, then position -1 in the .alpha.-helix is Asp m)if base 1 in the quadruplet is G, then position +2 is Asp;
n) if base 1 in the quadruplet is A, then position +2 is not Asp;
o) if base 1 in the quadruplet is C, then position +2 is not Asp;
p) if base 1 in the quadruplet is T, then position +2 is Ser or Thr.
a) if base 4 in the quadruplet is G, then position +6 in the .alpha.-helix is Arg; or position +6 is Ser or Thr and position ++2 is Asp;
b) if base 4 in the quadruplet is A, then position +6 in the .alpha.-helix is Gln and position + +2 is not Asp;
c) if base 4 in the quadruplet is T, then position +6 in the .alpha.-helix is Ser or Thr and position + +2 is Asp;
d) if base 4 in the quadruplet is C, then position +6 in the .alpha.-helix may be any amino acid, provided that position + +2 in the .alpha.-helix is not Asp;
e) if base 3 in the quadruplet is G, then position +3 in the .alpha.-helix is His:
f) if base 3 in the quadruplet is A, then position +3 in the .alpha.-helix is Asn;
g) if base 3 in the quadruplet is T, then position +3 in the .alpha.-helix is Ala, Ser or Val;
provided that if it is Ala, then the residues at -1 or +6 are small residues;
h) if base 3 in the quadruplet is C, then position +3 in the .alpha.-helix is Ser. Asp, Glu, Leu, Thr or Val;
i) if base 2 in the quadruplet is G, then position -1 in the .alpha.-helix is Ark:
j) if base 2 in the quadruplet is A, then position -1 in the .alpha.-helix is Gln;
k) if base 2 in the quadruplet is T, then position -1 in the .alpha.-helix is Asn or Gln;
l) if base 2 in the quadruplet is C, then position -1 in the .alpha.-helix is Asp m)if base 1 in the quadruplet is G, then position +2 is Asp;
n) if base 1 in the quadruplet is A, then position +2 is not Asp;
o) if base 1 in the quadruplet is C, then position +2 is not Asp;
p) if base 1 in the quadruplet is T, then position +2 is Ser or Thr.
4. A method according to any preceding claim. wherein the or each zinc finger has the general primary structure (A) X a C X2-4 C X2-3 F X c X X X X L X X H X X X b H - linker wherein X (including X a, X b and X c) is any amino acid.
A method according to claim 5 wherein X a is F/Y-X or P- F/Y-X.
6. A method according to claim 4 or claim 5 wherein X2-4, is selected from any one of:
S-X.E-X.K-X.T-X.P-X and R-X.
S-X.E-X.K-X.T-X.P-X and R-X.
7. A method according to any one of claims 4 to 6 wherein X b is T or I.
8. A method according to any one of claims 4 to 7 wherein 2-3; is G-K-A, G-K-C, G-K-S, G-K-G, M-R-N or M-R.
9. A method according to any one of claims 4 to 8 wherein the linker is T-G-E-K or T-G-E-K-P.
10. A method according to any one of claims 4 to 9 wherein position +9 is R or K.
11. A method according to any one of claims 4 to 10 wherein positions +1, +5 and +8 are not occupied by any one of the hydrophobic amino acids, F, W or Y.
12. A method according to claim 11 wherein positions +1, +5 and +8 are occupied by the residues K, T and Q respectively.
13. A method for preparing a nucleic acid binding protein of the Cys2-His2 zinc finder class capable of binding to a target nucleic acid sequence, comprising the steps of:
a) selecting a model zinc finger domain from the group consisting of naturally occurring zinc fingers and consensus zinc fingers; and b) mutating the finger according to the rules set in any one of claims 1 to 3.
a) selecting a model zinc finger domain from the group consisting of naturally occurring zinc fingers and consensus zinc fingers; and b) mutating the finger according to the rules set in any one of claims 1 to 3.
14. A method according to claim 13, wherein the model zinc finder is a consensus zinc finer whose structure is selected from the group consisting of the consensus structure P Y
KCPECGKSFSQKSDLVKHQRTHTG,and the consensus structure PYK
CSECGKAFSQKSNLTRHQRIHTGEKP.
42~
KCPECGKSFSQKSDLVKHQRTHTG,and the consensus structure PYK
CSECGKAFSQKSNLTRHQRIHTGEKP.
42~
15. A method according to claim 13 wherein the model zinc finger is a naturally occurring zinc finger whose structure is selected from one finger of a protein selected from the group consisting of Zif 268 (Elrod-Erickson et al., (1996) Structure 4:1171-1180), GLI
(Pavletich and Pabo, (1993) Science 261:1701-1707), Tramtrack (Fairall et al., (1993) Nature 366:483-487) and YY1 (Houbaviy et al., (1996) PNAS (USA) 93:13577-1382).
(Pavletich and Pabo, (1993) Science 261:1701-1707), Tramtrack (Fairall et al., (1993) Nature 366:483-487) and YY1 (Houbaviy et al., (1996) PNAS (USA) 93:13577-1382).
16. A method according to claim 15 wherein the model zinc finger is finder 2 of Zif 268.
17. A method according to any preceding claim wherein the binding protein comprises two or more zinc finger binding motifs, placed N-terminus to C-terminus.
18. A method according to claim 14, wherein the N-terminal zinc finger is preceded by a leader peptide having the sequence MAEEKP.
19. A method according to claim 14 or claim 15, wherein the nucleic acid binding protein is constructed by recombinant nucleic acid technology, the method comprising the steps of:
a) preparing a nucleic acid coding sequence encoding two or more zinc finger binding motifs as defined in any one of claims 5 to 13, placed N-terminus to C-terminus;
b) inserting the nucleic acid sequence into a suitable expression vector; and c) expressing the nucleic acid sequence in a host organism in order to obtain the nucleic acid binding protein.
a) preparing a nucleic acid coding sequence encoding two or more zinc finger binding motifs as defined in any one of claims 5 to 13, placed N-terminus to C-terminus;
b) inserting the nucleic acid sequence into a suitable expression vector; and c) expressing the nucleic acid sequence in a host organism in order to obtain the nucleic acid binding protein.
20. A method according to any preceding claim comprising the additional steps of subjecting the nucleic acid binding protein to one or more rounds of randomisation and selection in order to improve the characteristics thereof.
21. A method according to claim 20, wherein the randomisation and selection is carried out by phage display technology.
22. A method according to claim 21, comprising the steps of:
a) preparing a nucleic acid construct capable of expressing a fusion protein comprising the nucleic acid binding protein and a minor coat protein of a filamentous bacteriophage;
b) preparing further nucleic acid constructs capable of expressing a fusion protein comprising a selectively mutated nucleic acid binding protein and a minor coat protein of a filamentous bacteriophage;
c) causing the fusion proteins defined in steps (a) and (b) to be expressed on the surface of bacteriophage transformed with the nucleic acid constructs:
d) assaying the ability of the bacteriophage to bind the target nucleic acid sequence and selecting the bacteriophage demonstrating superior binding characteristics.
a) preparing a nucleic acid construct capable of expressing a fusion protein comprising the nucleic acid binding protein and a minor coat protein of a filamentous bacteriophage;
b) preparing further nucleic acid constructs capable of expressing a fusion protein comprising a selectively mutated nucleic acid binding protein and a minor coat protein of a filamentous bacteriophage;
c) causing the fusion proteins defined in steps (a) and (b) to be expressed on the surface of bacteriophage transformed with the nucleic acid constructs:
d) assaying the ability of the bacteriophage to bind the target nucleic acid sequence and selecting the bacteriophage demonstrating superior binding characteristics.
23. A method according to any one of claims 20 to 22 wherein the nucleic acid binding protein is selectively randomised at any one of positions +1, +5, +8, -1, +2, +3 or +6.
24. A method for determining the presence of a target nucleic acid molecule, comprising the steps of:
a) preparing a nucleic acid binding protein by the method of any preceding claim which is specific for the target nucleic acid molecule;
b) exposing a test system comprising the target nucleic acid molecule to the nucleic acid binding protein under conditions which promote binding, and removing any nucleic acid binding protein which remains unbound;
c) detecting the presence of the nucleic acid binding protein in the test system.
a) preparing a nucleic acid binding protein by the method of any preceding claim which is specific for the target nucleic acid molecule;
b) exposing a test system comprising the target nucleic acid molecule to the nucleic acid binding protein under conditions which promote binding, and removing any nucleic acid binding protein which remains unbound;
c) detecting the presence of the nucleic acid binding protein in the test system.
25. A method according to claim 24, wherein the presence of the nucleic acid binding protein in the test system is detected by means of an antibody.
26. A method according to claim 24 or claim 25 wherein the nucleic acid binding protein in use, is displayed on the surface of a filamentous bacteriophage and the presence of the nucleic acid binding protein is detected by detecting the bacteriophage or a component thereof.
27. A synthetic nucleic acid binding protein whose design incorporates a method according to any one of claims 1 to 23.
28. A nucleic acid encoding a nucleic acid binding protein according to claim 27.
29. A host cell transformed with a nucleic acid according to claim 28.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB9710809.6 | 1997-05-23 | ||
| GBGB9710809.6A GB9710809D0 (en) | 1997-05-23 | 1997-05-23 | Nucleic acid binding proteins |
| PCT/GB1998/001516 WO1998053060A1 (en) | 1997-05-23 | 1998-05-26 | Nucleic acid binding proteins |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CA2290886A1 true CA2290886A1 (en) | 1998-11-26 |
| CA2290886C CA2290886C (en) | 2010-08-31 |
Family
ID=10813050
Family Applications (3)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002290720A Expired - Lifetime CA2290720C (en) | 1997-05-23 | 1998-05-26 | Nucleic acid binding polypeptide library |
| CA002290717A Expired - Lifetime CA2290717C (en) | 1997-05-23 | 1998-05-26 | Nucleic acid binding proteins |
| CA2290886A Expired - Lifetime CA2290886C (en) | 1997-05-23 | 1998-05-26 | Nucleic acid binding proteins |
Family Applications Before (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002290720A Expired - Lifetime CA2290720C (en) | 1997-05-23 | 1998-05-26 | Nucleic acid binding polypeptide library |
| CA002290717A Expired - Lifetime CA2290717C (en) | 1997-05-23 | 1998-05-26 | Nucleic acid binding proteins |
Country Status (9)
| Country | Link |
|---|---|
| US (8) | US6866997B1 (en) |
| EP (5) | EP0983350B1 (en) |
| JP (4) | JP2001527417A (en) |
| AT (2) | ATE393822T1 (en) |
| AU (3) | AU7542398A (en) |
| CA (3) | CA2290720C (en) |
| DE (2) | DE69839425T2 (en) |
| GB (1) | GB9710809D0 (en) |
| WO (3) | WO1998053060A1 (en) |
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