Binding proteins for recognition of DNA

• US 6,013,453 A
• Filed: 08/25/1998
• Issued: 01/11/2000
• Est. Priority Date: 08/20/1994
• Status: Expired due to Term

• Current Assignee: UK Research and Innovation
• Original Assignee: Medical Research Council (Government of United Kingdom)
• Inventors: Choo, Yen, Klug, Aaron, Sanchez Garcia, Isidro
• Primary Examiner(s): Degen, Nancy
• Assistant Examiner(s): SANDALS, WILLIAM O
• Application Number:
  US09/139,762
• Time in Patent Office: 504 Days
• Field of Search: 435/6, 536/23.4
• US Class Current: 506/9
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C07K 14/4702 Regulators; Modulating activity
• CPC Class Codes: C07K 14/4705 stimulating, promoting or activating activity
• CPC Class Codes: C07K 19/00 Hybrid peptides , i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/09 containing a nuclear localisation signal
• CPC Class Codes: C07K 2319/30 Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
• CPC Class Codes: C07K 2319/81 containing a Zn-finger domain for DNA binding
• CPC Class Codes: C12N 15/1037 Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C40B 40/02 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

Abstract

Disclosed are libraries of DNA sequences encoding zinc finger binding motifs for display on a particle, together with methods of designing zinc finger binding polypeptides for binding to a particular target sequence and, inter alia, use of designed zinc finger polypeptides for various in vitro or in vivo applications.

Claims

1. A library of DNA sequences, each sequence encoding a zinc finger polypeptide for display on a viral particle, the zinc finger polypeptide comprising at least three zinc fingers, with one zinc finger having a partially randomized allocation of amino acids being positioned between two or more zinc fingers having a defined amino acid sequence, the partially randomized zinc finger having random allocation of amino acids at positions -1, +2, +3 and +6, position +1 being the first amino acid in the α

-helix of the zing finger.

2. A library according to claim 1, wherein the partially randomized zinc finger further has random allocation of amino acids in at least one of the positions +1, +5 and +8.

3. A library according to claim 1 or 2, wherein the encoded partially randomized zinc finger is a zinc finger of the Zif268 polypeptide.

4. A library according to claim 1 as a fusion with the DNA encoding the minor cost protein of bacteriophage fd.

5. A method of designing a zinc finger polypeptide for binding to a particular target DNA sequence, comprising the steps of:

comparing the binding to one or more DNA triplets of each of a plurality of zinc finger polypeptides having a partially randomized zinc finger positioned between two or more zinc fingers having defined amino acid sequence, the zinc finger polypeptides being encoded by a library in accordance with claim 1; and

selecting those nucleic acid sequences encoding randomized zinc fingers which bind to the target DNA sequence.

6. A method of designing a zinc finger polypeptide for binding to a particular target DNA sequence, comprising the steps of:

screening against at least a portion of the target DNA sequence a plurality of zinc finger polypeptides having a partially randomized zinc finger positioned between two or more zinc fingers having defined amino acid sequence, the portion of the target DNA sequence being sufficient to allow binding of some of the zinc finger polypeptides, the zinc finger polypeptides being encoded by a library in accordance with claim 1;

comparing the binding to one or more DNA triplets of each of plurality zinc finger polypeptides having a partially randomized zinc finger positioned between two or more zinc fingers having defined amino acid sequence; and

selecting those nucleic acid sequences encoding randomized zinc fingers which bind to the target DNA sequence.

7. A method of designing a zinc finger polypeptide for binding to a particular target DNA sequence, the method comprising the steps of:

screening against at least a portion of the target DNA sequence a plurality of zinc finger polypeptides having a partially randomized zinc finger positioned between two or more zinc fingers having defined amino acid sequence, the portion of the target DNA sequence being sufficient to allow binding of some of the zinc finger polypeptides, the zinc finger polypeptides being encoded by a library in accordance with claim 1;

comparing the binding to one or more DNA triplets of each of a plurality zinc finger polypeptides having a partially randomized zinc finger positioned between two or more zinc fingers having defined amino acid sequence;

selecting certain of the screened randomized zinc fingers for analysis of preferred binding characteristics;

and combining those sequences encoding desired zinc fingers to form a sequence encoding a zinc finger polypeptide which binds to the target DNA sequence.

8. A method for producing a zinc finger polypeptide for binding to a particular target DNA sequence, comprising the steps of:

screening against at least a portion of the target DNA sequence a plurality of zinc finger polypeptides having a partially randomized zinc finger positioned between two or more zinc fingers having defined amino acid sequence, the portion of the target DNA sequence being sufficient to allow binding of some of the zinc finger polypeptides, the plurality of zinc finger polypeptides being coded by a library in according with claim 1;

selecting those nucleic acid sequences encoding randomized zinc fingers which bind to the target DNA sequence; and

expressing the selected nucleic acid sequences to produce zinc finger polypeptides which bind to the target DNA sequence.

9. A method of designing a zinc finger polypeptide for binding to a particular target DNA sequence, comprising the steps of:

screening against at least a portion of the target DNA sequence a plurality of zinc finger polypeptides having a partially randomized zinc finger positioned between two or more zinc fingers having defined amino acid sequence, the portion of the target DNA sequence being sufficient to allow binding of some of the zinc finger polypeptides, the zinc finger polypeptides being encoded by a library in accordance with claim 1; and

selecting those nucleic acid sequences encoding randomized zinc fingers which bind to the target DNA sequence.

10. A method according to claim 9, wherein two or more rounds of screening are performed.

11. A method of designing a zinc finger polypeptide for binding to a particular DNA target sequence, wherein a plurality of sequences encoding individual zinc fingers selected by the method of claim 9 are randomly combined in the appropriate order to encode a plurality of zinc finger polypeptides, the zinc finger polypeptides being screened against the target sequence, that combination of zinc finger sequences encoding a zinc finger polypeptide which binds to the target DNA sequence selected for use.

12. A method of modifying a nucleic acid sequence of interest present in a sample mixture by binding thereto a zinc finger polypeptide, wherein the zinc finger polypeptide is designed in accordance with claim 9, comprising contacting the sample mixture with zinc finger polypeptide having affinity for at least a portion of the sequence of interest, so as to allow the zinc finger polypeptide to bind specifically to the sequence of interest.

13. A method according to claim 12, further comprising the step of separating the zinc finger polypeptide (and nucleic acid sequences specifically bound thereto) from the rest of the sample.

14. A method according to claim 12, wherein the zinc finger polypeptide is bound to a solid phase support.

15. A method according to claim 12, wherein the presence of the zinc finger polypeptide bound to the sequence of interest is detected by the addition of one or more detection reagents.

16. A method according to claim 12, wherein the DNA sequence of interest is present in an acrylamide or agarose gel matrix, or is present on the surface of a membrane.

17. A DNA library according to claim 1, consisting of 64 sequences, each sequence comprising a different one of the 64 possible permutations of a DNA triplet, the library being arranged in twelve sub-libraries, wherein for any one sub-library one base in the triplet is defined and the other two bases are randomized.

18. A library according to claim 17 wherein the sequences are biotinylated.

19. A library according to claim 17, wherein the sequences are associated with separation means.

20. A library according to claim 19, wherein the separation means is selected from the group consisting of:

a microtitre plate;

magnetic or non-magnetic beads or particles capable of sedimentation; and

an affinity chromatography column.

21. A kit for making a zinc finger polypeptide for binding to a DNA of interest, comprising:

a library of DNA sequences, each sequence encoding a zinc finger polypedtide for display on a viral particle, the zinc finger polypeptide comprising at least three zinc fingers, with one zinc finger having a partially randomized allocation of amino acids being positioned between two or more zinc fingers having a defined amino acid sequence, the partially randomized zinc finger having random allocation of amino acids at positions -1, +2, +3 and +6. position +1 being the first amino acid in the α

-helix of the zing finger;

a vector molecule suitable for accepting one or more sequences from the library; and

instructions for use.

22. A kit according to claim 21, wherein the vector directs the expression of the cloned sequences as a zinc finger polypeptide.

23. A kit according to claim 21, wherein the vector is capable of directing the expression of the cloned sequences as a zinc finger polypeptide displayed on the surface of a viral particle.

24. A kit according to claims 21 A library of DNA sequences, each sequence encoding a zinc finger polypeptide for display on a viral particle, the zinc finger polypeptide comprising at least three zinc fingers, with one zinc finger having a partially randomized allocation of amino acids being positioned between two or more zinc fingers having a defined amino acid sequence, the partially randomized zinc finger having random allocation of amino acids at positions -1, +2, +3 and +6, position +1 being the first amino acid in the α

-helix of the zing finger, further comprising a library consisting of 64 sequences, each sequence comprising a different one of the 64 possible permutations of a DNA triplet, the library being arranged in twelve sub-libraries, wherein for any one sub-library one base in the triplet is defined and the other two bases are randomized.

25. A kit according to claim 24, further comprising appropriate buffer solutions and/or reagents for detection of bound zinc fingers.

26. A kit according to claim 25, further comprising a vector suitable for accepting one or more sequences selected from the library of DNA sequences encoding zinc fingers.