SEQUENCE ENABLED REASSEMBLY (SEER) - A NOVEL METHOD FOR VISUALIZING SPECIFIC DNA SEQUENCES

• US 20090068164A1
• Filed: 05/05/2006
• Published: 03/12/2009
• Est. Priority Date: 05/05/2005
• Status: Abandoned Application

• Current Assignee: The Scripps Research Institute, Arizona Board of Regents (University of Arizona)
• Original Assignee: ARIZ BD of Regents On Behalf of The University of AZ, The Scripps Research Institute
• Inventors: Porter, Jason, Ooi, Aik T., Ghosh, Indraneel, Stains, Cliff L., Segal, David J., Barbas, Carlos F.
• Application Number:
  US11/913,592
• Publication Number:
  US 20090068164A1
• Time in Patent Office: Days
• US Class Current: 424/94.600
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C12N 15/1055 Protein x Protein interaction, e.g. two hybrid selection
• CPC Class Codes: C12N 15/1086 Preparation or screening of expression libraries, e.g. reporter assays
• CPC Class Codes: G01N 33/574 for cancer

Abstract

The present invention provides a nucleotide sequence detection system in which a reporter enzyme is split into two halves each half of which is associated with at least one zinc finger domain. Upon DNA binding to the specific sequence defined by the zinc finger domains associated with the respective halves, the split-protein reassembles to reconstitute a functional enzyme. As such, the present invention provides methods of using the nucleotide sequence detection system for various diagnostic and identification purposes.

Claims

1. A nucleotide sequence detection system comprising:

a first protein wherein said protein comprises at least sequence-specific DNA binding domain and the N-terminal oligomerization domain of a split-protein enzyme, wherein said at least one zinc finger domain is separated from said N-terminal oligomerization domain of said split-protein enzyme by a linker; and

a second protein wherein said protein comprises at least sequence-specific DNA binding domain and the C-terminal oligomerization domain of said split-protein enzyme, wherein said at least one zinc finger domain is separated from said C-terminal oligomerization domain of said split-protein enzyme by a linker.

2. The nucleotide sequence detection system of claim 1, wherein said sequence-specific DNA binding domain is selected from the group consisting of a helix-turn-helix protein, a miniature DNA binding protein, a methyl-cytosine binding domain, and a zinc finger domain.

3. The nucleotide sequence detection system of claim 2, wherein at least one of said first protein and said second protein contains at least one methyl-cytosine binding domain as said sequence-specific DNA binding domain.

4. The nucleotide sequence detection system of claim 1, wherein at least one of said first protein and said second protein contains at least one zinc finger domain as said sequence-specific DNA binding domain.

5. The nucleotide sequence detection system of claim 1, wherein each of said first protein and said second protein contain at least one zinc finger domain as said sequence-specific DNA binding domain.

6. The nucleotide sequence detection system of claim 5, wherein said at least one zinc finger domain of said first protein is contained within a zinc finger module which is derived from a zinc finger protein selected from the group consisting of Zif268, PBSII and PE1A.

7. The nucleotide sequence detection system of claim 5, wherein said at least one zinc finger domain of said second protein is contained within a zinc finger module which is derived from a zinc finger protein selected from the group consisting of Zif268, PBSII and PE1A.

8. The nucleotide sequence detection system of claim 5, wherein said at least one zinc finger domain of said first protein are located C-terminal to the N-terminal oligomerization domain of said split-protein enzyme.

9. The nucleotide sequence detection system of claim 5, wherein said at least one zinc finger domain of said second protein are located N-terminal to the C-terminal oligomerization domain of said split-protein enzyme.

10. The nucleotide sequence detection system of claim 1, wherein said split-protein enzyme reassembles to form a functional enzyme;

wherein said first protein binds the cognate nucleotide sequence for the sequence-specific DNA biding domain comprised therein,

wherein said second protein binds the cognate nucleotide sequence for the sequence-specific DNA biding domain comprised therein, and

wherein the cognate nucleotide sequence for said first protein is located 5′

to the cognate nucleotide sequence for said second protein.

11. The nucleotide sequence detection system of claim 10, wherein said split-protein enzyme is selected from the group consisting of beta-galactosidase, beta-lactamase, dihydrofolate reductase, green fluorescent protein, and luciferase, and variants or homologs thereof.

12. The nucleotide sequence detection system of claim 10, wherein said split-protein enzyme is a beta-lactamase, variants or homologs thereof.

13. The nucleotide sequence detection system of claim 10, wherein said split-protein enzyme is green fluorescent protein, variants or homologs thereof.

14. The nucleotide sequence detection system of claim 1, wherein said linker in said first protein ranges from 0 to 30 amino acids.

15. The nucleotide sequence detection system of claim 1, wherein said linker in said first protein is 15 amino acids.

16. The nucleotide sequence detection system of claim 1, wherein said linker in said second protein ranges from 0 to 30 amino acids.

17. The nucleotide sequence detection system of claim 1, wherein said linker in said second protein is 15 amino acids.

18. The nucleotide sequence detection system of claim 1, wherein said first protein has the sequence comprising SEQ ID NO:

16 and said second protein has the sequence comprising SEQ ID NO;

14.

19. The nucleotide sequence detection system of claim 1, wherein said first protein has the sequence comprising SEQ ID NO:

46 and said second protein has the sequence comprising SEQ ID NO;

44.

20. The nucleotide sequence detection system of claim 1, wherein said first protein has the sequence comprising SEQ ID NO:

48 and said second protein has the sequence comprising SEQ ID NO;

44.

21. The nucleotide sequence detection system of claim 1, wherein said first protein has the sequence comprising SEQ ID NO:

16 and said second protein has the sequence comprising SEQ ID NO;

52.

22. An isolated polynucleotide encoding said first protein of the nucleotide sequence detection system of claim 1.

23. The isolated polynucleotide of claim 22, wherein said polynucleotide is selected from the group consisting of SEQ ID NO:

15, SEQ ID NO;

45, and SEQ ID NO;

47.

24. An isolated polynucleotide encoding said second protein of the nucleotide sequence detection system of claim 1.

25. The isolated polynucleotide of claim 24, wherein said polynucleotide is selected from the group consisting of SEQ ID NO:

13, SEQ ID NO;

43, and SEQ ID NO;

51.

26. A kit comprising the nucleotide sequence detection system of claim 1 and a hybridization buffer.

27. The kit of claim 26, wherein said first protein and said second protein are in a lyophilized form.

28. A method of detecting the presence of a specific nucleotide sequence in a sample comprising a polynucleotide, wherein said method comprises:

contacting said sample with the nucleotide sequence detection system of claim 1 for a time and under conditions suitable to facilitate hybridization, wherein said nucleotide sequence detection system is tuned to detect said specific nucleotide sequence by the arrangement and number of sequence-specific DNA binding domains contained within said first protein and said second protein;

monitoring the formation of activity associated with the split-protein enzyme when in a reassembled state; and

correlating an observed positive activity from said monitoring to the presence of said specific sequence in said polynucleotide.

29. The method of claim 28, wherein said split-protein enzyme is green fluorescent protein and said monitoring comprises monitoring the fluorescence emission at 509 nm upon excitation at 395 nm.

30. The method of claim 28, wherein said split-protein enzyme is beta-lactamase and said monitoring comprises monitoring hydrolysis of a substrate selected from the group consisting of nitrocefin, CCF2, CCF4, CC2, C-mel, penicillin, ampicillin, and carbonicillin.

31. The method of claim 28, wherein said method is a method of detecting a genetic abnormality in a subject in need thereof.

32. The method of claim 28, wherein said method is a method of detecting single nucleotide polymorphism in a subject in need thereof.

33. The method of claim 28, wherein said method is a method of detecting shortening of telomeres in a subject in need thereof.

34. The method of claim 33, wherein said subject in need thereof is a subject having or suspected of having cancer.

35. The method of claim 33, wherein said subject in need thereof is a subject having or suspected of having an age related disease.

36. The method of claim 28, wherein said method is a method of determining the age of cells or cloned animals and said specific nucleotide sequence is the repeat sequence in telomeres.

37. The method of claim 28, wherein said method is a method of diagnosing cancer in a subject in need thereof and said specific nucleotide sequence is a unique marker for a specific type of cancer.

38. The method of claim 28, wherein said method is a method of identifying an infectious agent and said sample is selected from the group consisting of a tissue sample, a blood sample, a sera sample, a nasal swab, a vaginal swab, and a rectal swab.

39. The method of claim 28, wherein said method is a method of identifying an infectious agent and said sample is selected from the group consisting of food, beverage, and water.

40. The method of claim 28, wherein said method is a sample-to-source matching method wherein the specific nucleotide sequence represents a unique nucleotide sequence obtained from a biological sample of interest and said sample is obtained from a subject suspected to contain said unique nucleotide sequence.

41. The method of claim 40, wherein said biological sample of interest is selected from the group consisting of blood, hair, skin, sperm, and semen.

42. The method of claim 40, wherein said sample is selected from the group consisting of blood, hair, skin, sperm, and semen.

43. A method of treating eradicating a viral infection in a subject in need thereof, comprising:

tailoring the sequence specificity of said sequence-specific DNA binding domains of said nucleotide sequence detection system of claim 1 to the virus infecting said subject to a unique nucleic acid sequence thereto, wherein said split-protein enzyme facilitates hydrolysis of a substrate that becomes toxic to said virus upon hydrolysis;

administering an effective amount of said nucleotide sequence detection system of claim 1 to said subject; and

administering an effective amount of said substrate to said subject.

44. The method of claim 43, wherein said split-protein enzyme is beta-lactamase.

45. The method of claim 44, wherein said substrate is C-mel.

46. A method of treating cancer in a subject in need thereof, comprising:

tailoring the sequence specificity of said sequence-specific DNA binding domains of said nucleotide sequence detection system of claim 1 to a mutant oncogene in said subject to a unique nucleic acid sequence thereto, wherein said split-protein enzyme facilitates hydrolysis of a substrate that becomes toxic to said virus upon hydrolysis;

administering an effective amount of said nucleotide sequence detection system of claim 1 to said subject; and

administering an effective amount of said substrate to said subject.

47. The method of claim 46, wherein said split-protein enzyme is beta-lactamase.

48. The method of claim 47, wherein said substrate is C-mel.

49. A method of detecting the presence of specific sites of DNA methylation within a specific sequence of a polynucleotide of a subject in need thereof comprising:

tailoring the sequence specificity of said sequence-specific DNA binding domains of said nucleotide sequence detection system of claim 1 to a specific DNA sequence in said subject to a unique nucleic acid sequence thereto, wherein said sequence-specific DNA binding domain of at least one of said first protein and said second protein is a methyl binding domain;

delivering an effective amount of said nucleotide sequence detection system of claim 1 to a sample obtained from said subject;

monitoring the formation of activity associated with the split-protein enzyme when in a reassembled state; and

correlating an observed positive activity from said monitoring to the presence of DNA methylation within said specific sequence in said polynucleotide.

50. The method of claim 49, wherein said methyl binding domain is a methyl-cytosine binding domain.

51. The method of claim 50, wherein said first protein has the sequence comprising SEQ ID NO:

16 and said second protein has the sequence comprising SEQ ID NO;

52.

52. The method of claim 49, wherein the presence of said DNA methylation is correlated with a propensity for or a diagnosis of cancer.

53. A method for simultaneous detection the presence of multiple specific nucleotide sequences in a sample comprising a polynucleotide, wherein said method comprises:

contacting said sample with two or more different nucleotide sequence detection systems of claim 1 for a time and under conditions suitable to facilitate hybridization, wherein said nucleotide sequence detection systems are tuned to detect independent specific nucleotide sequences by the arrangement and number of sequence-specific DNA binding domains contained within said first protein and said second protein and wherein said split-protein enzyme for each nucleotide sequence detection system is distinct from any other,

monitoring the formation of activity associated with the split-protein enzymes when in a reassembled state; and

correlating an observed positive activity from said monitoring to the presence of said specific sequences in said polynucleotide.

54. The method of claim 53, wherein said wherein at least one of said split-protein enzymes is selected from the group consisting of beta-galactosidase, beta-lactamase, dihydrofolate reductase, green fluorescent protein, and luciferase, and variants or homologs thereof.

55. The method of claim 53, wherein at least one of said split-protein enzymes is a beta-lactamase, variants or homologs thereof.

56. The method of claim 53, wherein at least one of said split-protein enzymes is green fluorescent protein, variants or homologs thereof.

57. The method of claim 56, wherein at least one of said split-protein enzymes is selected from the group consisting of green fluorescent protein, cyan fluorescent protein, yellow fluorescent protein, red fluorescent protein, and reef coral fluorescent protein.

58. The method of claim 53, wherein said contacting is with three to five of said nucleotide sequence detection systems.