Fluorescent and DNA cleavage properties of peptide/dye conjugates

• US 6,348,317 B1
• Filed: 11/16/2000
• Issued: 02/19/2002
• Est. Priority Date: 11/18/1999
• Status: Expired due to Fees

• Current Assignee: Arizona Board of Regents (University of Arizona)
• Original Assignee: Arizona Board of Regents (University of Arizona)
• Inventors: Thompson, Martin, Woodbury, Neal W.
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US09/713,950
• Time in Patent Office: 460 Days
• Field of Search: 435/6, 435/7.1, 530/300, 530/350
• US Class Current: 435/6.11
• CPC Class Codes: C07K 14/721 Steroid/thyroid hormone superfamily, e.g. GR, EcR, androgen receptor, oestrogen receptor
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2563/125 the label being enzymatic, i.e. proteins, and non proteins, such as nucleic acid with enzymatic activity

Abstract

A method of identifying the presence or absence of a DNA molecule in a test sample comprising a specific DNA sequence is disclosed. In one embodiment, the method comprises the steps of mixing a test sample with a peptide/dye conjugate comprising a covalently linked peptide and a dye, wherein the peptide binds to the specific DNA sequence and wherein the peptide/dye conjugate will fluoresce if the peptide is bound to the specific DNA sequence, and measuring fluorescence, wherein specific fluorescence above background level indicates that the conjugate is bound to the specific DNA sequence. In another embodiment, the present invention is a method of cleaving a specific DNA molecule and a test sample. The method comprises mixing a test sample with a peptide dye conjugate comprising a covalently linked peptide and a dye, wherein the peptide binds to the specific DNA sequence and wherein the peptide dye conjugate will cleave if the peptide is bound to a specific DNA sequence.

Claims

1. A method of identifying the presence or absence of a DNA molecule that contains a specific DNA sequence, comprising the steps of:

(a) mixing a test sample with a peptide/dye conjugate comprising a covalently linked peptide and a dye, wherein the peptide binds to the specific DNA sequence and wherein the peptide/ dye conjugate will fluoresce only if the peptide is bound to the specific DNA sequence; and

(b) measuring fluorescence, wherein specific fluorescence above background level indicates that the conjugate is bound to the specific DNA sequence.

2. The method of claim 1, wherein the dye is a cyanine dye.

3. The method of claim 1, wherein the dye is selected from the group consisting of cyanine, phenanthridine and acridine dyes.

4. The method of claim 3, wherein the dye is oxazole yellow or thiozole orange.

5. The method of claim 1, wherein the peptide is a zinc finger.

6. The method of claim 1, wherein the peptide is a glucocorticoid receptor DNA binding domain zinc finger.

7. The method of claim 6, wherein the specific DNA sequence is a glucocorticoid receptor response element.

8. The method of claim 1, wherein the peptide is a helix-turn-helix motif.

9. The method of claim 1, wherein the peptide is a Tc3 transposase DNA binding domain helix-turn-helix motif.

10. The method of claim 9, wherein the specific DNA sequence is the Tc3 transposase DNA binding domain native consensus sequence.

11. The method of claim 1, wherein the peptide is a Hin recombinase DNA binding domain helix-turn-helix motif.

12. The method of claim 11, wherein the specific DNA sequence is the Hin recombinase DNA binding domain native consensus sequence.

13. The method of claim 1 further comprising the step of administering light to the conjugate after the conjugate has bound to the DNA sequence such that cleavage of the DNA results.

14. The method of claim 13, wherein the dye is selected from the group consisting of cyanine, phenanthridine and acridine dyes.

15. A peptide/dye conjugate comprising:

(a) a peptide that binds to a specific DNA sequence; and

(b) a dye capable of fluorescence only when the conjugate is bound to a specific DNA sequence, wherein the dye is covalently linked to the peptide.

16. The conjugate of claim 15, wherein the dye is selected from the group consisting of thiazole orange and oxazole yellow.

17. The conjugate of claim 15, wherein the dye is a cyanine dye.

18. The conjugate of claim 17, wherein the cyanine dye is oxazole yellow or thiozole orange.

19. The conjugate of claim 15, wherein the peptide is a zinc finger.

20. The conjugate of claim 15, wherein the peptide is a glucocorticoid receptor DNA binding domain zinc finger.

21. The conjugate of claim 20, wherein the peptide binds specifically to a glucocorticoid response element.

22. The conjugate of claim 20, wherein the dye is thiazole orange.

23. The conjugate of claim 15, wherein the peptide is a helix-turn-helix motif.

24. The conjugate of claim 15, wherein the peptide is a Tc3 transposase DNA binding domain helix-turn-helix motif.

25. The conjugate of claim 15, wherein the specific DNA sequence is the Hin recombinase DNA binding domain native consensus sequence.

26. The conjugate of claim 24, wherein the peptide binds specifically to the Tc3 transposase DNA binding domain native consensus sequence.