IRE-1α substrates

• US 8,017,331 B2
• Filed: 11/04/2005
• Issued: 09/13/2011
• Est. Priority Date: 11/04/2005
• Status: Active Grant

• Current Assignee: Fosun Orinove Pharmatech, Inc.
• Original Assignee: Mannkind Corporation
• Inventors: Patterson, John Bruce, Brumm, Duane, Stiles, Caryn, Volkmann, Kori Shallyn
• Primary Examiner(s): Babic; Christopher M.
• Application Number:
  US11/266,603
• Publication Number:
  US 20070105123A1
• Time in Patent Office: 2,139 Days
• Field of Search: None
• US Class Current: 435/6.1
• CPC Class Codes: C12Q 1/34 involving hydrolase
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides

Abstract

IRE-1α substrates useful for identifying agonists and antagonists of IRE-1α RNase activity.

Claims

1. A substrate for IRE-1α

, comprising;

(a) an oligonucleotide molecule comprising;

(i) an RNA loop consisting of a cleavage site for IRE-1α

; and

(ii) a nucleotide stem consisting of 4, 5, 6, 7, 8, 9, or 10 nucleotide base pairs; and

(b) a donor moiety and an acceptor moiety which exhibit a detectable resonance energy transfer when the donor moiety is excited, wherein the donor moiety is conjugated to one of the 5′

or 3′

ends of the oligonucleotide molecule and the acceptor moiety is conjugated to the other of the 5′

or 3′

ends of the oligonucleotide molecule.

3. The substrate of claim 1 wherein the oligonucleotide consists of SEQ ID NO:

3.

4. The substrate of claim 1 wherein the nucleotide stem comprises DNA.

5. The substrate of claim 1 wherein the nucleotide stem comprises RNA.

6. The substrate of claim 1 wherein the nucleotide stem comprises a nucleotide analog.

7. The substrate of claim 1 wherein at least one of the donor and acceptor moieties is a fluorescent protein.

8. The substrate of claim 1 wherein each of the donor and the acceptor moieties is a fluorescent protein.

9. The substrate of claim 1 wherein the acceptor moiety is a luminescent moiety.

10. The substrate of claim 9 wherein the luminescent moiety is selected from the group consisting of a luminescent protein and a lanthanide chelate.

11. The substrate of claim 1 wherein the acceptor moiety is selected from the group consisting of a fluorescent dye, a coumarin, a xanthene, a fluorescein, a fluorescent protein, a circularly permuted fluorescent protein, a rhodol, a rhodamine, a resorufin, a cyanine, a difluoroboradiazaindacene, a phthalocyanine, an indigo, a benzoquinone, an anthraquinone, an azo compound, a nitro compound, an indoaniline, a diphenylmethane, a triphenylmethane, and a zwitterionic azopyridinium compound.

12. The substrate of claim 1 wherein the donor moiety is selected from the group consisting of a fluorescent dye, a non-fluorescent dye, a coumarin, a xanthene, a rhodol, a rhodamines, a resorufin, a cyanine dye, a bimane, an acridine, an isoindole, a dansyl dye, an aminophthalic hydrazide, an aminophthalimide, an aminonaphthalimide, an aminobenzofuran, an aminoquinoline, a dicyanohydroquinone, a semiconductor fluorescent nanocrystal, a fluorescent protein, a circularly permuted fluorescent protein, and a fluorescent lanthanide chelate.

13. The substrate of claim 1 wherein the donor moiety is a fluorescent protein selected from the group consisting of a green fluorescent protein (GFP), a red fluorescent protein (RFP), a yellow fluorescent protein (YFP), a blue fluorescent protein (BFP), and a cyan fluorescent protein (CFP).

14. The substrate of claim 1 wherein the acceptor moiety is a luminescent protein.

15. The substrate of claim 14 wherein the luminescent protein is selected from the group consisting of an aequorin, an obelin, a lux protein, a luciferase protein, a phycobiliprotein, a pholasin, and a green fluorescent protein.

22. The substrate of claim 1 wherein at least one of the donor and acceptor moieties is a fluorescent dye.

23. The substrate of claim 22 wherein each of the donor and acceptor moieties is a fluorescent dye.

24. The substrate of claim 1 wherein the donor moiety is a xanthene.

25. The substrate of claim 24 wherein the xanthene is 6-carboxyfluorescein (FAM).

26. The substrate of claim 1 wherein the donor moiety is a cyanine.

27. The substrate of claim 26 wherein the cyanine is indodicarbocyanine 5 (Cy5).

28. The substrate of claim 1 wherein the acceptor moiety is a dark quencher.

29. The substrate of claim 28 wherein the dark quencher is a QSY™

dye.

30. The substrate of claim 28 wherein the dark quencher is a BLACK HOLE QUENCHER®

(BHQ®

).

40. The substrate of claim 1 wherein the RNA loop consists of a nucleotide sequence selected from the group consisting of:

5′

-CCGAAGC-3′

, 5′

-GCGAAGC-3′

, 5′

-ACGAAGC-3′

, 5′

-UCGAAGC-3′

, 5′

-CCGAAGC-3′

, 5′

-CGGAAGC-3′

, 5′

-CAGAAGC-3′

, 5′

-CUGAAGC-3′

, 5′

-CCGAAGC-3′

, 5′

-CCAAAGC-3′

, 5′

-CCUAAGC-3′

, 5′

-CCGAAGC-3′

, 5′

-CCGGAGC-3′

, 5′

-CCGUAGC-3′

, 5′

-CCGCAGC-3′

, 5′

-CCGAAGC-3′

, 5′

-CCGAGGC-3′

, 5′

-CCGAUGC-3′

, 5′

-CCGACGC-3′

, 5′

-CCGAAGC-3′

, 5′

-CCGAAAC-3′

, 5′

-CCGAAGC-3′

, 5′

-CCGAAGA-3′

, and 5′

-CCGAAGU-3′

.

46. The substrate of claim 1 wherein the 1 nucleotide stem consists of 4 nucleotide base pairs.

47. The substrate of claim 1 wherein the 1 nucleotide stem consists of 5 nucleotide base pairs.

48. The substrate of claim 1 wherein the 1 nucleotide stem consists of 6 nucleotide base pairs.

49. The substrate of claim 1 wherein the 1 nucleotide stem consists of 7 nucleotide base pairs.

50. The substrate of claim 1 wherein the 1 nucleotide stem consists of 8 nucleotide base pairs.

51. The substrate of claim 1 wherein the 1 nucleotide stem consists of 9 nucleotide base pairs.

52. The substrate of claim 1 wherein the 1 nucleotide stem consists of 10 nucleotide base pairs.

2. A substrate for IRE-1α

, comprising;

(a) an oligonucleotide molecule comprising;

(i) an RNA loop consisting of the nucleotide sequence 5′

-CCGCAGC-3′

; and

(ii) a nucleotide stem consisting of 4, 5, 6, 7, 8, 9, or 10 nucleotide base pairs; and

(b) a donor moiety and an acceptor moiety which exhibit a detectable resonance energy transfer when the donor moiety is excited, wherein the donor moiety is conjugated to one of the 5′

or 3′

ends of the oligonucleotide molecule and the acceptor moiety is conjugated to the other of the 5′

or 3′

ends of the oligonucleotide molecule.

41. The substrate of claim 2 wherein the donor moiety is a cyanine.

42. The substrate of claim 41 wherein the cyanine is indodicarbocyanine 5 (Cy5).

43. The substrate of claim 2 wherein the acceptor moiety is a dark quencher.

44. The substrate of claim 43 wherein the dark quencher is a QSY™

dye.

45. The substrate of claim 43 wherein the dark quencher is a BLACK HOLE QUENCHER®

(BHQ®

).

16. A method for detecting RNase activity of an IRE-1α

polypeptide, comprising;

(1) contacting the IRE-1α

polypeptide with a substrate, wherein the substrate comprises;

(a) an oligonucleotide molecule comprising;

(i) an RNA loop consisting of a cleavage site for IRE-1α

;

(ii) a nucleotide stem consisting of 4, 5, 6, 7, 8, 9, or 10 ribonucleotide base pairs; and

(b) a donor moiety and an acceptor moiety which exhibit a detectable resonance energy transfer when the donor moiety is excited, wherein the donor moiety is conjugated to one of the 5′

or 3′

ends of the oligonucleotide molecule and the acceptor moiety is conjugated to the other of the 5′

or 3′

ends of the oligonucleotide molecule; and

(2) detecting resonance energy transfer between the donor and acceptor moieties, whereby a change in the resonance energy transfer indicates RNase activity of the IRE-1α

polypeptide.

17. The method of claim 16 further comprising detecting the resonance energy transfer in the presence of a test compound.

18. The method of claim 16 wherein the IRE-1α

polypeptide and the substrate are in a cell-free system.

19. The method of claim 16 wherein the change in resonance energy is detected by determining a property selected from the group consisting of a molar extinction coefficient at an excitation wavelength, a quantum efficiency, an excitation spectrum, an emission spectrum, an excitation wavelength maximum, an emission wavelength maximum, a ratio of excitation amplitudes at two wavelengths, a ratio of emission amplitudes at two wavelengths, an excited state lifetime, anisotropy, a polarization of emitted light, resonance energy transfer, and a quenching of emission at a wavelength.

20. The method of claim 16 wherein the IRE-1α

polypeptide is a full-length IRE-1α

protein.

21. The method of claim 16 wherein the IRE-1α

polypeptide comprises a kinase domain.

31. The method of claim 16 wherein at least one of the donor and acceptor moieties is a fluorescent dye.

32. The method of claim 16 wherein each of the donor and acceptor moieties is a fluorescent dye.

33. The method of claim 16 wherein the acceptor moiety is a dark quencher.

34. The method of claim 16 wherein the acceptor moiety is selected from the group consisting of a QSY™

dye and a BLACK HOLE QUENCHER®

(BHQ®

).

35. The method of claim 16 wherein the acceptor moiety is selected from the group consisting of BHQ-1®

, BHQ-2, and BHQ-3®

.

36. The method of claim 16 wherein the donor moiety is selected from the group consisting of 6-carboxyfluorescein (FAM), 2′

7′

-dimethoxy-4′

5′

-dichloro-6-carboxyfluorescein (JOE), tetrachlorofluorescein (TET), 6-carboxyrhodamine (R6G), N,N,N;

N′

-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), sulforhodamine 101 (Texas Red), indodicarbocyanine 3 (Cy3), indodicarbocyanine 5 (Cy5), indodicarbocyanine 5.5 (Cy5.5), 3-1-carboxy-pentyl)-3′

-ethyl-5,5′

-dimethyloxacarbocyanine (CyA), 9-isothiocyanatoacridine, acridine orange, and N-(p-(2-benzoxazolyl)phenyl)maleimide.

37. The method of claim 36 wherein the donor moiety is indodicarbocyanine 5 (Cy5).

38. The method of claim 36 wherein the donor moiety is 6-carboxyfluorescein (FAM).

39. The method of claim 16 wherein the donor is a fluorescent dye and the acceptor is a dark quencher.