IRE-1alpha substrates

• US 20070105123A1
• Filed: 11/04/2005
• Published: 05/10/2007
• Est. Priority Date: 11/04/2005
• Status: Active Grant

• Current Assignee: Fosun Orinove Pharmatech, Inc.
• Original Assignee: Mannkind Corporation
• Inventors: Brumm, Duane, Stiles, Caryn, Patterson, John, Volkmann, Kori
• Granted Patent: US 8,017,331 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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 an oligonucleotide molecule which consists of (i) an RNA loop comprising a cleavage site for IRE-1α

; and

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

2. The substrate of claim 1 further comprising 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 further comprising a detectable label.

4. The substrate of claim 1 wherein the RNA loop comprises the nucleotide sequence 5′

-CCGCAGC-3′

.

5. The substrate of claim 1 which comprises SEQ ID NO:

1 or SEQ ID NO;

3.

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

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

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

9. The substrate of claim 2 wherein at least one of the donor and acceptor moieties is a fluorescent protein.

10. The substrate of claim 2 wherein each of the donor and the acceptor moieties is a fluorescent protein.

11. The substrate of claim 2 wherein the acceptor moiety is a luminescent moiety.

12. The substrate of claim 11 wherein the luminescent moiety is selected from the group consisting of a luminescent protein and a lanthanide chelate.

13. The substrate of claim 2 wherein the acceptor moiety is selected from the group consisting of 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.

14. The substrate of claim 2 wherein the donor moiety is selected from the group consisting of 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.

15. The substrate of claim 2 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).

16. The substrate of claim 2 wherein the acceptor moiety is a luminescent protein.

17. The substrate of claim 16 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.

18. A mutant substrate for IRE-1α

comprising an oligonucleotide molecule which consists of (i) an RNA loop with a mutant cleavage site for IRE-1α

; and

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

19. The mutant substrate of claim 18 wherein the RNA loop comprises the nucleotide sequence 5′

-CCCCAGC-3′

.

20. The mutant substrate of claim 18 further comprising 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.

21. The mutant substrate of claim 18 further comprising a detectable label.

22. 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 consisting of

(i) an RNA loop comprising 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.

23. The method of claim 22 further comprising detecting the resonance energy transfer in the presence of a test compound.

24. The method of claim 22 wherein the IRE-1α

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

25. The method of claim 22 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.

26. The method of claim 22 wherein the IRE-1α

polypeptide is a full-length IRE-1α

protein.

27. The method of claim 22 wherein the IRE-1α

polypeptide comprises a kinase domain.