IRE-1α substrates
IRE-1α substrates useful for identifying agonists and antagonists of IRE-1α RNase activity.
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 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′.
3. The substrate of claim 1 wherein the RNA loop consists of the nucleotide sequence 5′-CCGCAGC-3′.
4. The substrate of claim 1 wherein the oligonucleotide molecule consists of SEQ ID NO:3.
5. The substrate of claim 1 wherein the nucleotide stem comprises DNA.
6. The substrate of claim 1 wherein the nucleotide stem comprises RNA.
7. The substrate of claim 1 wherein the nucleotide stem comprises a nucleotide analog.
8. The substrate of claim 1 , further comprising a detectable label.
9. A method for detecting RNase activity of an IRE-1α polypeptide, comprising:
(1) contacting the IRE-1α polypeptide with 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; and
(2) detecting cleavage of the substrate.
10. The method of claim 9 further comprising contacting the IRE-1α polypeptide with a test compound.
11. The method of claim 9 wherein the IRE-1α polypeptide and the substrate are in a cell-free system.
12. The method of claim 9 wherein the IRE-1α polypeptide is a full-length IRE-1α protein.
13. The method of claim 9 wherein the IRE-1α polypeptide comprises a kinase domain.
14. The method of claim 9 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′.
15. The method of claim 9 wherein the RNA loop consists of the nucleotide sequence 5′-CCGCAGC-3′.
16. The method of claim 9 wherein the oligonucleotide molecule consists of SEQ ID NO:3.
17. The method of claim 9 wherein the nucleotide stem comprises DNA.
18. The method of claim 9 wherein the nucleotide stem comprises RNA.
19. The method of claim 9 wherein the nucleotide stem comprises a nucleotide analog.
20. The method of claim 9 wherein the substrate further comprising a detectable label.