ENDORIBONUCLEASE COMPOSITIONS AND METHODS OF USE THEREOF

• US 20130130248A1
• Filed: 11/07/2012
• Published: 05/23/2013
• Est. Priority Date: 05/10/2010
• Status: Active Grant

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Doudna, Jennifer A., Wiedenheft, Blake, Haurwitz, Rachel E., Jinek, Martin
• Granted Patent: US 9,115,348 B2
• Time in Patent Office: Days
• US Class Current: 435/6.11
• CPC Class Codes: C07K 1/22 Affinity chromatography or related techniques based upon selective absorption processes
• CPC Class Codes: C07K 14/8117 Bovine/basic pancreatic trypsin inhibitor (BPTI, aprotinin)
• CPC Class Codes: C12N 15/1003 Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6823 Release of bound markers
• CPC Class Codes: C12Q 2521/313 Type II endonucleases, i.e. cutting outside recognition site

Abstract

The present disclosure provides variant Csy4 endoribonucleases, nucleic acids encoding the variant Csy4 endoribonucleases, and host cells genetically modified with the nucleic acids. The variant Csy4 endoribonucleases find use in a variety of applications, which are also provided. The present disclosure also provides methods of detecting a specific sequence in a target polyribonucleotide; and methods of regulating production of a target RNA in a eukaryotic cell.

Claims

1. A variant Csy4 endoribonuclease comprising an amino acid sequence having at least about 95% amino acid sequence identity to the amino acid sequence set forth in FIG. 6, wherein the endoribonuclease comprises an amino acid substitution at His-29, wherein the variant Csy4 endoribonuclease is enzymatically inactive in the absence of imidazole, and wherein the variant Csy4 endoribonuclease is activatable in the presence of imidazole.

2. The variant Csy4 endoribonuclease of claim 1, wherein the amino acid substitution is a His29 to Ala29 substitution.

3. The variant Csy4 endoribonuclease of claim 1, wherein the variant Csy4 endoribonuclease comprises a moiety that provides a detectable signal.

4. The variant Csy4 endoribonuclease of claim 1, wherein the moiety that provides a detectable signal is a fluorophore, a quantum dot, an enzyme other than the endoribonuclease, or a nanoparticle.

5. The variant Csy4 endoribonuclease of claim 1, wherein the endoribonuclease is immobilized on an insoluble support.

6. The variant Csy4 endoribonuclease of claim 5, wherein the insoluble support is a bead.

7. The variant Csy4 endoribonuclease of claim 1, wherein, in the variant Csy4 endoribonuclease binds an RNA substrate comprising the nucleotide sequence 5′

-GUUCACUGCCGUAUAGGCAGCUAAGAAA-3′

(SEQ ID NO;

1).

8. A nucleic acid comprising a nucleotide sequence encoding the variant Csy4 endoribonuclease of claim 1.

9. A recombinant expression vector comprising a nucleotide sequence encoding the variant Csy4 endoribonuclease of claim 1.

10. The recombinant expression vector of claim 9, wherein the nucleotide sequence encoding the variant Csy4 endoribonuclease is operably linked to a promoter.

11. The recombinant expression vector of claim 10, wherein the promoter is an inducible promoter.

12. An in vitro genetically modified host cell comprising the recombinant expression vector of claim 9.

13. A kit for purifying a target RNA present in a mixed population of nucleic acids, the kit comprising:

the variant Csy4 endoribonuclease of claim 1.

14. The kit of claim 13, further comprising a recombinant expression vector comprising, in order from 5′

to 3′ and

in operable linkage;

a) a nucleotide sequence encoding an RNA substrate that is specifically bound by the variant Csy4 endoribonuclease of claim 1;

b) a multiple cloning site suitable for insertion of a nucleic acid encoding the target RNA.

15. The kit of claim 14, wherein the nucleotide sequence encoding the RNA substrate is operably linked to a promoter.

16. The kit of claim 15, wherein the promoter is an inducible promoter.

17. The kit of claim 14, wherein the RNA substrate comprises the nucleotide sequence 5′

-GUUCACUGCCGUAUAGGCAGCUAAGAAA-3′

(SEQ ID NO;

1).

18. The kit of claim 14, wherein the recombinant expression vector comprises, inserted into the multiple cloning site, a nucleotide sequence encoding the target RNA.

19. The kit of claim 13, further comprising imidazole.

20. The kit of claim 13, further comprising one or more wash buffers.

21. The kit of claim 13, further comprising a positive control expression vector.

22. The kit of claim 13, wherein the variant Csy4 endoribonuclease is immobilized on an insoluble support.

23. A method of isolating a target RNA present in a mixed population of nucleic acids, the method comprising:

a) contacting a mixed population of nucleic acids with the variant Csy4 endoribonuclease of claim 1, where the variant Csy4 endoribonuclease is immobilized on an insoluble support, wherein the mixed population of nucleic acids comprises a tagged target RNA comprising a recognition nucleotide sequence that is specifically bound by the immobilized variant Csy4 endoribonuclease, forming a tagged target RNA-immobilized variant Csy4 endoribonuclease complex, wherein said contacting is carried out in a binding solution lacking imidazole;

b) adding imidazole to the binding solution to a final concentration of from about 100 mM to about 500 mM, thereby forming a reactivation solution that enzymatically reactivates the immobilized variant Csy4 endoribonuclease, wherein the reactivated immobilized variant Csy4 endoribonuclease cleaves the target RNA from the tag; and

c) collecting the released target RNA.

24. The method of claim 23, further comprising a wash step carried out after step (a) and before step (b).

25. A method of isolating a polypeptide that binds a target RNA, the method comprising:

a) contacting an immobilized complex with a liquid solution comprising a polypeptide that binds the target RNA, wherein the immobilized complex comprises the variant Csy4 endoribonuclease and a tagged target RNA comprising a recognition nucleotide sequence that is specifically bound by the variant Csy4 endoribonuclease, wherein said contacting results in binding of the polypeptide to the target RNA, wherein said contacting is carried out in a binding solution lacking imidazole; and

b) eluting the bound polypeptide.

26. A method of regulating production of a target RNA in a eukaryotic cell, the method comprising contacting a genetically modified host cell with an agent that activates an inducible promoter, wherein the genetically modified host cell is genetically modified with a recombinant expression vector comprising a nucleotide sequence encoding an enzymatically active sequence-specific Csy4 endoribonuclease that catalyzes cleavage at a sequence-specific cleavage site in a substrate polyribonucleotide, wherein the enzyme-encoding nucleotide sequence is operably linked to the inducible promoter, wherein, upon activation of the inducible promoter, the enzyme is produced in the cell and cleaves said target RNA from a precursor RNA.

27. The method of claim 26, wherein the target RNA species is a regulatory RNA.

28. The method of claim 26, wherein cleavage of said target RNA from a precursor RNA inactivates the precursor RNA.

29. A method of detecting a specific sequence in a target polyribonucleotide, the method comprising:

a) contacting the target polyribonucleotide with a oligonucleotide probe comprising the specific sequence and an enzymatically active sequence-specific Csy4 endoribonuclease under conditions that favor duplex formation between the oligonucleotide probe and the target polyribonucleotide, wherein the duplex is cleaved by the Csy4 endoribonuclease; and

b) detecting specific binding between the oligonucleotide probe and the target polyribonucleotide, wherein detection of duplex formation between the oligonucleotide probe and the target polyribonucleotide indicates the presence of the specific sequence in the target polyribonucleotide.

30. The method of claim 29, wherein the oligonucleotide probe is linked to a peptide, wherein the peptide is released upon cleavage of the duplex by the Csy4 endoribonuclease, and where the detection step comprises detection of the released peptide.

31. The method of claim 29, wherein the released peptide is detected by binding to an antibody specific for the peptide.

32. The method of claim 31, wherein the antibody is immobilized.

33. The method of claim 29, wherein the target polyribonucleotide is immobilized on a solid support.

34. The method of claim 29, wherein the target polyribonucleotide is a polyribonucleotide of a pathogen.

35. The method of claim 29, wherein the enzymatically active Csy4 endoribonuclease comprises an amino acid sequence of a Csy4 amino acid sequence depicted in FIG. 5.