Endoribonuclease compositions and methods of use thereof
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.
1. A variant Csy4 endoribonuclease comprising an amino acid sequence having at least 95% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:8, wherein the variant Csy4 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 3 , 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 kit for purifying a target RNA present in a mixed population of nucleic acids, the kit comprising:
the variant Csy4 endoribonuclease of claim 1 .
9. The kit of claim 8 , 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.
10. The kit of claim 9 , wherein the nucleotide sequence encoding the RNA substrate is operably linked to a promoter.
11. The kit of claim 10 , wherein the promoter is an inducible promoter.
12. The kit of claim 9 , wherein the RNA substrate comprises the nucleotide sequence 5′-GUUCACUGCCGUAUAGGCAGCUAAGAAA-3′ (SEQ ID NO:1).
13. The kit of claim 9 , wherein the recombinant expression vector comprises, inserted into the multiple cloning site, a nucleotide sequence encoding the target RNA.
14. The kit of claim 8 , further comprising imidazole.
15. The kit of claim 8 , further comprising one or more wash buffers.
16. The kit of claim 8 , further comprising a positive control expression vector.
17. The kit of claim 8 , wherein the variant Csy4 endoribonuclease is immobilized on an insoluble support.
18. 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.
19. The method of claim 18 , further comprising a wash step carried out after step (a) and before step (b).