Kits and methods for generating 5′ capped RNA
View Patent ↗The present invention relates to kits and methods for efficiently generating 5′ capped RNA having a modified cap nucleotide and for use of such modified-nucleotide-capped RNA molecules. In particular, the present invention provides kits and methods for capping RNA using a modified cap nucleotide and a capping enzyme system, such as poxvirus capping enzyme. The present invention finds use for in vitro production of 5′-capped RNA having a modified cap nucleotide and for in vitro or in vivo production of polypeptides by in vitro or in vivo translation of such modified-nucleotide-capped RNA. The invention also provides methods and kits for capturing or isolating uncapped RNA comprising primary RNA transcripts or RNA having a 5′-diphosphate, and methods and kits for using a capping enzyme system and modified cap nucleotides for labeling uncapped RNA comprising primary RNA transcripts or RNA having a 5′-diphosphate with detectable dye or enzyme moieties.
1. A composition for obtaining a modified nucleotide-capped RNA comprising:
i) a capping enzyme system comprising an RNA guanyltransferase that exhibits motif I consisting of the amino acid sequence KxDGxx; and
ii) a modified cap nucleotide, wherein the modified cap nucleotide:
A) is 3′-deoxyguanosine-5′-triphosphate or comprises a modified 2′- or 3′-deoxyguanosine-5′-triphosphate, wherein the 2′- or 3′-deoxy position of the sugar moiety is substituted by a group other than a hydroxyl group or a hydrogen, in particular wherein the 2′- or 3′-deoxy position of the sugar moiety is substituted with an amino, an azido, a fluorine, a methoxy, a thiol, or a mercapto group, or wherein the O 6 -oxygen of the guanine base is replaced with a thiol or mercapto group;
B) comprises a modified guanosine-5′-triphosphate, wherein the 2′- or 3′-hydroxyl group of the ribose is substituted with an alkyl group, or wherein the O 6 -oxygen of the guanine is substituted with an alkyl group or is replaced with a thiol or mercapto group; or
C) is selected from: N1-methyl-GTP; O 6 -methyl-GTP; 6-thio-GTP; 2′-O-methyl-GTP; 3′-O-methyl-GTP; 2′-amino-2′-dGTP; 3′-amino-3′-dGTP; 2′-azido-2′-dGTP; 3′-azido-3′-dGTP; 2′-F-2′-dGTP; 3′-F-3′-dGTP; 3′-dGTP; 2′-amino-2′,3′-ddGTP; 3′-amino-2′,3′-ddGTP; 2′-azido-2′,3′-ddGTP; and 3′-azido-2′,3′-ddGTP.
2. The composition of claim 1 , wherein the RNA guanyltransferase comprises a motif I that exhibits the amino acid sequence KTDG(I/V)x, wherein the sixth amino acid of said motif I is P, G, F, S or L.
3. The composition of claim 1 , further comprising uncapped RNA that exhibits a 5′ diphosphate.
4. The composition of claim 1 , further comprising polypeptide having RNA triphosphatase activity.
5. The composition of claim 4 , further comprising uncapped RNA that exhibits a 5′ triphosphate.
6. The composition of claim 5 , wherein the uncapped RNA is:
(i) a primary RNA transcript from a human, animal, plant, or fungal organism, organ, tissue, or cell, or from extracellular fluid from a patient or organism with a condition;
(ii) primary RNA from an in vitro transcription reaction;
(iii) obtained following fractionation of RNA from a biological source by subtractive hybridization and digestion, and RNA amplification that yields sense RNA;
(iv) primary RNA from a prokaryotic source;
(v) derived from a condition comprising a tumor or cancer cell; or
(vi) derived from a pathogen, or from a eukaryotic cell that is infected by a bacterial, viral or fungal pathogen.
7. The composition of claim 1 , further comprising S-adenosyl-methionine or S-adenosyl-ethionine.
8. The composition of claim 7 , further comprising a polypeptide having guanine-7-methyltransferase activity.
9. The composition of claim 8 , further comprising modified-nucleotide capped RNA, wherein the 5′ cap nucleotide comprises a modified guanine nucleoside-5′-triphosphate of claim 1 (b).
10. The composition of claim 9 , further comprising an enzyme having mRNA (nucleoside-2′-O—) methyltransferase activity.
11. The composition of claim 10 , wherein the enzyme having mRNA (nucleoside-2′-O—) methyltransferase activity is:
(i) encoded by a poxvirus DNA;
(ii) encoded a vaccinia virus DNA;
(iii) is purified from virions;
(iv) is purified from a recombinant source;
(v) is purified from E. coli cells that express a poxvirus gene that is cloned in a plasmid or other vector;
(vi) is purified from E. coli cells that express the vaccinia gene that is cloned in a plasmid or other vector;
(vii) is encoded by a yeast or fungal gene; or
(viii) is encoded by a nematode, mammalian or other metazoan gene, whether from a wild-type or recombinant source.
12. The composition of claim 9 , wherein the modified-nucleotide capped RNA exhibits a cap with a cap1 structure, wherein the 5′ penultimate nucleotide has a 2′-O-methyl group.
13. The composition of claim 1 further comprising poly(A) polymerase.
14. The composition of claim 13 , wherein said poly(A) polymerase is (i) Escherichia coli poly(A) polymerase; (ii) yeast poly(A) polymerase; or (iii) is recombinant poly(A) polymerase encoded by the E. coli pcnB gene.
15. The composition of claim 9 , wherein the modified-nucleotide capped RNA further exhibits a poly(A) tail.
16. The composition of claim 12 , wherein the modified-nucleotide capped RNA that exhibits a cap with a cap1 structure further exhibits a poly(A) tail.
17. The composition of claim 1 , further comprising a component that improves the efficiency of capping uncapped RNA that forms a duplex involving at least the 5′-terminal nucleotide, wherein said component consists of at least one of the following: (i) dimethylglycine (betaine); (ii) a single-stranded binding protein; (iii) an RNA helicase; (iv) an RNA polymerase; or (v) a DNA polymerase.
18. The composition of claim 9 , wherein the modified-nucleotide capped RNA further comprises a biotin moiety.
19. The composition of claim 12 , wherein the modified-nucleotide capped RNA that exhibits a cap with a cap1 structure further comprises a biotin moiety.
20. The composition of claim 15 , wherein the modified-nucleotide capped RNA that exhibits a poly(A) tail further comprises a biotin moiety.
21. The composition of claim 1 , further comprising an uncapped RNA comprising a primary RNA transcript or an RNA that has a 5′-diphosphate.