Kits and methods for generating 5′ capped RNA

A method for obtaining a modified nucleotide-capped RNA, the method comprising the steps of:

• (a) providing:
• i) an uncapped RNA comprising a primary RNA transcript or an RNA that has a 5′-diphosphate;
• ii) a capping enzyme system comprising an RNA guanyltransferase that exhibits motif I consisting of the amino acid sequence KxDGxx;
• and
• iii) a modified cap nucleotide, wherein the modified cap nucleotide:
• A) 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, or wherein the 06-oxygen of the guanine base is replaced with a thiol or mercapto group, 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;
• 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 O6-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;
• O6-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;
• and
• (b) contacting the uncapped RNA with the capping enzyme system and the modified cap nucleotide under conditions wherein modified nucleotide-capped RNA is synthesized.