Kits and methods for generating 5' capped RNA

Abstract

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. The invention is used to obtain novel compositions 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 for a variety of research, therapeutic, and commercial applications. The invention also provides methods and kits for capturing or isolating uncapped RNA comprising primary RNA transcripts or RNA having a 5′-diphosphate, such as RNA synthesized in vitro or obtained from a biological source, including prokaryotic mRNA that is in a mixture with other prokaryotic and/or eukaryotic nucleic acids. The method for capturing modified-nucleotide-capped RNA also provides methods and kits for obtaining only type-specific or condition-specific modified-nucleotide-capped RNA by cap-dependent subtraction of that portion of the captured modified-nucleotide-capped RNA in cells of one type or condition that is the same as RNA in cells of another type or condition. The invention further provides 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 method for obtaining a modified-nucleotide-capped RNA, the method comprising, 
 (a) providing: 
 i) an uncapped RNA comprising a primary RNA transcript or an RNA that has a 5′-diphosphate;  
 ii) a capping enzyme system; and  
 iii) a modified cap nucleotide; 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.    
     
     
         2 . The method of  claim 1 , wherein the uncapped RNA comprising a primary RNA transcript or an RNA that has a 5′-diphosphate is selected from the group consisting of: 
 (i) prokaryotic mRNA;    (ii) uncapped eukaryotic primary mRNA;    (iii) RNA from an in vitro transcription reaction using an RNA polymerase;    (iv) RNA from an in vitro replication reaction using a replicase;    (v) RNA from an in vivo transcription reaction, wherein the RNA polymerase is expressed in a prokaryotic or eukaryotic cell that contains a DNA template that is functionally joined downstream of an RNA polymerase promoter that binds the RNA polymerase;    (vi) RNA from an in vivo replication reaction using a replicase;    (vii) RNA from an RNA amplification reaction;    (viii) eukaryotic small nuclear (snRNA); and    (ix) micro RNA (miRNA).    
     
     
         3 . The method of  claim 1 , wherein the uncapped RNA has a poly(A) tail, wherein the uncapped RNA having a poly(A) tail is obtained: 
 (i) from an in vivo source;    (ii) by in vitro transcription of a DNA template that encodes the poly(A) tail; or    (iii) by in vitro polyadenylation of the uncapped RNA using poly(A) polymerase in the presence of ATP.    
     
     
         4 . The method of  claim 1 , wherein the capping enzyme system comprises: 
 (i) a conserved motif I consisting of the amino acid sequence KxDGxx;    (ii) the conserved motif I of (i) wherein the sixth (6th) amino acid of said motif I is not arginine;    (iii) the conserved motif I of point (i) wherein the sixth (6th) amino acid of said motif I is selected from the group consisting of phenylalanine, serine, and leucine;    (iv) a poxvirus capping enzyme system; or (v) a vaccinia capping enzyme system.    
     
     
         5 . The method of  claim 1 , wherein the modified cap nucleotide comprises: (i) a modified 2′- or 3′-deoxyguanosine-5′-triphosphate wherein the respective 2′- or 3′-deoxy position of the deoxyribose sugar moiety is substituted with a group comprising an amino group, an azido group, a fluorine group, a methoxy group, or a thiol group, or wherein the O6 oxygen of the guanine base is replaced by a thiol (or mercapto) group or methylthio (or methylmercapto) group; or (ii) a modified guanosine-5′-triphosphate wherein the 2′ or 3′ hydroxyl group of the ribose sugar or the O6 oxygen of the guanine base is substituted with an alkyl group, or wherein the O6 oxygen is replaced by a thiol (or mercapto) group or methylthio (or methylmercapto) group; (iii) 3′-deoxyguanosine; or (iv) a modified cap nucleotide selected from the group consisting of O 6 -Me-GTP; 6-thio-GTP; 6-methylthio-GTP; 2′-O-Me-GTP; 3′-O-Me-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.  
     
     
         6 . The method of  claim 1 , wherein the step of contacting the uncapped RNA comprising a primary RNA transcript or RNA that has a 5′-diphosphate with the capping enzyme system and the modified cap nucleotide under conditions wherein modified-nucleotide-capped RNA is synthesized is carried out in the presence of S-adenosyl-methionine or S-adenosyl-ethionine.  
     
     
         7 . The method of claims  1 , wherein the method additionally comprises a step selected from the group consisting of: 
 a) contacting the modified-nucleotide-capped RNA with mRNA (nucleoside-2′-O-)methyltransferase and S-adenosyl-methionine under conditions wherein the 2′-hydroxyl of the 5′-penultimate nucleotide is methylated and a modified-nucleotide-capped RNA with a cap I structure is obtained;    b) contacting the modified-nucleotide-capped RNA with poly(A) polymerase and ATP under conditions wherein modified-nucleotide-capped RNA having a poly(A) tail is obtained;    c) translating said modified-nucleotide-capped RNA into protein;    d) incubating the modified-nucleotide-capped RNA in an in vitro translation system under conditions wherein protein encoded by the modified-nucleotide-capped RNA is obtained; or    e) transforming eukaryotic cells with said modified-nucleotide-capped RNA, wherein the modified-nucleotide-capped RNA is translated into protein in vivo.    
     
     
         8 . The method of  claim 1 , wherein the modified cap nucleotide is a guanine nucleoside-5′-triphosophate having 2′ or 3′ substituent comprising an amino, an azido, or a thiol group, the method additionally provides an affinity tag reagent, and the method additionally comprises the step of contacting the modified-nucleotide-capped RNA obtained with the affinity tag having the reactive moiety under conditions wherein modified-nucleotide-capped RNA that is joined to the affinity tag is obtained.  
     
     
         9 . The method of  claim 10 , wherein the affinity tag reagent is a biotinylation reagent and the affinity tag is biotin.  
     
     
         10 . A method for cap-dependent capture of uncapped RNA comprising primary RNA transcripts or RNA having a 5′-diphosphate, the method comprising: 
 (i) providing a sample comprising the uncapped RNA; a capping enzyme system; a modified cap nucleotide, wherein the modified cap nucleotide contains a chemical binding moiety to facilitate binding to an affinity-tag-binding molecule; and a surface, to which the affinity-tag-binding molecule is attached;    (ii) contacting the uncapped RNA with the capping enzyme system and the modified cap nucleotide under conditions wherein modified-nucleotide-capped RNA is synthesized;    (iii) contacting the modified-nucleotide-capped RNA with reagents and under conditions that facilitate binding of the modified cap nucleotide to the surface to which the affinity-tag-binding molecule is attached; and    (iv) contacting the modified-nucleotide-capped RNA to the surface to which the affinity-tag-binding molecule is attached under conditions wherein the modified-nucleotide-capped RNA is bound to the surface, thereby capturing the modified-nucleotide-capped RNA.    
     
     
         11 . The method of  claim 10 , wherein the chemical binding moiety of the modified cap nucleotide provided in step (i) comprises an affinity tag that is capable of binding the affinity-tag-binding molecule and step (iii) comprises incubating the modified-nucleotide-capped RNA in a buffer and under conditions that facilitate binding of the modified cap nucleotide to surface to which the affinity-tag-binding molecule is attached.  
     
     
         12 . The method of  claim 10 , wherein the modified cap nucleotide provided in step (i) contains a chemical binding moiety comprising an amino, an azido, or a thiol group and step (iii) comprises contacting the modified-nucleotide-capped RNA with an affinity tag reagent under conditions wherein the affinity tag is chemically joined to the chemical binding moiety, and further incubating the modified-nucleotide-capped RNA in a buffer and under conditions that facilitate binding of the modified cap nucleotide to the surface to which the affinity-tag-binding molecule is attached.  
     
     
         13 . The method of  claim 10 , wherein the modified-nucleotide-capped RNA that is bound to the surface in step (iv) is from uncapped RNA provided in step (i) which comprises primary RNA transcripts or RNA having a 5′-diphosphate derived from a first sample comprising one or more cells of a first type or condition, and wherein the method further comprises the steps of: 
 (v) providing a second sample comprising RNA derived from a second sample comprising one or more cells of a second type or condition;    (vi) contacting the RNA derived from the second sample comprising one or more cells of a second type or condition with one or more primers that anneal to the RNA and an RNA-dependent DNA polymerase under conditions wherein first-strand cDNA that is complementary to the RNA from the second sample is synthesized, provided that, the first-strand cDNA has such a polarity or complementarity that, if the same RNA is present in the first sample, it will be complementary to the first-strand cDNA prepared from the RNA derived from the second sample;    (vii) contacting the captured modified-nucleotide-capped RNA comprising RNA derived from the first sample with the first-strand cDNA prepared from the RNA derived from the second sample under conditions wherein the first-strand cDNA from the second sample anneals to complementary captured modified-nucleotide-capped RNA from the first sample; and    (viii) contacting the nucleic acids in step (iii) with RNase H under conditions wherein RNA that is annealed to DNA is digested, thereby subtracting from the captured modified-nucleotide-capped RNA derived from the first sample those modified-nucleotide-capped RNA molecules or nucleic acid sequences that are also present in the RNA derived from the second sample.    
     
     
         14 . The method of  claim 10 , wherein the method for further comprises the step of: 
 (v) contacting the modified-nucleotide-capped RNA that is bound to the surface with tobacco acid pyrophosphatase or yeast decapping enzyme under conditions wherein the triphosphate between the modified cap nucleotide and the 5′-penultimate nucleotide of the modified-nucleotide-capped RNA is cleaved, thereby de-capping the modified-nucleotide-capped RNA and releasing the de-capped RNA therefrom.    
     
     
         15 . A method for synthesizing modified-nucleotide-capped RNA having a methylated 2′-hydroxyl group in the 5′-penultimate nucleotide, said method comprising: providing an m 2   7,3′-O G-capped RNA or an m 2   7,2′-O G-capped RNA having a cap 0 structure from an in vitro transcription reaction using a dinucleotide cap analog; and contacting the modified-nucleotide-capped RNA having a cap 0 structure with mRNA (nucleoside-2′-O-)methyltransferase and S-adenosyl-methionine under conditions wherein a modified-nucleotide-capped RNA with a cap I structure is obtained.  
     
     
         16 . A kit for obtaining a modified-nucleotide-capped RNA, the kit comprising: (a) a capping enzyme system; and (b) a modified cap nucleotide.  
     
     
         17 . The kit of  claim 16 , wherein the capping enzyme system is a capping enzyme comprising: 
 (i) a conserved motif I consisting of the amino acid sequence KxDGxx;    (ii) the conserved motif I of point (i) wherein the sixth (6th) amino acid of said motif I is not arginine;    (iii) the conserved motif I of point (i) wherein the sixth (6th) amino acid of said motif I is selected from the group consisting of phenylalanine, serine, and leucine;    (iv) a poxvirus capping enzyme system; or    (v) a vaccinia capping enzyme system.    
     
     
         18 . The kit of  claim 14 , wherein the modified cap nucleotide comprises: (i) a modified 2′- or 3′-deoxyguanosine-5′-triphosphate wherein the respective 2′- or 3′-deoxy position of the deoxyribose sugar moiety is substituted with a group comprising an amino group, an azido group, a fluorine group, a methoxy group, or a thiol group, or wherein the O6 oxygen of the guanine base is replaced by a thiol (or mercapto) group or methylthio (or methylmercapto) group; or (ii) a modified guanosine-5′-triphosphate wherein the 2′ or 3′ hydroxyl group of the ribose sugar or the O6 oxygen of the guanine base is substituted with an alkyl group, or wherein the O6 oxygen is replaced by a thiol (or mercapto) group or methylthio (or methylmercapto) group; (iii) 3′-deoxyguanosine; or (iv) a modified cap nucleotide selected from the group consisting of O 6 -Me-GTP; 6-thio-GTP; 6-methylthio-GTP; 2′-O-Me-GTP; 3′-O-Me-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.  
     
     
         19 . The kit of  claim 14 , wherein the modified cap nucleotide is selected from the group consisting of: N 1 -Me-GTP; O 6 -Me-GTP; 2′-O-Me-GTP; 3′-O-Me-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; 2′-dGTP; 3′-dGTP; 3′-OMe-GTP; 2′-OMe-GTP; 3′-dGTP; 2′-F-2′-dGTP; 2′-amino-2′-dGTP; 2′-azido-2′-dGTP; N 1 -methyl-GTP; and O 6 -methyl-GTP.  
     
     
         20 . The kit of  claim 17 , wherein the kit additionally comprises: 
 (i) S-adenosyl-methionine or S-adenosyl-ethionine;    (ii) mRNA (nucleoside-2′-O-)methyltransferase;    (iii) poly(A) polymerase;    (iv) an in vitro translation system selected from the group consisting of a plant, an animal, and a yeast or fungal cell-free extract;    (v) a eukaryotic cell selected from the group consisting of: (1) a human, or animal dendritic cell, (2) a human, or animal macrophage, (3) a human, or animal epithelial cell, and (4) an artificially generated human or animal antigen presenting cell (5) a human or animal oocyte, (6) an animal, plant, or fungal somatic cell, (7) and a human or animal T lymphocyte;    (vi) media and reagents for incubating the eukaryotic cell with polypeptides obtained by in vitro translation of modified-nucleotide-capped RNA and growing the cells;    (vii) media and reagents for incubating the eukaryotic cell with the modified-nucleotide-capped RNA and growing the cells;    (viii) one or more adjuvants or compositions for enhancing or modulating an immune response in a human or animal;    (ix) an affinity-tag-binding molecule that is attached to a surface;    (x) an affinity-tag-binding molecule that is labeled with a detectable dye or an enzyme moiety that uses a substrate that results in a detectable signal under enzymatic reaction conditions;    (xi) RNase H; or    (xii) tobacco acid pyrophosphatase or a decapping enzyme.    
     
     
         21 . The composition obtained using the methods of any of  claim 1 .  
     
     
         22 . The composition of  claim 21 , wherein said composition is contained in a eukaryotic cell.