Methods and Compositions for Increased Capping Efficiency of Transcribed RNA

Abstract

Methods and compositions for capping RNA in an in vitro transcription mixture are provided that include a thermostable RNA polymerase variant and a cap analog such that when a DNA template is added to the mixture, and the mixture is then incubated under conditions for in vitro transcription, capped RNA is produced.

1 - 16 . (canceled) 
     
     
         17 . A kit comprising a compound according to Formula 1 and an RNA polymerase variant, wherein Formula 1 comprises: 
       
         
           
           
               
               
           
         
         R 1  and/or R 2 ═O-alkyl, halogen, a linker, hydrogen or a hydroxyl; 
         R 3 =guanine, adenine, cytosine, uridine, guanine analog, adenine analog, cytosine analog, or uridine analog; 
         R4=N 1 (p-Nx) where N is a nucleoside or a modified nucleoside where the nucleoside is selected from adenine, uridine, guanine, or cytidine or analogs of adenine, uridine, guanine, or cytidine, and the modified nucleoside is selected from N 6 -methyladenine, N 1 -methyladenine, N 6 -2′-Q-dimethyladenosine, pseudouridine, N 1 -methylpseudouridine, 5-iodouridine, 4-thiouridine, 2-thiouridine, S-methyluridine, pseudoisocytosine, 5-methoxycytosine, 2-thiocytosine, 5-hydroxycytisine, N 4 -methylcytosine, 5-hydroxymethylcytosine, hypoxanthine, N 1 -methylguanine, N 1 -methylguanine, 1-methyl-guanosine, N 2 -methyl-guanosine (m 2 G), N 2 ,N 2 -dimethyl-guanosine (m 2,2 G), 2-methyl-2′-Q-methyl-guanosine (m 2 Gm), N 2 ,N 2 -dimethyl-2′-O-methyl-guanosine (m 2,2 Gm), 1-methyl-2′-O-methyl-guanosine, N 2 ,N 7 -dimethyl-2′-O-methyl-guanosine (m 2,7 Gm), or isoguanineadenine 
         wherein: 
         (i) x can be any integer from 0-8,
 wherein the sugar in the nucleotides may be selected from ribose, deoxyribose, and comprise of modifications including 2′-Q-alkyl, 2′-O-methoxyethyl, 2′-Q allyl, 2′-O alkylamine, 2′-fluororibose, or 2′-deoxyribose; 
 the phosphate groups in one or more nucleotides can be substituted for phosphorothioates, phosphorodithioate, alkylphosphonate, amylphosphonate, or N-phosphoramidate linkages; 
 
         (ii) the polynucleotide cap can be a salt or solvated form; and 
         (iii) the polynucleotide cap can be a single stereoisomer or plurality of stereoisomers of one or more of the compounds described by Formula 1 or a salt or salts thereof. 
       
     
     
         18 . A kit comprising:
 (a) a cap analog;   (b) an RNA polymerase comprising: (i) an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1; and (ii) an amino acid substitution at one or more positions corresponding to positions 388 and 567 of SEQ ID NO:1; and (iii) instructions for use including for achieving at least 95% capping, using an incubation temperature in the range of at least 30° C. to about 70° C.   
     
     
         19 . The kit according to  claim 18 , wherein the cap analog has the formula: 
       
         
           
           
               
               
           
         
         R 1  and/or R 2 ═O-alkyl, halogen, a linker, hydrogen or a hydroxyl; 
         R 3 =guanine, adenine, cytosine, uridine, guanine analog, adenine analog, cytosine analog, or uridine analog; 
         R4═N 1 (p-Nx) where N is a nucleoside or a modified nucleoside where the nucleoside is selected from adenine, uridine, guanine, or cytidine or analogs of adenine, uridine, guanine, or cytidine, and the modified nucleoside is selected from N 6 -methyladenine, N 1 -methyladenine, N 6 -2′-O-dimethyladenosine, pseudouridine, N 1 -methylpseudouridine, 5-iodouridine, 4-thiouridine, 2-thiouridine, 5-methyluridine, pseudoisocytosine, 5-methoxycytosine, 2-thiocytosine, 5-hydroxycytosine, N 4 -methylcytosine, 5-hydroxymethylcytosine, hypoxanthine, N 1 -methylguanine, O 6 -methylguanine, 1-methyl-guanosine, N 2 -methyl-guanosine (m 2 G), N 2 ,N 2 -dimethyl-guanosine (m 2,2 G), 2-methyl-2′-O-methyl-guanosine (m 2 Gm), N 2 ,N 2 -dimethyl-2′-O-methyl-guanosine (m 2,2 Gm), 1-methyl-2′-O-methyl-guanosine, N 2 ,N 7 -dimethyl-2′-O-methyl-guanosine (m 2,7 Gm), or isoguanineadenine 
         wherein: 
         (i) x can be any integer from 0-8,
 wherein the sugar in the nucleotides may be selected from ribose, deoxyribose, and comprise of modifications including 2′-O-alkyl, 2′-O-methoxyethyl, 2′-O allyl, 2′-O alkylamine, 2′-fluororibose, or 2′-deoxyribose; 
 the phosphate groups in one or more nucleotides can be substituted for phosphorothioates, phosphorodithioate, alkylphosphonate, arylphosphonate, or N-phosphoramidate linkages; 
 
         (ii) the polynucleotide cap can be a salt or solvated form; and 
         (iii) the polynucleotide cap can be a single stereoisomer or plurality of stereoisomers of one or more of the compounds described by Formula 1 or a salt or salts thereof. 
       
     
     
         20 . The kit according to  claim 18 , wherein the sugars in N 1  and N 2  are independently, for each position, selected from ribose and deoxyribose, and optionally comprise modifications including 2′-O-alkyl, 2′-O-methoxyethyl, 2′-O allyl, 2′-O alkylamine, 2′-fluororibose, and 2′-deoxyribose. 
     
     
         21 . The kit according to  claim 18 , wherein the bases in N 1  and N 2  are independently, for each position, selected from adenine, uridine, guanine, or cytidine or analogs of adenine, uridine, guanine, or cytidine. 
     
     
         22 . The kit according to  claim 18 , wherein the cap analog is a dinucleotide cap analog or trinucleotide cap analog. 
     
     
         23 . The kit according to  claim 18 , wherein the RNA polymerase comprises an amino acid substitution at positions corresponding to positions 388 and 567 of SEQ ID NO:1. 
     
     
         24 . The kit according to  claim 23 , wherein the RNA polymerase further comprises an amino acid substitution of at least one position corresponding to positions selected from 109, 205,534, and 618 of SEQ ID NO:1. 
     
     
         25 . The kit according to  claim 23 , wherein the RNA polymerase further comprises an amino acid substitution at at least two positions corresponding to positions selected from 109, 205,534 and 618 of SEQ ID NO:1. 
     
     
         26 . The kit according to  claim 18 , wherein the RNA polymerase further comprises an amino acid substitution at positions corresponding to positions 109,205,534 and 618 of SEQ ID NO:1. 
     
     
         27 . A composition comprising:
 rNTPs,   a cap analog, and   an RNA polymerase comprising:   an RNA polymerase comprising: (i) an amino acid sequence is at least 90% sequence identity to SEQ ID NO: 1; and (ii) an amino acid substitution at one or more positions corresponding to positions 388 and 567 of SEQ ID NO:1.   
     
     
         28 . The composition according to  claim 27 , wherein the cap analog has the formula 
       
         
           
           
               
               
           
         
         R 1  and/or R 2 ═O-alkyl, halogen, a linker, hydrogen or a hydroxyl; 
         R 3 =guanine, adenine, cytosine, uridine, guanine analog, adenine analog, cytosine analog, or uridine analog; 
         R4═N 1 (p-Nx) where N is a nucleoside or a modified nucleoside where the nucleoside is selected from adenine, uridine, guanine, or cytidine or analogs of adenine, uridine, guanine, or cytidine, and the modified nucleoside is selected from N 6 -methyladenine, N 1 -methyladenine, N 6 -2′-O-dimethyladenosine, pseudouridine, N 1 -methylpseudouridine, 5-iodouridine, 4-thiouridine, 2-thiouridine, 5-methyluridine, pseudoisocytosine, 5-methoxycytosine, 2-thiocytosine, 5-hydroxycytosine, N 4 -methylcytosine, 5-hydroxymethylcytosine, hypoxanthine, N 1 -methylguanine, O 6 -methylguanine, 1-methyl-guanosine, N 2 -methyl-guanosine (m 2 G), N 2 ,N 2 -dimethyl-guanosine (m 2,2 G), 2-methyl-2′-O-methyl-guanosine (m 2 Gm), N 2 ,N 2 -dimethyl-2′-O-methyl-guanosine (m 2,2 Gm), 1-methyl-2′-O-methyl-guanosine, N 2 ,N 7 -dimethyl-2′-O-methyl-guanosine (m 27 Gm), or isoguanineadenine, 
         wherein: 
         (i) x can be any integer from 0-8,
 wherein the sugar in the nucleotides may be selected from ribose, deoxyribose, and comprise of modifications including 2′-O-alkyl, 2′-O-methoxyethyl, 2′-O allyl, 2′-O alkylamine, 2′-fluororibose, or 2′-deoxyribose; 
 the phosphate groups in one or more nucleotides can be substituted for phosphorothioates, phosphorodithioate, alkylphosphonate, arylphosphonate, or N-phosphoramidate linkages; 
 
         (ii) the polynucleotide cap can be a salt or solvated form; and 
         (iii) the polynucleotide cap can be a single stereoisomer or plurality of stereoisomers of one or more of the compounds described by Formula 1 or a salt or salts thereof. 
       
     
     
         29 . The composition according to  claim 27 , wherein the sugars in N 1  and N 2  are independently, for each position, selected from ribose and deoxyribose, and optionally comprise modifications including 2′-O-alkyl, 2′-O-methoxyethyl, 2′-O allyl, 2′-O alkylamine, 2′-fluororibose, and 2′-deoxyribose. 
     
     
         30 . The composition according to  claim 27 , wherein the bases in N 1  and N 2  are independently, for each position, selected from adenine, uridine, guanine, or cytidine or analogs of adenine, uridine, guanine, or cytidine. 
     
     
         31 . The composition according to  claim 27 , wherein the cap analog is a dinucleotide cap analog or trinucleotide cap analog. 
     
     
         32 . The composition according to  claim 27 , wherein the RNA polymerase comprises an amino acid substitution at positions corresponding to positions 388 and 567 of SEQ ID NO:1. 
     
     
         33 . The composition according to  claim 32 , wherein the RNA polymerase further comprises an amino acid substitution of at least one position corresponding to positions selected from 109, 205, 534, and 618 of SEQ ID NO:1. 
     
     
         34 . The composition according to  claim 32 , wherein the RNA polymerase comprises an amino acid substitution at at least two positions corresponding to positions selected from 109, 205, 534 and 618 of SEQ ID NO:1. 
     
     
         35 . The composition according to  claim 27 , wherein the RNA polymerase further comprises an amino acid substitution at positions corresponding to positions 109,205,534 and 618 of SEQ ID NO:1. 
     
     
         36 . The composition according to  claim 27 , further comprising a nucleic acid template.