Synthesis and use of anti-reverse mRNA cap analogues

Abstract

The ability to synthesize capped RNA transcripts in vitro has been of considerable value in a variety of applications. However, one-third to one-half of the caps have, until now, been incorporated in the reverse orientation. Such reverse caps impair the translation of in vitro-synthesized mRNAs. Novel cap analogues, such as P 1 -3′-deoxy-7-methylguanosine-5′ P 3 -guanosine-5′ triphosphate and P 1 -3′-O,7-dimethylguanosine-5′ P 3 -guanosine-5′ triphosphate, have been designed that are incapable of being incorporated into RNA in the reverse orientation. Transcripts produced with SP6 polymerase using “anti-reverse” cap analogues were of the predicted length. Analysis of the transcripts indicated that reverse caps were not formed. The in vitro translational efficiency of transcripts with the novel “anti-reverse” cap analogues was significantly higher than that of transcripts formed with conventional caps.

1 . A composition comprising  
     
       
         
         
             
             
         
       
     
     wherein: 
 X is selected from the group consisting of H, OH, OCH 3 , and OCH 2 CH 3 ;  
 Y is selected from the group consisting of H, OH, OCH 3 , and OCH 2 CH 3 ;  
 n is 1,2,3,4, or5;  
 wherein, if Y is OH and n is 1, then X is neither H nor OH; and  
 B is selected from the group consisting of  
                     
 
   
   
       2 . A composition as recited in  claim 1 , wherein X is OH, Y is H, and n is 1.  
   
   
       3 . A composition as recited in  claim 1 , wherein X is OH, Y is OCH 3 , and n is 1.  
   
   
       4 . An RNA molecule whose 5′ end incorporates a composition as recited in  claim 1 .  
   
   
       5 . An RNA molecule whose 5′ end incorporates a composition as recited in  claim 2 .  
   
   
       6 . An RNA molecule whose 5′ end incorporates a composition as recited in  claim 3 .  
   
   
       7 . A method for synthesizing an RNA molecule as recited in  claim 4  in vitro; said method comprising reacting ATP, CTP, UTP, GTP, a composition as recited, and a polynucleotide template; in the presence an RNA polymerase; under conditions conducive to transcription by the RNA polymerase of the polynucleotide template into an RNA copy; whereby some of the RNA copies will incorporate the composition as recited to make an RNA molecule as recited.  
   
   
       8 . A method for synthesizing an RNA molecule as recited in  claim 5  in vitro; said method comprising reacting ATP, CTP, UTP, GTP, a composition as recited, and a polynucleotide template; in the presence an RNA polymerase; under conditions conducive to transcription by the RNA polymerase of the polynucleotide template into an RNA copy; whereby some of the RNA copies will incorporate the composition as recited to make an RNA molecule as recited.  
   
   
       9 . A method for synthesizing an RNA molecule as recited in  claim 6  in vitro; said method comprising reacting ATP, CTP, UTP, GTP, a composition as recited, and a polynucleotide template; in the presence an RNA polymerase; under conditions conducive to transcription by the RNA polymerase of the polynucleotide template into an RNA copy; whereby some of the RNA copies will incorporate the composition as recited to make an RNA molecule as recited.  
   
   
       10 . A method for synthesizing a protein or a peptide in vitro, said method comprising translating an RNA molecule as recited in  claim 4  in a cell-free protein synthesis system, wherein the RNA molecule comprises an open reading frame, under conditions conducive to translating the open reading frame of the RNA molecule into the protein or peptide encoded by the open reading frame.  
   
   
       11 . A method for synthesizing a protein or a peptide in vitro, said method comprising translating an RNA molecule as recited in  claim 5  in a cell-free protein synthesis system, wherein the RNA molecule comprises an open reading frame, under conditions conducive to translating the open reading frame of the RNA molecule into the protein or peptide encoded by the open reading frame.  
   
   
       12 . A method for synthesizing a protein or a peptide in vitro, said method comprising translating an RNA molecule as recited in  claim 6  in a cell-free protein synthesis system, wherein the RNA molecule comprises an open reading frame, under conditions conducive to translating the open reading frame of the RNA molecule into the protein or peptide encoded by the open reading frame.  
   
   
       13 . A composition comprising  
     
       
         
         
             
             
         
       
       wherein n is 1, 2, 3, 4, or 5.  
     
   
   
       14 . An RNA molecule whose 5′ end incorporates a composition as recited in  claim 13 .  
   
   
       15 . A method for synthesizing an RNA molecule as recited in  claim 14  in vitro; said method comprising reacting ATP, CTP, UTP, GTP, a composition as recited, and a polynucleotide template; in the presence an RNA polymerase; under conditions conducive to transcription by the RNA polymerase of the polynucleotide template into an RNA copy; whereby some of the RNA copies will incorporate the composition as recited to make an RNA molecule as recited.  
   
   
       16 . A method for synthesizing a protein or a peptide in vitro, said method comprising translating an RNA molecule as recited in  claim 14  in a cell-free protein synthesis system, wherein the RNA molecule comprises an open reading frame, under conditions conducive to translating the open reading frame of the RNA molecule into the protein or peptide encoded by the open reading frame.  
   
   
       17 . A composition comprising  
     
       
         
         
             
             
         
       
     
     wherein: 
 X is selected from the group consisting of H, OH, OCH 3 , and OCH 2 CH 3 ;  
 Y is selected from the group consisting of H, OH, OCH 3 , and OCH 2 CH 3 ;  
 n is 1, 2, 3, 4, or 5;  
 and Z is selected from the group consisting of C 1  to C 4  substituted or unsubstituted alkyl, C 6  to C 8  substituted or unsubstituted aryl, or C 1  to C 4  substituted or unsubstituted alkoxy;  
 and wherein, if Z is CH 3  and Y is OH and n is 1, then X is neither H nor OH.  
 
   
   
       18 . An RNA molecule whose 5′ end incorporates a composition as recited in  claim 17 .  
   
   
       19 . A method for synthesizing an RNA molecule as recited in  claim 18  in vitro; said method comprising reacting ATP, CTP, UTP, GTP, a composition as recited, and a polynucleotide template; in the presence an RNA polymerase; under conditions-conducive to transcription by the RNA polymerase of the polynucleotide template into an RNA copy; whereby some of the RNA copies will incorporate the composition as recited to make an RNA molecule as recited.  
   
   
       20 . A method for synthesizing a protein or a peptide in vitro, said method comprising translating an RNA molecule as recited in  claim 18  in a cell-free protein synthesis system, wherein the RNA molecule comprises an open reading frame, under conditions conducive to translating the open reading frame of the RNA molecule into the protein or peptide encoded by the open reading frame.  
   
   
       21 . A composition comprising  
     
       
         
         
             
             
         
       
     
     wherein: 
 X is selected from the group consisting of H, OH, OCH 3 , and OCH 2 CH 3 ;  
 Y is selected from the group consisting of H, OH, OCH 3 , and OCH 2 CH 3 ;  
 R is selected from the group consisting of H, CH 3 , CH 2 C 6 H 5 , CH 2 COC 6 H 5 , CH 2 CH 2 CH 2 OH, CH 2 CH═CH 2 , C 1  to C 4  substituted or unsubstituted alkyl, and C 6  to C 8  substituted or unsubstituted aryl;  
 n is 1, 2, 3, 4, or 5;  
 Z is selected from the group consisting of C 1  to C 4  substituted or unsubstituted alkyl, C 6  to C 8  substituted or unsubstituted aryl, or C 1  to C 4  substituted or unsubstituted alkoxy; and  
 B is selected from the group consisting of guanine, adenine, uridine, and cytosine;  
 and wherein, if Z is CH 3 , and R is H, and Y is OH, and n is 1, and B is guanine; then X is neither H nor OH.  
 
   
   
       22 . An RNA molecule whose 5′ end incorporates a composition as recited in  claim 21 .  
   
   
       23 . A method for synthesizing an RNA molecule as recited in  claim 22  in vitro; said method comprising reacting ATP, CTP, UTP, GTP, a composition as recited, and a polynucleotide template; in the presence an RNA polymerase; under conditions conducive to transcription by the RNA polymerase of the polynucleotide template into an RNA copy; whereby some of the RNA copies will incorporate the composition as recited to make an RNA molecule as recited.  
   
   
       24 . A method for synthesizing a protein or a peptide in vitro, said method comprising translating an RNA molecule as recited in  claim 22  in a cell-free protein synthesis system, wherein the RNA molecule comprises an open reading frame, under conditions conducive to translating the open reading frame of the RNA molecule into the protein or peptide encoded by the open reading frame.