Modification of RNA, Producing an Increased Transcript Stability and Translation Efficiency

Abstract

It was the object of the present invention to provide RNA with increased stability and translation efficiency and means for obtaining such RNA. It should be possible to obtain increased grades of expression by using said RNA in gene therapy approaches.

1 . A nucleic acid molecule comprising in the 5′→3′ direction of transcription:
 (a) a promoter; 
 (b) a transcribable first nucleic acid sequence or a second nucleic acid sequence for introducing the transcribable first nucleic acid sequence; 
 (c) a third nucleic acid sequence which, when transcribed under the control of the promoter, codes for a nucleotide sequence of at least 20 consecutive A nucleotides in the transcript; and 
 (d) a recognition sequence for a type IIS restriction endonuclease. 
 
     
     
         2 . The nucleic acid molecule of  claim 1 , wherein the first and third nucleic acid sequences are under control of the promoter and are transcribable into a common transcript, and wherein, in the common transcript, the nucleic acid sequence transcribed from the third nucleic acid sequence increases the translation efficiency and/or the stability of the nucleic acid sequence transcribed from the first nucleic acid sequence. 
     
     
         3 . The nucleic acid molecule of  claim 1 , wherein the third nucleic acid sequence, when transcribed under control of the promoter, codes for a nucleotide sequence of at least 40 consecutive A nucleotides in the transcript. 
     
     
         4 . The nucleic acid molecule of  claim 1 , wherein the third nucleic acid sequence, when transcribed under control of the promoter, codes for a nucleotide sequence of at least 80 consecutive A nucleotides in the transcript. 
     
     
         5 . The nucleic acid molecule of  claim 1 , wherein the third nucleic acid sequence, when transcribed under control of the promoter, codes for a nucleotide sequence of at least 100 consecutive A nucleotides in the transcript. 
     
     
         6 . The nucleic acid molecule of  claim 1 , wherein the third nucleic acid sequence, when transcribed under control of the promoter, codes for a nucleotide sequence of about 120 consecutive A nucleotides in the transcript. 
     
     
         7 . The nucleic acid molecule of  claim 1 , wherein the nucleic acid molecule comprises a sequence such that cleavage with the type IIS restriction endonuclease yields a cleaved nucleic acid molecule having a T nucleotide at an end of the strand that serves as a template for the nucleotide sequence of at least 20 consecutive A nucleotides encoded by the third nucleic acid sequence. 
     
     
         8 . The nucleic acid molecule of  claim 1 , which is a closed circular molecule prior to cleavage with the type IIS restriction endonuclease and a linear molecule after cleavage with the type IIS restriction endonuclease. 
     
     
         9 . The nucleic acid molecule of  claim 1 , wherein the cleavage site of the type IIS restriction endonuclease is located within the third nucleic acid sequence. 
     
     
         10 . The nucleic acid molecule of  claim 1 , wherein the recognition sequence for the type IIS restriction endonuclease is located at a distance of 5-26 base pairs downstream of the 3′ end of the third nucleic acid sequence. 
     
     
         11 . The nucleic acid molecule of  claim 1 , wherein the recognition sequence for the type IIS restriction endonuclease is located at a distance of 24-26 base pairs downstream of the 3′ end of the third nucleic acid sequence. 
     
     
         12 . The nucleic acid molecule of  claim 1 , wherein the type IIS restriction endonuclease is selected from the group consisting of SapI, EciI, BpiI, AarI, AloI, BaeI, BbvCI, PpiI and PsrI, BsrD1, BtsI, EarI, BmrI, BsaI, BsmBI, FauI, BbsI, BciVI, BfuAI, BspMI, BseRI, EciI, BtgZI, BpuEI, BsgI, MmeI, CspCI, BaeI, BsaMI, Mva1269I, PctI, Bse3DI, BseMI, Bst6I, Eam1104I, Ksp632I, BfiI, Bso31I, BspTNI, Eco31I, Esp3I, BfuI, Acc36I, AarI, Eco57I, Eco57MI, GsuI, AloI, Hin4I, PpiI, and PsrI. 
     
     
         13 . The nucleic acid molecule of  claim 1 , wherein the first nucleic acid sequence codes for a peptide or protein. 
     
     
         14 . The nucleic acid molecule of  claim 1 , wherein the second nucleic acid sequence comprises a multiple cloning site. 
     
     
         15 . A nucleic acid molecule produced by linearization of the nucleic acid molecule of  claim 1  with the type IIS restriction endonuclease. 
     
     
         16 . A method of transcribing in vitro an RNA molecule, comprising the steps:
 (i) coupling a first nucleic acid sequence which, when transcribed, codes for a nucleotide sequence of at least 20 consecutive A nucleotides, at the 3′ end of a second nucleic acid sequence which is transcribable into said RNA molecule;   (ii) cleaving the first nucleic acid sequence with a type IIS restriction endonuclease and   (iii) after cleaving the first nucleic acid sequence with the type IIS restriction endonuclease, transcribing in vitro the nucleic acid obtained after steps (i) and (ii);   such that said step of transcribing forms a transcript which comprises the nucleic acid sequences transcribed from the second nucleic acid sequence and a 3′-terminal nucleotide sequence of at least 20 consecutive A nucleotides, wherein the 3′-terminal nucleotide of said transcript is an A nucleotide.   
     
     
         17 . The method of  claim 16 , wherein the recognition sequence for the type IIS restriction endonuclease is located at a distance of 5-26 base pairs downstream of the 3′ end of the first nucleic acid sequence. 
     
     
         18 . The method of  claim 16 , wherein the recognition sequence for the type IIS restriction endonuclease is located at a distance of 24-26 base pairs downstream of the 3′ end of the first nucleic acid sequence. 
     
     
         19 . The method of  claim 16 , wherein the type IIS restriction endonuclease is selected from the group consisting of SapI, EciI, BpiI, AarI, AloI, BaeI, BbvCI, PpiI and PsrI, BsrD1, BtsI, EarI, BmrI, BsaI, BsmBI, FauI, BbsI, BciVI, BfuAI, BspMI, BseRI, EciI, BtgZI, BpuEI, BsgI, MmeI, CspCI, BaeI, BsaMI, Mva1269I, PctI, Bse3DI, BseMI, Bst6I, Eam1104I, Ksp632I, BfiI, Bso31I, BspTNI, Eco31I, Esp3I, BfuI, Acc36I, AarI, Eco57I, Eco57MI, GsuI, AloI, Hin4I, PpiI, and PsrI. 
     
     
         20 . The method of  claim 16 , wherein the second nucleic acid sequence codes for a peptide or protein.