Compositions inducing cleavage of RNA motifs

Abstract

Disclosed are compositions inducing cleavage of an RNA substrate, as well as their use for inducing cleavage of RNA substrates in vitro and in vivo. The compositions contain part of an active center, with the other part of the active center provided by the RNA substrate. The subunits of the active center region of the compositions are nucleotides and/or nucleotide analogues. The disclosed compositions also have flanking regions contributing to the formation of a specific hybridization with an RNA substrate. Preferred compositions form, in combination with an RNA substrate, a structure resembling a hammerhead structure. The active center of the disclosed compositions is characterized by the presence of I 15.1 which allows cleavage of RNA substrates having C 16.1 .

We claim:  
     
         1 . A composition that induces cleavage of an RNA substrate, the composition comprising: 
       5′-Z 1 -Z 2 -Z 3 -3′ wherein Z 1  and Z 3  are oligomeric sequences which (1) are comprised of nucleotides, nucleotide analogues, or both, or (2) are oligonuleotide analogues, wherein the oligomeric sequences specifically interact with the RNA substrate by hybridization.    wherein Z 2  consists of   5′-X 12 X 13 X 14 X 15.1 -3′, or5′-X 12 X 13 X 14 X 15.1 -3′,   wherein Z 2  is comprised of nucleotides, nucleotide analogues, or both, wherein the nucleotides and nucleotide analogues each have the structure                          wherein each B is independently adenin-9-yl, cytosin-1-yl, guanin-9-yl, uracil-1-yl, uracil-5-yl, hypoxanthin-9-yl, thymin-1-yl, 5-methylcytosin-1-yl, 2,6-diaminopurin-9-yl, purin-9-yl, 7-deazaadenin-9-yl, 7-deazaguanin-9-yl, 5-propynylcytosin-1-yl, 5-propynyluracil-1-yl, isoguanin-9-yl, 2-aminopurin-9-yl, 6-methyluracil-1-yl, 4-thiouracil-1-yl, 2-pyrimidone-1-yl, quinazoline-2,4-dione-1-yl, xanthin-9-yl, N 2 -dimethylguanin-9yl or a functional equivalent thereof,    wherein each V is independently an O, S, NH, or CH 2  group,    wherein each W is independently selected from the group consisting of —H, —OH, —COOH, —CONH 2 , —CONHR 1 , —CONR 1 R 2 , —NH 2 , —NHR 1 , —NR 1 R 2 , —NHCOR 1 , —SH, SR 1 , —F, —ONH 2 , —ONHR 1 , —ONR 1 R 2 , —NHOH, —NHOR 1 , —NR 2 OH, —NR 2 OR 1 , substituted or unsubstituted C 1 -C 10  straight chain or branched alkyl, substituted or unsubstituted C 2 -C 10  straight chain or branched alkenyl, substituted or unsubstituted C 2 -C 10  straight chain or branched alkynyl, substituted or unsubstituted C 1 -C 10  straight chain or branched alkoxy, substituted or unsubstituted C 2 -C 10  straight chain or branched alkenyloxy, and substituted or unsubstituted C 2 -C 10  straight chain or branched alkynyloxy, wherein the substituents are independently halogen, cyano, amino, carboxy, ester, ether, carboxamide, hydroxy, or mercapto, wherein R 1  and R 2  are, independently, substituted or unsubstituted alkyl, alkenyl, or alkynyl groups, where the substituents are independently halogen, cyano, amino, carboxy, ester, ether, carboxamide, hydroxy, or mercapto,    wherein D and E are residues which together form a phosphodiester or phosphorothioate diester bond between adjacent nucleosides or nucleoside analogues or together form an analogue of an internucleosidic bond,    wherein in X 15.1 , B is hypoxanthin-9-yl or a functional equivalent thereof,    wherein in X 12 , B is independently guanin-9-yl, hypoxanthin-9-yl or 7-deazaguanin-9-yl;    wherein in X 13  and X 14 , B is independently adenin-9-yl, 2,6-diaminopurin-9-yl, purin-9-yl or 7-deazaadenin-9-yl;    wherein in X^  12 , B is independently adenin-9-yl, cytosin-1-yl, guanin-9-yl, uracil-1-yl, uracil-5-yl, hypoxanthin-9-yl, thymin-1-yl, 5-methylcytosin-1-yl, 2,6-diaminopurin-9-yl, purin-9-yl, 7-deazaadenin-9-yl, 7-deazaguanin-9-yl, 5-propynylcytosin-1-yl, 5-propynyluracil-1-yl, isoguanin-9-yl, 2-aminopurin-9-yl, 6-methyluracil-1-yl, 4-thiouracil-1-yl, 2-pyrimidone-1-yl, quinazoline-2,4-dione-1-yl, xanthin-9-yl, N 2 -dimethylguanin-9-yl or a functional equivalent thereof.    
     
     
         2 . The composition of  claim 1  wherein the RNA substrate comprises 
       5′-Z 3 -C 16.1 -X 17 -S-Z 4 -Z 1 ′-3′, wherein Z 1 ′ and Z 3 ′ interact with Z 1  and Z 3 , wherein C 16.1  is cytidine, wherein X 17  is adenosine, guanosine, cytidine, or uridine, wherein S comprises a sequence capable of forming a hairpin structure,    wherein cleavage occurs 3′ of X 17 ,    wherein Z 4  consists of   5′-X 3 X 4 X 5 X 6 X 7 X 8 X 9 -3′, or5′-X 3 X 4 X 5 X 6 X 7 X 8 X 9 ^ -3′   wherein X 5  and X 8  are guanosine, wherein X 6  and X 9  are adenosine, wherein X 4  is uridine, wherein X 3  is cytidine, and wherein X 7  and X 9^  are independently adenosine, guanosine, cytidine, or uridine.    
     
     
         3 . The composition of  claim 2  wherein X 17  is adenosine, cytidine, or uridine.  
     
     
         4 . The composition of  claim 1 , wherein Z 1  and Z 3  do not contain any pyrimidines that are ribonucleotides.  
     
     
         5 . The composition of  claim 1 , wherein Z 1  and Z 3  do not contain any ribonucleotides.  
     
     
         6 . The composition of  claim 1 , wherein Z 1  and Z3 are comprised of nucleotides, nucleotide analogues, or both, wherein the nucleotides and nucleotide analogues each have the structure  
       
         
           
           
               
               
           
         
         wherein each B is independently adenin-9-yl, cytosin-1-yl, guanin-9-yl, uracil-1-yl, uracil-5-yl, hypoxanthin-9-yl, thymin-1-yl, 5-methylcytosin-1-yl; 2,6-diaminopurin-9-yl, purin-9-yl, 7-deazaadenin-9-yl, 7-deazaguanin-9-yl, 5-propynylcytosin-1-yl, 5-propynyluracil-1-yl, isoguanin-9-yl, 2-aminopurin-9-yl, 6-methyluracil-1-yl, 4-thiouracil-1-yl, 2-pyrimidone-1-yl, quinazoline-2,4-dione-1-yl, xanthin-9-yl, N 2 -dimethylguanin-9-yl or a functional equivalent thereof,  
         wherein each V is independently an 0, S, NH, or CH 2  group,  
         wherein each W is independently selected from the group consisting of substituted or unsubstituted C 1 -C 10  straight chain or branched alkyl, C 2 -C 10  straight chain or branched alkenyl, C 2 -C 10  straight chain or branched alkynyl, C 1 -C 10  straight chain or branched alkoxy, C 2 -C 10  straight chain or branched alkenyloxy, and C 2 -C 10  straight chain or branched alkynyloxy, wherein D and E are residues which together form a phosphodiester or phosphorothioate diester bond between adjacent nucleosides or nucleoside analogues or together form an analogue of an internucleosidic bond.  
       
     
     
         7 . The composition of  claim 1 , wherein Z 1  and Z 3  each independently contain from 3 to 40 nucleotides, nucleotide analogues, or a combination.  
     
     
         8 . The composition of  claim 1 , wherein Z 2  contains one or several nucleotide analogues wherein each W is independently selected from the group consisting of C 1 -C 5  straight chain or branched alkyl, C 2 -C 5  straight chain or branched alkenyl, C 2 -C 5  straight chain or branched alkynyl, C 1 -C 5  straight chain or branched alkoxy, C 2 -C 5  straight chain or branched alkenyloxy, and C 2 -C 5  straight chain or branched C 2 -C 5  alkynyloxy.  
     
     
         9 . The composition of  claim 1 , wherein the free 3′ end is protected against exonuclease degradation.  
     
     
         10 . The composition of  claim 1 , wherein in X^  12  W is independently NH 2 , OH-substituted C 2 -C 4  alkyl, OH-substituted C 2 -C 4  alkenyl, OH-substituted C 1 -C 4  alkoxy or OH-substituted C 2 -C 4  alkenyloxy.  
     
     
         11 . The composition of  claim 10 , wherein in X^  12  W is independently NH 2 , methoxy, 2-hydroxyethoxy, allyloxy or allyl.  
     
     
         12 . The composition of  claim 1 , wherein X 12  is a ribonucleotide.  
     
     
         13 . The composition of  claim 1 , wherein X 13  and X 14 , or a combination is a nucleotide analogue in which each W is independently C 1 -C 4  alkyl, C 2 -C 4  alkenyl, C 1 -C 4  alkoxy, C 2 -C 4  alkenyloxy, OH-substituted C 1 -C 4  alkyl, OH-substituted C 2 -C 4  alkenyl, OH-substituted C 1 -C 4  alkoxy, or OH-substituted C 2 -C 4  alkenyloxy.  
     
     
         14 . The composition of  claim 13 , wherein X 13  and X 14 , or a combination is a nucleotide analogue in which each W is independently methoxy, 2-hydroxyethoxy or allyloxy.  
     
     
         15 . The composition of  claim 1 , wherein the RNA substrate is selected from the group consisting of human dopamine D2 receptor mRNA, human brain cholecystokinin receptor mRNA, human serotonin 5-HT3 receptor mRNA, human alpha-2-macroglobulin receptor RNA, human tyrosine kinase-type receptor (HER2) mRNA, human interleukin 2 receptor beta chain mRNA, human MAD-3 mRNA, human bcl-1 mRNA, human bcl-2 mRNA, human cyclin F mRNA, human cyclin G1 mRNA, human bleomycin hydrolase mRNA, human acute myeloid leukemia 1 oncogene mRNA, human polycystic kidney disease 1 protein (PKD1) mRNA, transcripts of the bovine viral diarrhea virus, transcripts of the foot and mouth disease virus 3D gene and transcripts of the Epstein-Barr virus.  
     
     
         16 . The composition of  claim 1 , wherein X 15.1  is a ribonucleotide.  
     
     
         17 . A method for the specific cleavage of an RNA substrate, the method comprising bringing into contact the composition of  claim 1  and the RNA substrate.  
     
     
         18 . The method of  claim 17 , wherein the RNA substrate is selected from the group consisting of human dopamine D2 receptor mRNA, human brain cholecystokinin receptor mRNA, human serotonin 5-HT3 receptor mRNA, human alpha-2-macroglobulin receptor RNA, human tyrosine kinase-type receptor (HER2) mRNA, human interleukin 2 receptor beta chain mRNA, -human MAD-3 mRNA, human bcl-1 mRNA, human bcl-2 mRNA, human cyclin F mRNA, human cyclin G1 mRNA, human bleomycin hydrolase mRNA, human acute myeloid leukemia 1 oncogene mRNA, human polycystic kidney disease 1 protein (PKD1) mRNA, transcripts of the bovine viral diarrhea virus, transcripts of the foot and mouth disease virus 3D gene and transcripts of the Epstein-Barr virus.  
     
     
         19 . A method of identifying the function of a gene, the method comprising 
 bringing into contact the composition of  claim 1  and a cell containing the gene, wherein the composition reduces expression of the gene, and    observing any change in the cell.    
     
     
         20 . A method of treating a disease that is associated with an RNA molecule, the method comprising administering to a subject having the disease the composition of  claim 1 , wherein the RNA substrate is the RNA molecule associated with the disease.  
     
     
         21 . The method of claim  20  wherein the RNA molecule is an RNA molecule that is overexpressed.