Nicked or gapped nucleic acid molecules and uses thereof

Abstract

The present disclosure provides meroduplex (nicked or gapped) ribonucleic acid molecules (mdRNA) that decreases or silences target gene expression. An mdRNA of this disclosure comprises at least three strands that combine to form at least two non-overlapping double-stranded regions separated by a nick or gap wherein one strand is complementary to a target gene RNA. In addition, the meroduplex may have one or more modifications or substitutions, such as nucleotide base, sugar, terminal cap structure, internucleotide linkage, or any combination of such modifications. Also provided are methods of decreasing expression of a target gene in a cell or in a subject to treat a disease related to altered expression of a target gene.

1 .- 64 . (canceled) 
     
     
         65 . A ribonucleic acid, comprising:
 (a) an A strand having a length from 15 to 30 nucleotides,   (b) an S1 strand having a length from 5 to 25 nucleotides, and   (c) an S2 strand having a length from 5 to 25 nucleotides;   i. wherein said S1 strand and said A strand anneal to form a first double-stranded region having a length of from 5 base pairs to 15 base pairs,   ii. wherein said S2 strand and said A strand anneal to form a second double-stranded region having a length of from 3 base pairs to 25 base pairs,   iii. wherein the sum of the lengths of said first double-stranded region and said second double-stranded region is from 19 base pairs to 30 base pairs,   iv. wherein said first double-stranded region is separated from said second double stranded region by a nick between said S1 strand and said S2 strand, and   v. wherein said ribonucleic acid has 0, 1, or 2 overhangs.   
     
     
         66 . The ribonucleic acid of  claim 65  wherein said A strand has a length from 18 to 25 nucleotides. 
     
     
         67 . The ribonucleic acid of  claim 65  wherein said A strand has a length of from 25 to 30 nucleotides. 
     
     
         68 . The ribonucleic acid of  claim 65  wherein one or both of said S1 strand and said S2 strand have a length of from 5 to 13 nucleotides. 
     
     
         69 . The ribonucleic acid of  claim 65  wherein one or both of said S1 strand and said S2 strand have a length of from 15 to 25 nucleotides. 
     
     
         70 . The ribonucleic acid of  claim 65  wherein the sum of the lengths of said S1 strand and said S2 strand is from 18 nucleotides to 25 nucleotides. 
     
     
         71 . The ribonucleic acid of  claim 65  wherein one or both of said double-stranded regions have a length of from 5 base pairs to 13 base pairs. 
     
     
         72 . The ribonucleic acid of  claim 65 , comprising one or two overhangs wherein each overhang independently have a length of from 1 nucleotides to 5 nucleotides. 
     
     
         73 . The ribonucleic acid of  claim 72  wherein one or both of said overhangs are 3′ overhangs. 
     
     
         74 . The ribonucleic acid of  claim 65  wherein one or more nucleotides have a modified sugar. 
     
     
         75 . The ribonucleic acid of  claim 65  wherein one or more nucleotides have a 2′ sugar bridge. 
     
     
         76 . The ribonucleic acid of  claim 65  wherein one or more nucleotides have a modified internucleoside linkage. 
     
     
         77 . The ribonucleic acid of  claim 76  wherein one or more of said modified internucleoside linkages are phosphorothioate internucleoside linkages. 
     
     
         78 . The ribonucleic acid of  claim 65  wherein one or more nucleotides are locked nucleic acid nucleotides. 
     
     
         79 . The ribonucleic acid of  claim 65  wherein one or more nucleotides have a 2′-sugar substitution. 
     
     
         80 . The ribonucleic acid of  claim 65  wherein one or more nucleotides have a G clamp. 
     
     
         81 . The ribonucleic acid of  claim 65  wherein one or more nucleotides have a terminal cap substituent. 
     
     
         82 . The ribonucleic acid of  claim 65  wherein one or more nucleotides have a 2′-methoxy modification or a fluoro modification. 
     
     
         83 . The ribonucleic acid of  claim 65  wherein one or more pyrimidine of said ribonucleic acid are pyrimidine nucleosides according to Formula I or Formula II: 
       
         
           
           
               
               
           
         
         wherein: 
         R 1  and R 2  are each independently a —H, —OH, —OCH 3 , —OCH 2 , OCH 2 CH 3 , 
         —OCH 2 CH 2 OCH 3 , halogen, substituted or unsubstituted C 1 -C 10  alkyl, alkoxy, alkoxyalkyl, hydroxyalkyl, carboxyalkyl, alkylsulfonylamino, aminoalkyl, dialkylamino, alkylaminoalkyl, dialkylaminoalkyl, haloalkyl, trifluoromethyl, cycloalkyl, (cycloalkyl)alkyl, substituted or unsubstituted C 2 -C 10  alkenyl, substituted or unsubstituted —O-allyl, —O—CH 2 CH═CH 2 , —O—CH═CHCH 3 , substituted or unsubstituted C 2 -C 10  alkynyl, carbamoyl, carbamyl, carboxy, carbonylamino, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, —NH 2 , —NO 2 , —C≡N, or heterocyclo group, 
         R 3  and R 4  are each independently a hydroxyl, a protected hydroxyl, a phosphate, or an internucleoside linking group, and
 R 5  and R 8  are independently O or S. 
 
       
     
     
         84 . The ribonucleic acid of  claim 83  wherein one or more pyrimidines nucleosides of said ribonucleic acid are pyrimidine nucleosides according to Formula I wherein R 1  is methyl and R 2  is —OH. 
     
     
         85 . The ribonucleic acid of  claim 83  wherein one or more pyrimidines nucleosides of said ribonucleic acid are pyrimidine nucleosides according to Formula I wherein R 2  is —OCH 3  or fluoro. 
     
     
         86 . A composition comprising the ribonucleic acid of  claim 65  and a liposome, a hydrogel, a cyclodextrin, a biodegradable nanocapsule, a bioadhesive microsphere, or a proteinaceous vector.