Modified polynucleotides for use in rna interference

Abstract

Methods and compositions for performing RNA interference comprising a wide variety of stabilized siRNAs suitable for use in serum-containing media and for in vivo applications, such as therapeutic applications, are provided. These siRNAs permit effective and efficient applications of RNA interference to applications such as diagnostics and therapeutics through the use of one or more modifications including orthoesters, terminal conjugates, modified linkages and 2′ modified nucleotides. Uniquely modified siRNAs have been developed that reduces off-target effects incurred in gene-silencing. The modifications include phosphorylation of the first 5′ terminal antisense nucleotide; 2′ carbon modifications of the first and second or first, second, and third 5′ terminal antisense nucleotides; and optionally 2′ carbon modifications of the first and second or first, second, and third 5′ terminal sense nucleotide. Control and exaequo molecules are also provided. siRNA molecules and related control, trackability and exaequo agents with specific stability modifications were developed.

1 . A double stranded polyribonucleotide comprising:
 (a) a sense strand comprising:   a first nucleotide closest to the 5′ end of the sense strand, said first nucleotide having a first 2′-O-alkyl modification; and   a second nucleotide next closest to the 5′ end of the sense strand, said second nucleotide having a second 2′-O-alkyl modification;   (b) an antisense strand comprising a phosphate group on the 5′ end, wherein all pyrimidine nucleotides on said antisense stand comprise a 2′ halogen modification;   wherein the sense strand and the antisense strand are capable of forming a duplex of between 18 and 30 base pairs; wherein said antisense strand is at least 80% complementary to said sense strand; and wherein said antisense strand is at least 80% complementary to a target nucleic acid.   
     
     
         2 . The double stranded polyribonucleotide of  claim 1 , wherein the sense strand and antisense strand are capable of forming a duplex of between 19 and 25 base pairs. 
     
     
         3 . The double stranded polyribonucleotide of  claim 1 , wherein said target nucleic acid is a mRNA. 
     
     
         4 . The double stranded polyribonucleotide of  claim 1  wherein said first 2′-O-alkyl modification and said second 2′-O-alkyl modification are each independently selected from the group consisting of a 2′-O-methyl modification, a 2′-O-ethyl modification, a 2′-O-propyl modification, a 2′-O-isopropyl modification, a 2′-O-butyl modification, and a 2′-O-isobutyl modification. 
     
     
         5 . The double stranded polyribonucleotide of  claim 1  wherein said first 2′-O-alkyl modification and said second 2′-O-alkyl modification are both 2′-O-methyl modifications. 
     
     
         6 . The double stranded polyribonucleotide of  claim 1  wherein said 2′ halogen modification is a 2′-F modification. 
     
     
         7 . The double stranded polyribonucleotide of  claim 1 , further comprising a conjugate. 
     
     
         8 . The double stranded polyribonucleotide of  claim 7 , wherein said conjugate is a detectable label. 
     
     
         9 . The double stranded polyribonucleotide of  claim 8 , wherein said detectable label is attached to said first 5′ terminal sense nucleotide. 
     
     
         10 . The double stranded polyribonucleotide of  claim 8 , wherein said detectable label is a fluorescent dye. 
     
     
         11 . The double stranded polyribonucleotide of  claim 7  wherein said conjugate is cholesterol. 
     
     
         12 . The double stranded polyribonucleotide of  claim 11  wherein said cholesterol is attached to said sense strand. 
     
     
         13 . The double stranded polyribonucleotide of  claim 12  wherein said cholesterol is attached to the 3′ end of said sense strand. 
     
     
         14 . The double stranded polyribonucleotide of  claim 1 , further comprising at least one phosphorothioate internucleotide linkage. 
     
     
         15 . The double stranded polyribonucleotide of  claim 1 , further comprising:
 a 3′ overhang on the 3′ end of the antisense strand comprising a first overhang nucleotide and a second overhang nucleotide; and   a blunt end at the 5′ terminus of the antisense strand and the 3′ terminus of the sense strand.   
     
     
         16 . The double stranded polyribonucleotide of  claim 15  wherein the first overhang nucleotide is attached to the double stranded polyribonucleotide by a phosphorothioate internucleotide linkage, and the first overhang nucleotide and the second overhang nucleotide are attached to each other by a phosphorothioate internucleotide linkage. 
     
     
         17 . A method of gene silencing comprising introducing a double stranded polyribonucleotide into a cell that is expressing or is capable of expressing a target nucleic acid, wherein said double stranded polyribonucleotide comprises:
 (a) a sense strand comprising:   a first nucleotide closest to the 5′ end of the sense strand, said first nucleotide having a first 2′-O-alkyl modification; and   a second nucleotide next closest to the 5′ end of the sense strand, said second nucleotide having a second 2′-O-alkyl modification;   (b) an antisense strand comprising a phosphate group on the 5′ end, wherein all pyrimidine nucleotides on said antisense stand comprise a 2′ halogen modification;   wherein the sense strand and the antisense strand are capable of forming a duplex of between 18 and 30 base pairs; wherein said antisense strand is at least 80% complementary to said sense strand; and wherein said antisense strand is at least 80% complementary to said target nucleic acid.