Reduced size self-delivering rnai compounds

Abstract

The present invention relates to RNAi constructs with minimal double-stranded regions, and their use in gene silencing. RNAi constructs associated with the invention include a double stranded region of 8-14 nucleotides and a variety of chemical modifications, and are highly effective in gene silencing.

What is claimed is: 
     
         1 .- 91 . (canceled) 
     
     
         92 . An isolated double stranded nucleic acid molecule comprising a guide strand and a passenger strand,
 wherein the isolated double stranded nucleic acid molecule includes a double stranded region and a single stranded region, wherein the single stranded region is at the 3′ end of the guide strand and is 4-12 nucleotides long, wherein the single stranded region contains 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 phosphorothioate modifications, and wherein at least 40% of the nucleotides of the isolated double stranded nucleic acid molecule are modified.   
     
     
         93 . The isolated double stranded nucleic acid molecule of  claim 92 , wherein the guide strand has a minimal length of 16 nucleotides. 
     
     
         94 . The isolated double stranded nucleic acid molecule of  claim 92 , wherein the single stranded region is at least 6 or at least 7 nucleotides long. 
     
     
         95 . The isolated double stranded nucleic acid molecule of  claim 92 , wherein each nucleotide within the single stranded region has a phosphorothioate modification. 
     
     
         96 . The isolated double stranded nucleic acid molecule of  claim 92 , wherein at least one of the nucleotides of the isolated double stranded molecule that is modified comprises a 2′ O-methyl or a 2′-fluoro modification and/or wherein at least one of the nucleotides of the isolated double stranded nucleic acid molecule that is modified comprises a hydrophobic modification. 
     
     
         97 . The isolated double stranded nucleic acid molecule of  claim 92 , wherein a hydrophobic conjugate is attached to the double stranded nucleic acid molecule. 
     
     
         98 . The isolated double stranded nucleic acid molecule of  claim 97 , wherein the hydrophobic conjugate is attached at the 3′ end of the double stranded nucleic acid molecule. 
     
     
         99 . The isolated double stranded nucleic acid molecule of  claim 93 , wherein the guide strand is 16-28 nucleotides long. 
     
     
         100 . The isolated double stranded nucleic acid molecule of  claim 92 , wherein the passenger strand is 8-16 nucleotides long 
     
     
         101 . An isolated asymmetric nucleic acid molecule comprising:
 a first polynucleotide wherein the first polynucleotide is complementary to a second polynucleotide and a target gene; and   a second polynucleotide, wherein the second polynucleotide is at least 6 nucleotides shorter than the first polynucleotide,   wherein the first polynucleotide includes a single stranded region of 6, 7, 8, 9, 10, 11 or 12 nucleotides, wherein the single stranded region of the first polynucleotide contains 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 phosphorothioate modifications, wherein the asymmetric nucleic acid molecule also includes a double stranded region, and wherein at least 50% of C and U nucleotides in the double stranded region are 2′ O-methyl modified or 2′-fluoro modified.   
     
     
         102 . The isolated double stranded nucleic acid molecule of  claim 101 , wherein the single stranded region is 6 or 7 nucleotides long and/or wherein each nucleotide within the single stranded region has a phosphorothioate modification. 
     
     
         103 . An isolated double stranded nucleic acid molecule comprising:
 a guide strand of 17-21 nucleotides in length that has complementarity to a target gene, and a passenger strand of 8-16 nucleotides in length,   wherein the guide strand and the passenger strand form the double stranded nucleic acid molecule having a double stranded region and a single stranded region, wherein the guide strand has a 3′ single stranded region of 4-12 nucleotides in length, wherein the single stranded region comprises 2-12 phosphorothioate modifications, wherein at least 40% of the nucleotides of the double stranded nucleic acid are modified, wherein if the passenger strand is 16 nucleotides long, then one end of the double-stranded nucleic acid molecule is blunt, and wherein at least one modification is a hydrophobic base modification and/or the double stranded nucleic acid molecule is linked to a hydrophobic conjugate.   
     
     
         104 . The isolated double stranded nucleic acid molecule of  claim 103 , wherein the hydrophobic base modification comprises a hydrophobic modification of a pyrimidine base, optionally at position 4 or 5, optionally, wherein the hydrophobic base modification is selected from the group consisting of a phenyl, 4-pyridyl, 2-pyridyl, indolyl, isobutyl, tryptophanyl (C 8 H 6 N)CH 2 CH(NH 2 )CO), methyl, butyl, aminobenzyl, and naphthyl modification of a uridine or cytidine. 
     
     
         105 . The isolated double stranded nucleic acid molecule of  claim 103 , wherein the hydrophobic conjugate is a small molecule, optionally wherein the small molecule is a sterol-type molecule, optionally wherein the sterol-type molecule is cholesterol. 
     
     
         106 . The isolated double stranded nucleic acid molecule of  claim 103 , wherein the hydrophobic conjugate is attached to the double stranded nucleic acid molecule through a linker, optionally wherein the linker is a TEG linker. 
     
     
         107 . A method for inhibiting the expression of a target gene in a mammalian cell, comprising contacting the mammalian cell with the isolated double stranded nucleic acid molecule of  claim 92 . 
     
     
         108 . The method of  claim 92 , wherein one end of the isolated double stranded nucleic acid molecule is blunt. 
     
     
         109 . The isolated double stranded nucleic acid molecule of  claim 101 , wherein the guide strand has a minimal length of 16 nucleotides. 
     
     
         110 . The isolated double stranded nucleic acid molecule of  claim 101 , wherein one end of the isolated double stranded nucleic acid molecule is blunt.