Conjugates For Use In Hepatocyte Free Uptake Assays

Abstract

The present invention provides methods of identifying oligomeric compounds, such as siRNA and double-stranded RNA compounds, having bioactivity in vivo, and kits.

1 - 33 . (canceled) 
     
     
         34 . A method for reducing the level of target mRNA in a cell comprising:
 contacting the cell with a single-stranded sense oligonucleotide consisting of 10 to 40 linked nucleosides; and   contacting the cell with a single-stranded antisense oligonucleotide consisting of 10 to 40 linked nucleosides at least one hour after contacting the cell with the sense oligonucleotide;
 wherein the antisense oligonucleotide is complementary to the target mRNA, the sense and antisense oligonucleotides are fully complementary to each other, the contacting occurs in the absence of a transfection reagent, and the sense oligonucleotide is a symmetric gapped oligomeric compound, 
   
       thereby reducing the level of target mRNA in the cell. 
     
     
         35 . The method of  claim 34  wherein the cell is a mammalian tissue-derived cell. 
     
     
         36 . The method of  claim 34  wherein the cell is a rodent primary hepatocyte or a primate primary hepatocyte. 
     
     
         37 . The method of  claim 34  wherein the cell is contacted with the antisense oligonucleotide at least two hours after the cell is contacted with the sense oligonucleotide. 
     
     
         38 . The method of  claim 34  wherein each of the nucleosides of the antisense oligonucleotide comprises a β-D-ribofuranose sugar group. 
     
     
         39 . The method of  claim 34  wherein at least one nucleoside of the antisense oligonucleotide comprises a 2′-substituent selected from the group consisting of —F, —O—CH 2 CH 2 —O—CH 3 , —O—CH 3 , —O—CH 2 —CH═CH 2  or —O—CH 2 —CH—CH 2 —NH(R j ), where R j  is H or C 1 -C 10  alkyl. 
     
     
         40 . The method of  claim 34  wherein each nucleoside of the sense oligonucleotide comprises a 2′ sugar modification that conveys 3′-endo sugar conformational geometry. 
     
     
         41 . The method of  claim 34  wherein the 3′-terminus of at least one of the sense and antisense oligonucleotides, independently, comprises a stabilizing or conjugate group. 
     
     
         42 . The method of  claim 41  wherein the stabilizing group is a capping group or a dTdT dimer. 
     
     
         43 . The method of  claim 34  wherein at least one of the sense and antisense oligonucleotides comprises a 5′-phosphate group. 
     
     
         44 . The method of  claim 34  wherein the 5′-terminus of at least one of the sense and antisense oligonucleotides, independently, comprises a stabilizing or conjugate group. 
     
     
         45 . The method of  claim 44  wherein the stabilizing group is a capping group. 
     
     
         46 . The method of  claim 34  wherein at least one of the sense and antisense oligonucleotides comprises at least one terminal cap moiety. 
     
     
         47 . The method of  claim 46  wherein the terminal cap moiety is attached to one or both of the 3′-terminal and 5′-terminal ends of the at least one oligonucleotide. 
     
     
         48 . The method of  claim 46  wherein the terminal cap moiety is an inverted deoxy abasic moiety. 
     
     
         49 . The method of  claim 34  wherein each of the internucleoside linking groups of at least one of the sense oligonucleotide and the antisense oligonucleotide is, independently, a phosphorothioate. 
     
     
         50 . The method of  claim 34  wherein the sense and antisense oligonucleotides are capable of hybridizing to form a duplex having 3′-dTdT overhangs. 
     
     
         51 . The method of  claim 34  wherein the sense and antisense oligonucleotides are capable of hybridizing to form a duplex having blunt ends.