Double-stranded oligonucleotides

Abstract

Antisense sequences, including duplex RNAi compositions, which possess improved properties over those taught in the prior art are disclosed. The invention provides optimized antisense oligomer compositions and method for making and using the both in in vitro systems and therapeutically. The invention also provides methods of making and using the improved antisense oligomer compositions.

1 . A double-stranded oligonucleotide composition having the structure:  
       
         
           
           
               
               
           
         
       
       wherein 
 (1) N is a nucleomonomer in complementary oligonucleotide strands of equal length and where the sequence of Ns corresponds to a target gene sequence and  
 (2) X and Y are each independently selected from a group consisting of nothing; from about 1 to about 20 nucleotides of 5′ overhang; from about I to about 20 nucleotides of 3′ overhang; and a loop structure consisting from about 4 to about 20 nucleomonomers, where the nucleomonomers are selected from the group consisting of G and A.  
 
     
     
         2 . A double-stranded oligonucleotide composition having the structure:  
       
         
           
           
               
               
           
         
       
       wherein 
 (1) oligoA is an oligonucleotide of a number of nucleomonomers;  
 (2) oligoB is an oligonucleotide that has the same number of nucleomonomers as oligoA and that is complementary to oligoA;  
 (3) either oligoA or oligoB corresponds to a target gene sequence;  
 (4) X is selected from a group consisting of (a) nothing; (b) an oligonucleotide of about 1 to about 20 nucleotides covalently bonded to the 5′ end of oligoA and constituting a 5′ overhang; (c) an oligonucleotide of about 1 to about 20 nucleotides covalently bonded to the 3′ end of oligoB and constituting a 3′ overhang; (d) and an oligonucleotide of about 4 to about 20 nucleomonomers covalently bonded to the 3′ end of oligoB and the 5′ end of oligoA and constituting a loop structure, where the nucleomonomers are selected from the group consisting of G and A and  
 (5) Y is selected from a group consisting of (a) nothing; (b) an oligonucleotide of about 1 to about 20 nucleotides covalently bonded to the 5′ end of oligoB and constituting a 5′ overhang; (c) an oligonucleotide of about 1 to about 20 nucleotides covalently bonded to the 3′ end of oligoA and constituting a 3′ overhang; (d) and an oligonucleotide of about 4 to about 20 nucleomonomers covalently bonded to the 3′ end of oligoA and the 5′ end of oligoB and constituting a loop structure, where the nucleomonomers are selected from the group consisting of G and A.  
 
     
     
         3 . The composition of  claim 1 , wherein the number of nucleomonomers in each strand of the duplex is between about 12 and about 40.  
     
     
         4 . The composition of  claim 2 , wherein the number of nucleomonomers in each strand oligoA and oligoB is between about 12 and about 40.  
     
     
         5 . The composition of  claim 1 , wherein the number of nucleomonomers in each strand of the duplex is about 27.  
     
     
         6 . The composition of  claim 2 , wherein the number of nucleomonomers in each strand of oligoA and oligoB is about 27.  
     
     
         7 . The composition of  claim 1 , wherein X is a sequence of about 4 to about 20 nucleomonomers which form a loop, wherein the nucleomonomers are selected from the group consisting of G and A.  
     
     
         8 . The composition of  claim 2 , wherein X or Y is a sequence of about 4 to about 20 nucleomonomers that forms a loop, wherein the nucleomonomers are selected from the group consisting of G and A.  
     
     
         9 . The composition of  claim 8 , wherein two of the adjacent Ns are unlinked.  
     
     
         10 . The composition of  claim 8 , wherein the nucleotide sequence of the loop is GAAA.  
     
     
         11 . A double-stranded oligonucleotide composition having the structure: 
         5′-(Z) 2-8 -(N) 15-40 (M) 2-8 -3′ 3′-(Z) 2-8 -(N) 15-40 (M) 2-8 -5′ 
       wherein 
 (1) each of N, Z, and M is independently a nucleomonomer;  
 (2) both of the sequences of Ns are complementary oligonucleotide strands of equal length having between about 15 and about 40 nucleomonomers;  
 (3) at least one of the sequences of Ns, optionally with some or all of the flanking Ms or Zs, corresponds to a target gene sequence;  
 (4) both of the sequences of Zs are complementary oligonucleotide strands of between about 2 and about 8 nucleomonomers in length; and  
 (5) both of the sequences of Ms are complementary oligonucleotide strands of between about 2 and about 8 nucleomonomers in length.  
 
     
     
         12 . The composition of  claim 11 , wherein each Z and M nucleomonomer is selected from the group consisting of C and G.  
     
     
         13 . The composition of  claim 12  wherein, the sequence of Z or M is CC, GG, CG, GC, CCC, GGG, CGG, GCC, GCG, CGC, CGGG, GCCC, CCCC, GGGG, GCGC, CGCG, GGGC, CCCG, CGGG, GCCC, GGCC, or CCGG.  
     
     
         14 . A double-stranded oligonucleotide composition having the structure:  
       
         
           
           
               
               
           
         
       
       wherein 
 (1) N is a nucleomonomer in complementary oligonucleotide strands of equal length and where the sequence of Ns corresponds to a target gene sequence and  
 (2) X is selected from the group consisting of nothing; 1-20 nucleotides of 5′ overhang; 1-20 nucleotides of 3′ overhang; a loop structure consisting of from about 4 to about 20 nucleomonomers, where the nucleomonomers are selected from the group consisting of G and A, and  
 (3) where M is a nucleomonomer in complementary oligonucleotide strands of between about 2 and about 8 nucleomonomers in length which optionally correspond to the target sequence.  
 
     
     
         15 . A double-stranded oligonucleotide composition having the structure:  
       
         
           
           
               
               
           
         
       
       wherein 
 (1) oligoA is 5′-(N) 15-40 -(M) 2-8 -3′ and oligoB is 5′-(N) 15-40 -(M) 2-8 -3′, wherein each of N and M is independently a nucleomonomer;  
 (2) both of the sequences of Ns are complementary oligonucleotide strands of equal length having between about 15 and 40 nucleomonomers;  
 (3) at least one of the sequences of Ns, optionally with some or all of the flanking Ms, corresponds to a target gene sequence;  
 (4) X is selected from a group consisting of (a) nothing; (b) an oligonucleotide of about 1 to about 20 nucleotides covalently bonded to the 5′ end of oligoA and constituting a 5′ overhang; (c) an oligonucleotide of about 1 to about 20 nucleotides covalently bonded to the 3′ end of oligoB and constituting a 3′ overhang; (d) and an oligonucleotide of about 4 to about 20 nucleomonomers covalently bonded to the 3′ end of oligoB and the 5′ end of oligoA and constituting a loop structure, where the nucleomonomers are selected from the group consisting of G and A; and  
 (5) both of the sequences of Ms are complementary oligonucleotide strands of between about 2 and about 8 nucleomonomers in length.  
 
     
     
         16 . The composition of  claim 15 , wherein M nucleomonomer is selected from the group consisting of C and G.  
     
     
         17 . The composition of  claim 16 , wherein the sequence of M is CC, GG, CG, GC, CCC, GGG, CGG, GCC, GCG, CGC, CGGG, GCCC, CCCC, GGGG, GCGC, CGCG, GGGC, CCCG, CGGG, GCCC, GGCC, or CCGG.  
     
     
         18 . A double-stranded oligonucleotide composition having the structure:  
       
         
           
           
               
               
           
         
       
       wherein 
 (1) N is a nucleomonomer in complementary oligonucleotide strands of equal length and which correspond to a target gene sequence and  
 (2) Y is selected from the group consisting of nothing; 1-20 nucleotides of 5′ overhang; 1-20 nucleotides of 3′ overhang; a loop consisting of a sequence of from about 4 to about 20 nucleomonomers, where the nucleomonomers are all either Gs or A's and  
 (3) where Z is a are nucleomonomer in complementary oligonucleotide strands of between about 2 and about 8 nucleomonomers in length and which comprise a sequence which can optionally correspond to the target sequence.  
 
     
     
         19 . A double-stranded oligonucleotide composition having the structure:  
       
         
           
           
               
               
           
         
       
       wherein 
 (1) oligoA is 5′-(Z) 2-8 -(N) 12-40 -3′ and oligoB is 5′-(Z) 2-8 -(N) 12-40 -3′, wherein each of N and Z is independently a nucleomonomer;  
 (2) both of the sequences of Ns are complementary oligonucleotide strands of equal length having between about 12 and 40 nucleomonomers;  
 3) at least one of the sequences of Ns, optionally with some or all of the flanking Zs, corresponds to a target gene sequence;  
 (4) Y is selected from a group consisting of (a) nothing; (b) an oligonucleotide of about 1 to about 20 nucleotides covalently bonded to the 5′ end of oligoB and constituting a 5′ overhang; (c) an oligonucleotide of about 1 to about 20 nucleotides covalently bonded to the 3′ end of oligoA and constituting a 3′ overhang; (d) and an oligonucleotide of about 4 to about 20 nucleomonomers covalently bonded to the 3′ end of oligoA and the 5′ end of oligoB and constituting a loop structure, where the nucleomonomers are selected from the group consisting of G and A; and  
 (5) both of the sequences of Zs are complementary oligonucleotide strands of between about 2 and about 8 nucleomonomers in length.  
 
     
     
         20 . The composition of  claim 19 , wherein the Z nucleomonomers are selected from the group consisting of C and G.  
     
     
         21 . The composition of  claim 20 , wherein the sequence of Z is CC, GG, CG, GC, CCC, GGG, CGG, GCC, GCG, CGC, CGGG, GCCC, CCCC, GGGG, GCGC, CGCG, GGGC, CCCG, CGGG, GCCC, GGCC, or CCGG.  
     
     
         22 . A method of regulating gene expression in a cell, comprising contacting a cell with the double-stranded duplex oligonucleotide composition of  claim 1 , to thereby regulate gene expression in a cell.  
     
     
         23 . A method of increasing the nuclease resistance of an antisense sequence, comprising forming a double-stranded oligonucleotide composition of  claim 1 , such that a double-stranded duplex is formed, wherein the nuclease resistance of the antisense sequence is increased compared to a control composition.