Compositions and methods for optimizing cleavage of rna by rnase h

Abstract

The present invention provides compositions and methods for the optimization of cleavage of RNA species by RNase H. In some embodiments, the invention provides oligonucleotides that possess two or more regions of differing conformation, and at least one transitional nucleobase positioned between the regions that is capable of modulating transfer of the helical conformation characteristic of the region bound to the 3′ hydroxy thereof, to the region bound to the 5′ hydroxyl thereof.

What is claimed is: 
     
         1 .- 32 . (canceled) 
     
     
         33 . A method of modulating the concentration of a targeted RNA molecule in a eukaryotic cell comprising the step of contacting said cell with an oligonucleotide having
 a) a first region of nucleotides of one conformation which, when bound to said targeted RNA, forms a substrate for cleavage by an RNase;   b) a second region of nucleotides having a different conformation which, when bound to said targeted RNA molecule does not form a substrate for cleavage by an RNase, and   c) a transition moiety which modulates the transmission of the conformation of said second region into said first region.   
     
     
         34 . The method of  claim 33 , wherein the first region comprises deoxynucleotides. 
     
     
         35 . The method of  claim 34 , wherein the second region comprises 2′-O-alkoxyalkyl ribonucleotides. 
     
     
         36 . The method of  claim 35 , wherein the 2′-O-alkoxyalkyl ribonucleotides are 2′-O-methoxyethyl ribonucleotides. 
     
     
         37 . The method of  claim 36 , wherein the internucleotide linkages in the first or second regions are phosphorothioates. 
     
     
         38 . The method of  claim 36 , further comprising a third region of nucleotides having a conformation different than the conformation of said first region, said third region is positioned 3′ to said first region and when bound to said targeted RNA molecule does not form a substrate for cleavage by an RNase. 
     
     
         39 . The method of  claim 38 , wherein said third region has the same conformation as the second region. 
     
     
         40 . The method of  claim 39 , wherein the transition moiety is interspersed at the junction between the first and second region of the oligonucleotide. 
     
     
         41 . The method of  claim 39 , wherein the transition moiety is interspersed at the junction between the first and third region of the oligonucleotide. 
     
     
         42 . The method of  claim 39 , wherein the transition moiety is positioned between said first and said second regions. 
     
     
         43 . The method of  claim 40 , wherein the transition moiety is a region of 2-10 nucleotides comprising at least one:
 i. modified nucleotide, or   ii. flexible hydrocarbon internucleotide linker.   
     
     
         44 . The method of  claim 43 , wherein the modified nucleotide is selected from a modified base nucleotide, a modified sugar nucleotide, a modified or unmodified sugar abasic nucleotide, a THF nucleotide, or an acyclic nucleotide. 
     
     
         45 . The method of  claim 43 , wherein the flexible hydrocarbon internucleotide linker is C 3 -C 6  alkylene. 
     
     
         46 . The method of  claim 44 , wherein the modified base nucleotide comprises a modified base moiety which does not form hydrogen bonds with the bases of the targeted RNA molecule and can optionally it stack with adjacent bases. 
     
     
         47 . The method of  claim 46 , wherein the modified base moiety is a universal base, a promiscuous base, a size expanded base or a fluorinated base. 
     
     
         48 . The method of  claim 47 , wherein the modified base moiety is tetrafluoroindolyl. 
     
     
         49 . The method of  claim 44 , wherein the modified sugar nucleotide is a 2′-ara-modified nucleotide. 
     
     
         50 . The method of  claim 49 , wherein the 2′-ara-modified nucleotide is a 2′-ara-fluoro nucleotide. 
     
     
         51 . The method of  claim 44 , wherein the modified sugar moiety is an acyclic sugar analog. 
     
     
         52 . The method of  claim 41 , wherein the modified nucleotide is selected from a modified base nucleotide, a modified sugar nucleotide, a modified or unmodified sugar abasic nucleotide, a THF nucleotide, or an acyclic nucleotide.