Gapped 2' modified oligonucleotides

Abstract

Oligonucleotides and other macromolecules are provided that have increased nuclease resistance, substituent groups for increasing binding affinity to complementary strand, and sub-sequences of 2′-deoxy-erythro-pentofuranosyl nucleotides that activate RNase H enzyme. Such oligonucleotides and macromolecules are useful for diagnostics and other research purposes, for modulating protein in organisms, and for the diagnosis, detection and treatment of other conditions susceptible to antisense therapeutics.

1 . An oligonucleotide comprising a sequence of nucleotide units capable of specifically hybridizing to a strand of nucleic acid, wherein: 
 at least one of said nucleotide units is functionalized to increase nuclease resistance of said oligonucleotide;    at least one of said nucleotide units bears a substituent group that increases binding affinity of said oligonucleotide to said strand of nucleic acid; and    a plurality of said nucleotide units have 2′-deoxy-erythro-pentofuranosyl sugar moieties, said 2′-deoxy-erythro-pentofuranosyl nucleotide units being consecutively located in said sequence of nucleotide units.    
     
     
         2 . The oligonucleotide of  claim 1  wherein said substituent group for increasing binding affinity comprises a 2′-substituent group.  
     
     
         3 . The oligonucleotide of  claim 2  wherein said 2′-substituent group is fluoro, C1-C9 alkoxy, C1-C9 aminoalkoxy, allyloxy, imidazolealkoxy and poly(ethylene glycol).  
     
     
         4 . The oligonucleotide of  claim 1  wherein each of said nucleotide units is a phosphorothioate or phosphorodithioate nucleotide.  
     
     
         5 . The oligonucleotide of  claim 1  wherein the 3′ terminal nucleotide unit of said oligonucleotide includes a nuclease resistance modifying group on at least one of the 2′ or the 3′ positions of said nucleotide unit.  
     
     
         6 . The oligonucleotide of  claim 1  wherein: 
 a plurality of said nucleotide units bear substituent groups that increases binding affinity of said oligonucleotide to said strand of nucleic acid, said substituent-bearing nucleotides being divided into a first nucleotide unit sub-sequence and a second nucleotide unit sub-sequence; and    said plurality of 2′-deoxy-erythro-pentofuranosyl nucleotide units is positioned in said sequence of nucleotide units between said first nucleotide unit sub-sequence and said second nucleotide unit sub-sequence.    
     
     
         7 . The oligonucleotide of  claim 1  wherein: 
 a plurality of said nucleotide units bear substituent groups that increase binding affinity of said oligonucleotide to said complementary strand of nucleic acid; and    at least a portion of said substituent-bearing nucleotide are consecutively located at one of the 3′ terminus or the 5′ terminus of said oligonucleotide.    
     
     
         8 . The oligonucleotide of  claim 1  wherein at least five of said nucleotide units have 2′-deoxy-erythro-pentofuranosyl sugar moieties, said at least five 2′-deoxy-erythro-pentofuranosyl nucleotide units being consecutively located in said sequence of nucleotide units.  
     
     
         9 . The oligonucleotide of  claim 1  wherein from one to about eight of said nucleotide units bear a substituent group that increases the binding affinity of said oligonucleotide to said complementary strand, said substituent-bearing nucleotide units being consecutively located in said sequence of nucleotide units.  
     
     
         10 . The oligonucleotide of  claim 1  wherein: 
 from one to about eight of said nucleotide units bear a substituent group for increasing the binding affinity of said oligonucleotide to said complementary strand, said substituent-bearing nucleotide units being consecutively located in said sequence of nucleotide units; and    at least five of said nucleotide units have 2′-deoxy-erythro-pentofuranosyl sugar moieties, said at least five 2′-deoxy-erythro-pentofuranosyl nucleotide units being consecutively located in said sequence of nucleotide units.    
     
     
         11 . An oligonucleotide comprising a sequence of phosphorothioate nucleotides capable of specifically hybridizing to a strand of nucleic acid, wherein: 
 a plurality of said nucleotides bear a substituent group that increases binding affinity of said oligonucleotide to said strand of nucleic acid; and    a plurality of said nucleotides have 2′-deoxy-erythro-pentofuranosyl sugar moieties.    
     
     
         12 . The oligonucleotide of  claim 11  wherein said substituent group for increasing binding affinity comprises a 2′-substituent group.  
     
     
         13 . The oligonucleotide of  claim 12  wherein said 2′-substituent group is fluoro, C1-C9 alkoxy, C1-C9 aminoalkoxy or allyloxy.  
     
     
         14 . The oligonucleotide of  claim 12  including: a further plurality of said nucleotides bearing 2′-substituent groups; 
 said 2′-deoxy-erythro-pentofuranosyl nucleotides being positioned in said oligonucleotide between groups of nucleotides having said 2′-substituent group located thereon.    
     
     
         15 . The oligonucleotide of  claim 11  wherein said substituent-bearing nucleotides are located at one of the 3′ terminus or the 5′ terminus of said oligonucleotide.  
     
     
         16 . An oligonucleotide comprising a sequence of phosphorothioate nucleotides capable of specifically hybridizing to a strand of nucleic acid, wherein: 
 a first portion of said nucleotides have 2′-deoxy-2′-fluoro, 2′-methoxy, 2′-ethoxy, 2′-propoxy, 2′-aminopropoxy or 2′-allyloxy pentofuranosyl sugar moieties; and    a further portion of said nucleotides have 2′-deoxy-erythro-pentofuranosyl sugar moieties.    
     
     
         17 . An oligonucleotide of  claim 16  wherein said first portion of said nucleotides are located at either the 3′ terminus or the 5′ terminus of said oligonucleotide.  
     
     
         18 . An oligonucleotide of  claim 17  including: 
 an additional portion of said nucleotides having 2′-deoxy-2′-fluoro, 2′-methoxy, 2′-ethoxy, 2′-propoxy, 2′-aminopropoxy or 2′-allyloxy pentofuranosyl sugar moieties; and    said further portion of said nucleotides positioned in said oligonucleotide between said first portion of nucleotides and said additional portion of said nucleotides.    
     
     
         19 . A method of treating an organism having a disease characterized by the undesired production of a protein comprising contacting the organism with an oligonucleotide having a sequence of nucleotides capable of specifically hybridizing to a strand of nucleic acid coding for said protein at least one of the nucleotides being functionalized to increase nuclease resistance of the oligonucleotide, a plurality of the nucleotides having a substituent group located thereon to increase binding affinity of the oligonucleotide to the strand of nucleic acid, and a plurality of the nucleotides having 2′-deoxy-erythro-pentofuranosyl sugar moieties.  
     
     
         20 . The method of  claim 19  wherein each of said nucleotides is a phosphorothioate nucleotide.  
     
     
         21 . The method of  claim 19  wherein said substituent group is a 2′-substituent group.  
     
     
         22 . The method of  claim 21  wherein said 2′-substituent group is fluoro, alkoxy, aminoalkoxy or allyloxy.  
     
     
         23 . A pharmaceutical composition comprising: 
 an pharmaceutically effective amount of an oligonucleotide having a sequence of nucleotides capable of specifically hybridizing to a strand of nucleic acid, at least one of the nucleotides being functionalized to increase nuclease resistance of the oligonucleotide, a plurality of the nucleotides having a substituent group located thereon to increase binding affinity of the oligonucleotide to a complementary strand of nucleic acid; a plurality of the nucleotides having 2′-deoxy-erythro-pentofuranosyl sugar moieties; and    a pharmaceutically acceptable diluent or carrier.    
     
     
         24 . A method of modifying in vitro a sequence-specific nucleic acid, comprising contacting a test solution containing RNase H and said nucleic acid with an oligonucleotide having a sequence of nucleotides capable of specifically hybridizing to a strand of nucleic acid where at least one of the nucleotides is functionalized to increase nuclease resistance of the oligonucleotide, where a plurality of the nucleotides have a substituent group located thereon to increase binding affinity of the oligonucleotide to a complementary strand of nucleic acid, and where a plurality of the nucleotides have 2′-deoxy-erythro-pentofuranosyl sugar moieties.  
     
     
         25 . A method of concurrently enhancing hybridization and RNase H activation in a organism comprising contacting the organism with an oligonucleotide having a sequence of nucleotides capable of specifically hybridizing to a complementary strand of nucleic acid and where at least one of the nucleotides is functionalized to increase nuclease resistance of the oligonucleotide, where a plurality of the nucleotides have a substituent group located thereon to increase binding affinity of the oligonucleotide to a complementary strand of nucleic acid, and where a plurality of the nucleotides have 2′-deoxy-erythro-pentofuranosyl sugar moieties.  
     
     
         26 . A macromolecule comprising a plurality of nucleosides linked by covalent linkages in a sequence that is hybridizable to a complementary nucleic acid, wherein: 
 said nucleosides are selected from α-nucleosides, β-nucleosides including 2′-deoxy-erythro-pentofuranosyl β-nucleosides, 4′-thionucleosides and carbocyclic-nucleosides;    said linkages are selected from charged phosphorous linkages, neutral phosphorous linkages or non-phosphorous linkages; and    said sequence of linked nucleosides contains at least two nucleoside regions, wherein: 
 a first of said regions includes nucleosides selected from said α-nucleosides linked by charged and neutral 3′-5′ phosphorous linkages, said α-nucleosides linked by charged and neutral 2′-5′ phosphorous linkages, said α-nucleosides linked by non-phosphorous linkages, said 4′-thionucleosides linked by charged and neutral 3′-5′ phosphorous linkages, said 4′-thionucleosides linked by charged and neutral 2′-5′ phosphorous linkages, said 4′-thionucleosides linked by non-phosphorous linkages, said carbocyclic-nucleosides linked by charged and neutral 3′-5′ phosphorous linkages, said carbocyclic-nucleosides linked by charged and neutral 2′-5′ phosphorous linkages, said carbocyclic-nucleosides linked by non-phosphorous linkages, said β-nucleosides linked by charged and neutral 2′-5′ linkages, and said β-nucleosides linked by non-phosphorous linkages; and  
 a second of said regions consists of said 2′-deoxy-erythro-pentofuranosyl β-nucleosides linked by charged 3′-5′ phosphorous linkages having a negative charge at physiological pH.  
   
     
     
         27 . A macromolecule of  claim 26  wherein said second region includes at least 3 of said 2′-deoxy-erythro-pentofuranosyl β-nucleosides.  
     
     
         28 . A macromolecule of  claim 26  wherein said second nucleoside region is position between said first nucleoside region and a third nucleoside region, said third nucleoside region including nucleosides selected from said α-nucleosides linked by charged and neutral 3′-5′ phosphorous linkages, said α-nucleosides linked by charged and neutral 2′-5′ phosphorous linkages, said α-nucleosides linked by non-phosphorous linkages, said 4′-thionucleosides linked by charged and neutral 3′-5′ phosphorous linkages, said 4′-thionucleosides linked by charged and neutral 2′-5′ phosphorous linkages, said 4′-thionucleosides linked by non-phosphorous linkages, said carbocyclic-nucleosides linked by charged and neutral 3′-5′ phosphorous linkages, said carbocyclic-nucleosides linked by charged and neutral 2′-5′ phosphorous linkages, said carbocyclic-nucleosides linked by non-phosphorous linkages, said β-nucleosides linked by charged and neutral 2′-5′ linkages, and said β-nucleosides linked by non-phosphorous linkages.  
     
     
         29 . A macromolecule of  claim 26  wherein said charged phosphorous linkages include phosphodiester, phosphorothioate, phosphorodithioate, phosphoroselenate or phosphorodiselenate linkages.  
     
     
         30 . A macromolecule of  claim 26  wherein said charged phosphorous linkages is phosphodiester or phosphorothioate.  
     
     
         31 . A macromolecule of  claim 26  wherein said neutral phosphorous linkages include alkyl and aryl phosphonates, alkyl and aryl phosphoroamidites, alkyl and aryl phosphotriesters, hydrogen phosphonate and boranophosphate linkages.  
     
     
         32 . A macromolecule of  claim 26  wherein said non-phosphorous linkages include peptide linkages, hydrazine linkages, hydroxy-amine linkages, carbamate linkages, morpholine linkages, carbonate linkages, amide linkages, oxymethyleneimine linkages, hydrazide linkages, silyl linkages, sulfide linkages, disulfide linkages, sulfone linkages, sulfoxide linkages, sulfonate linkages, sulfonamide linkages, formacetal linkages, thioformacetal linkages, oxime linkages and ethylene glycol linkages.  
     
     
         33 . A macromolecule of  claim 26  wherein said first nucleoside region includes at least two α-nucleoside linked by a charged or neutral 3′-5′ phosphorous linkages.  
     
     
         34 . A macromolecule comprising a plurality of units linked by covalent linkages in a sequence that is hybridizable to a complementary nucleic acid, wherein: 
 said units are selected from nucleosides and nucleobases: 
 said nucleosides are selected from α-nucleosides, β-nucleosides including 2′-deoxy-erythro-pentofuranosyl β-nucleosides, 4′-thionucleosides, and carbocyclic-nucleosides;  
 said nucleobases are selected from purin-9-yl and pyrimidin-1-yl heterocyclic bases;  
 said linkages are selected from charged 3′-5′ phosphorous, neutral 3′-5′ phosphorous, charged 2′-5′ phosphorous, neutral 2′-5′ phosphorous or non-phosphorous linkages; and  
 said sequence of linked units is divided into at least two regions, wherein: 
 a first of said regions includes said nucleobases linked by non-phosphorous linkages and nucleobases that are attached to phosphate linkages via non-sugar tethering groups, and nucleosides selected from said α-nucleosides linked by charged and neutral 3′-5′ phosphorous linkages, said α-nucleosides linked by charged and neutral 2′-5′ phosphorous linkages, said α-nucleosides linked by non-phosphorous linkages, said 4′-thionucleosides linked by charged and neutral 3′-5′ phosphorous linkages, said 4′-thionucleosides linked by charged and neutral 2′-5′ phosphorous linkages, said 4′-thionucleosides linked by non-phosphorous linkages, said carbocyclic-nucleosides linked by charged and neutral 3′-5′ phosphorous linkages, said carbocyclic-nucleosides linked by charged and neutral 2′-5′ phosphorous linkages, said carbocyclic-nucleosides linked by non-phosphorous linkages, said β-nucleosides linked by charged and neutral 2′-5′ linkages, and said β-nucleosides linked by non-phosphorous linkages; and  
 a second of said regions includes said 2′-deoxy-erythro-pentofuranosyl β-nucleosides linked by charged 3′-5′ phosphorous linkages having a negative charge at physiological pH.  
 
   
     
     
         35 . The macromolecule of  claim 34  wherein said first region includes at least two nucleobases linked by a non-phosphate linkage.  
     
     
         36 . The macromolecule of  claim 35  wherein said non-phosphate linkage is a peptide linkage.  
     
     
         37 . The macromolecule of  claim 35  wherein said second region is positioned between said first region and a third region, said third region including said nucleobases linked by non-phosphorous linkages and nucleobases that are attached to phosphate linkages via a non-sugar tethering moiety, and nucleosides selected from said α-nucleosides linked by charged and neutral 3′-5′ phosphorous linkages, said α-nucleosides linked by charged and neutral 2′-5′ phosphorous linkages, said α-nucleosides linked by non-phosphorous linkages, said 4′-thionucleosides linked by charged and neutral 3′-5′ phosphorous linkages, said 4′-thionucleosides linked by charged and neutral 2′-5′ phosphorous linkages, said 4′-thionucleosides linked by non-phosphorous linkages, said carbocyclic-nucleosides linked by charged and neutral 3′-5′ phosphorous linkages, said carbocyclic-nucleosides linked by charged and neutral 2′-5′ phosphorous linkages, said carbocyclic-nucleosides linked by non-phosphorous linkages, said β-nucleosides linked by charged and neutral 2′-5′ linkages, and said β-nucleosides linked by non-phosphorous linkages.  
     
     
         38 . A macromolecule of  claim 35  wherein said nucleobases are selected from adenine, guanine, cytosine, uracil, thymine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl adenine, 2-propyl and other alkyl adenine, 5-halo uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudo uracil), 4-thiouracil, 8-halo, amino, thiol, thiolalkyl, hydroxyl and other 8 substituted adenine and guanine, or 5-trifluoromethyl uracil and cytosine.  
     
     
         39 . A macromolecule comprising a plurality of units linked by covalent linkages in a sequence that is hybridizable to a complementary nucleic acid, wherein: 
 said units are selected from nucleosides and nucleobases;    said nucleosides are selected from α-nucleosides, β-nucleosides, 4′-thionucleosides and carbocyclic-nucleosides;    said nucleobases are selected from purin-9-yl and pyrimidin-1-yl heterocyclic bases;    said linkages are selected from charged phosphorous, neutral phosphorous or non-phosphorous linkages; and    said sequence of linked units is divided into at least two regions, wherein: 
 a first of said regions includes said α-nucleosides linked by charged and neutral 3′-5′ phosphorous linkages, said α-nucleosides linked by charged and neutral 2′-5′ phosphorous linkages, said α-nucleosides linked by non-phosphorous linkages, said 4′-thionucleosides linked by charged and neutral 3′-5′ phosphorous linkages, said 4′-thionucleosides linked by charged and neutral 2′-5′ phosphorous linkages, said 4′-thionucleosides linked by non-phosphorous linkages, said carbocyclic-nucleosides linked by charged and neutral phosphorous linkages, said carbocyclic-nucleosides linked by non-phosphorous linkages, said β-nucleosides linked by charged and neutral 3′-5′ linkages, said β-nucleosides linked by charged and neutral 2′-5′ linkages, and said β-nucleosides linked by non-phosphorous linkages; and  
 a second of said regions including said nucleobases linked by non-phosphorous linkages and nucleobases that are attached to phosphate linkages via a non-sugar tethering moiety.  
   
     
     
         40 . The macromolecule of  claim 38  wherein said non-phosphate linkage is a peptide linkage.  
     
     
         41 . A macromolecule of  claim 38  including a plurality of said first regions.  
     
     
         42 . A macromolecule of  claim 38  including a plurality of said second regions.  
     
     
         43 . A macromolecule of  claim 41  including a plurality of said first regions.  
     
     
         44 . A method of treating an organism having a disease characterized by the undesired production of a protein comprising contacting the organism with a compound of  claim 34 .  
     
     
         45 . A pharmaceutical composition comprising a pharmaceutically effective amount of a compound of  claim 34  and a pharmaceutically acceptable diluent or carrier.  
     
     
         46 . A method of modifying in vitro a sequence-specific nucleic acid, comprising contacting a test solution containing a RNase H and said nucleic acid with a compound of  claim 34 .  
     
     
         47 . A method of treating an organism having a disease characterized by the undesired production of a protein comprising contacting the organism with a compound of  claim 39 .  
     
     
         48 . A pharmaceutical composition comprising a pharmaceutically effective amount of a compound of  claim 39  and a pharmaceutically acceptable diluent or carrier.