Human RNase H1 oligonucleotide compositions thereof

Abstract

The present invention provides oligonucleotides that can serve as substrates for human Type 2 RNase H. The present invention is also directed to methods of using these oligonucleotides in enhancing antisense oligonucleotide therapies.

1 . A mixed sequence oligonucleotide comprising at least 12 nucleotides in length and having a 3′ end and a 5′ end and divided into a first portion and a further portion, 
 said first portion comprising nucleotides that support cleavage of a complementary target RNA by human RNase H1 polypeptide,    said further portion comprising nucleotides that do not support said cleavage by said RNase H1,    wherein said first portion comprises at least 6 contiguous nucleotides and is positioned in said oligonucleotide such that at least one of said at least 6 contiguous nucleotides is 8 to 12 nucleotides from the 3′ end of said oligonucleotide.    
     
     
         2 . A mixed sequence oligonucleotide comprising at least 12 nucleotides and having a 3′ end and a 5′ end and divided into a first portion and a further portion, 
 said first portion supports cleavage of a complementary target RNA by a purified or isolated human RNase H1 polypeptide,    said further portion does not support said cleavage by said purified or isolated RNase H1 and where    said first portion comprises at least 6 contiguous nucleotides and is positioned in said oligonucleotide such that at least one of said at least 6 contiguous nucleotides is 8 to 12 nucleotides from the 3′ end of said oligonucleotide.    
     
     
         3 . The oligonucleotide of  claim 2  comprising from about 12 to about 50 nucleotides.  
     
     
         4 . The oligonucleotide of  claim 2  comprising from about 12 to about 25 nucleotides.  
     
     
         5 . A mixed sequence oligonucleotide comprising at least 12 nucleotides and having a 3′ end and a 5′ end and divided into a first portion and a further portion, 
 said first portion supports cleavage of a complementary target RNA by human RNase H1 polypeptide,    said further portion does not support said cleavage by said RNase H1;    wherein:    said first portion comprises at least 6 nucleotides and is positioned in said oligonucleotide such that at least one of said at least 6 nucleotides is 8 to 12 nucleotides from the 3′ end of said oligonucleotide;    said nucleotides of said first portion have B-form conformational geometry and are joined together in a continuous sequence, and    at least one of said nucleotides having B-form conformational geometry is not a 2′-deoxyribonucleotide.    
     
     
         6 . A mixed sequence oligonucleotide comprising at least 12 nucleotides and having a 3′ end and a 5′ end and divided into a first portion and a further portion, 
 said first portion supports cleavage of a complementary target RNA by human RNase H1 polypeptide,    said further portion does not support said cleavage by said RNase H1;    wherein:    said first portion comprises at least 6 contiguous nucleotides and is positioned in said oligonucleotide such that at least one of said at least 6 contiguous nucleotides is 8 to 12 nucleotides from the 3′ end of said oligonucleotide; and    each of said nucleotides of said first portion is, independently, a 2′-SCH 3  ribonucleotide, a 2′-NH 2  ribonucleotide, a 2′-NH(C 1 -C 2  alkyl) ribonucleotide, a 2′-N(C 1 -C 2  alkyl) 2  ribonucleotide, a 2′-CF 3  ribonucleotide, a 2′=CH 2  ribonucleotide, a 2′=CHF ribonucleotide, a 2′=CF 2  ribonucleotide, a 2′-CH 3  ribonucleotide, a 2′-C 2 H 5  ribonucleotide, a 2′-CH═CH 2  ribonucleotide or a 2′-CCH ribonucleotide.    
     
     
         7 . The oligonucleotide of  claim 2  wherein at least one of said nucleotides of said first portion is a 2′-deoxyribonucleotide.  
     
     
         8 . The oligonucleotide of  claim 2  wherein said nucleotides of said first portion are joined together in said continuous sequence by phosphorothioate linkages.  
     
     
         9 . The oligonucleotide of  claim 2  wherein said further portion includes a plurality of nucleotides, at least some of said nucleotides comprise a 2′ substituent group wherein each substituent group is, independently, hydroxyl, C 1 -C 20  alkyl, C 2 -C 20  alkenyl, C 2 -C 20  alkynyl, halogen, amino, thiol, keto, carboxyl, nitro, nitroso, nitrile, trifluoromethyl, trifluoromethoxy, O-alkyl, O-alkenyl, O-alkynyl, S-alkyl, S-alkenyl, S-alkynyl, NH-alkyl, NH-alkenyl, NH-alkynyl, N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl, S-aralkyl, NH-aralkyl, N-phthalimido, imidazole, azido, hydrazino, hydroxylamino, isocyanato, sulfoxide, sulfone, sulfide, disulfide, silyl, aryl, heterocycle, carbocycle, intercalator, reporter molecule, conjugate, polyamine, polyamide, polyalkylene glycol, or polyether; 
 or each substituent group has one of formula I or II:                          wherein:    Z 0  is O, S or NH;    J is a single bond, O or C(═O);    E is C 1 -C 10  alkyl, N(R 1 )(R 2 ), N═C(R 1 )(R 2 ), or has one of formula III or IV;                          each R 6 , R 7 , R 8 , R 9  and R 10  is, independently, hydrogen, C(O)R 11 , substituted or unsubstituted C 1 -C 10  alkyl, substituted or unsubstituted C 2 -C 10  alkenyl, substituted or unsubstituted C 2 -C 10  alkynyl, alkylsulfonyl, arylsulfonyl, a chemical functional group or a conjugate group, wherein the substituent groups are selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl;    or optionally, R 7  and R 8 , together form a phthalimido moiety with the nitrogen atom to which they are attached;    or optionally, R 9  and R 10 , together form a phthalimido moiety with the nitrogen atom to which they are attached;    each R 11  is, independently, substituted or unsubstituted C 1 -C 10  alkyl, trifluoromethyl, cyanoethyloxy, methoxy, ethoxy, t-butoxy, allyloxy, 9-fluorenylmethoxy, 2-(trimethylsilyl)-ethoxy, 2,2,2-trichloroethoxy, benzyloxy, butyryl, iso-butyryl, phenyl or aryl;    each R 1  and R 2  is, independently, H, a nitrogen protecting group, substituted or unsubstituted C 1 -C 10  alkyl, substituted or unsubstituted C 2 -C 10  alkenyl, substituted or unsubstituted C 2 -C 10  alkynyl, wherein said substitution is OR 3 , SR 3 , NH 3   + , N(R 3 )(R 4 ), guanidino or acyl where said acyl is an acid amide or an ester;    or R 1  and R 2 , together, are a nitrogen protecting group or are joined in a ring structure that optionally includes an additional heteroatom selected from N and O;    or R 1 , T and L, together, are a chemical functional group;    each R 3  and R 4  is, independently, H, C 1 -C 10  alkyl, a nitrogen protecting group, or R 3  and R 4 , together, are a nitrogen protecting group;    or R 3  and R 4  are joined in a ring structure that optionally includes an additional heteroatom selected from N and O;    Z 4  is OX, SX, or N(X) 2 ;    each X is, independently, H, C 1 -C 8  alkyl, C 1 -C 8  haloalkyl, C(═NH)N(H)R 5 , C(═O)N(H)R 5  or OC(═O)N(H)R 5 ;    R 5  is H or C 1 -C 8  alkyl;    Z 1 , Z 2  and Z 3  comprise a ring system having from about 4 to about 7 carbon atoms or having from about 3 to about 6 carbon atoms and 1 or 2 hetero atoms wherein said hetero atoms are selected from oxygen, nitrogen and sulfur and wherein said ring system is aliphatic, unsaturated aliphatic, aromatic, or saturated or unsaturated heterocyclic;    Z 5  is alkyl or haloalkyl having 1 to about 10 carbon atoms, alkenyl having 2 to about 10 carbon atoms, alkynyl having 2 to about 10 carbon atoms, aryl having 6 to about 14 carbon atoms, N(R 1 )(R 2 ) OR, halo, SR 1  or CN;    each q 1  is, independently, an integer from 1 to 10;    each q 2  is, independently, 0 or 1;    q 3  is 0 or an integer from 1 to 10;    q 4  is an integer from 1 to 10; and    q 5  is 0, 1 or 2;    provided that when q 3  is 0, q 4  is greater than 1.    
     
     
         10 . The oligonucleotide of  claim 2  wherein said further portion comprises a plurality of nucleotides and wherein each of said nucleotides of said further portion is, independently, a 2′-F ribonucleotide, a 2′-O-(C 1 -C 6  alkyl) ribonucleotide, or a 2′-O-(C 1 -C 6  substituted alkyl) ribonucleotide wherein the substitution is C 1 -C 6  ether, C 1 -C 6  thioether, amino, amino(C 1 -C 6  alkyl) or amino(C 1 -C 6  alkyl) 2 .  
     
     
         11 . The oligonucleotide of  claim 2  wherein said further portion comprises a plurality of nucleotides and wherein at least two of said nucleotides of said further portion are joined together in a continuous sequence that is positioned 3′ to said first portion.  
     
     
         12 . The oligonucleotide of  claim 2  wherein said further portion comprises a plurality of nucleotides and wherein at least two of said nucleotides of said further portion are joined together in a continuous sequence that is positioned 5′ to said first portion.  
     
     
         13 . The oligonucleotide of  claim 2  wherein said further portion comprises a plurality of nucleotides and wherein at least two of said nucleotides of said further portion are joined together in a continuous sequence that is positioned 3′ to said first portion and at least two of said further portion are joined together in a continuous sequence that is positioned 5′ to said first portion.  
     
     
         14 . The oligonucleotide of  claim 2  wherein said further portion comprises a plurality of nucleotides and wherein at least four of said nucleotides of said further portion are joined together in a continuous sequence that is positioned 3′ to said first portion.  
     
     
         15 . The oligonucleotide of  claim 2  wherein said further portion comprises a plurality of nucleotides and wherein at least four of said nucleotides of said further portion are joined together in a continuous sequence that is positioned 5′ to said first portion.  
     
     
         16 . The oligonucleotide of  claim 2  wherein said further portion comprises a plurality of nucleotides and wherein at least four of said nucleotides of said further portion are joined together in a continuous sequence that is positioned 3′ to said first portion and at least four of said nucleotides of said further portion are joined together in a continuous sequence that is positioned 5′ to said first portion.  
     
     
         17 . A mixed sequence oligonucleotide comprising at least 8 oligonucleotides and having a 2′-OH arabinonucleotide sequence of at least 6 nucleotides and where at least one of said arabinonucleotides is positioned 8 to 12 nucleotides from the 3′ end of said oligonucleotide, and wherein said oligonucleotide supports cleavage of a complementary target RNA by human RNase H1 polypeptide.  
     
     
         18 . A mixed sequence oligonucleotide comprising at least 8 oligonucleotides and having a a 2′-F arabinonucleotide sequence of at least 6 nucleotides and where at least one of said 2′-F arabinonucleotides is positioned 8 to 12 nucleotides from the 3′ end of said oligonucleotide, and wherein said oligonucleotide supports cleavage of a complementary target RNA by human RNase H1 polypeptide.  
     
     
         19 . A mixed sequence oligonucleotide comprising 8 to 25 nucleotides and having a 2′-OH arabinonucleotide sequence wherein at least one of the nucleotides of said sequence is positioned 8 to 12 nucleotides from the 3′ end of said oligonucleotide, wherein said oligonucleotide supports cleavage of a complementary target RNA by human RNase H1 polypeptide.  
     
     
         20 . A mixed sequence oligonucleotide comprising 8 to 25 nucleotides and having a 2′-F arabinonucleotide sequence wherein at least one of the nucleotides of said sequence is positioned 8 to 12 nucleotides from the 3′ end of said oligonucleotide, wherein said oligonucleotide supports cleavage of a complementary target RNA by human RNase H1 polypeptide.  
     
     
         21 . A chimeric oligonucleotide comprising 8 to 25 nucleotides and having a portion that supports cleavage of a complementary target RNA by human RNase H1 polypeptide wherein said portion supporting said cleavage is at least 6 contiguous nucleotides in length and is positioned in said oligonucleotide such that at least one of said at least 6 contiguous nucleotides is positioned 8 to 12 nucleotides from the 3′ end of said oligonucleotide, wherein said oligonucleotide supports cleavage of a complementary target RNA by human RNase H1 polypeptide.  
     
     
         22 . The oligonucleotide of  claim 21  wherein said RNase H1 polypeptide is a purified or isolated polypeptide.  
     
     
         23 . A method comprising contacting an oligonucleotide according to  claim 2  with RNA or DNA in vitro.  
     
     
         24 . A method comprising contacting an oligonucleotide according to  claim 5  with RNA or DNA in vitro.  
     
     
         25 . A method comprising contacting an oligonucleotide according to  claim 6  with RNA or DNA in vitro.  
     
     
         26 . A method comprising contacting an oligonucleotide according to  claim 17  with RNA or DNA in vitro.  
     
     
         27 . A method comprising contacting an oligonucleotide according to  claim 18  with RNA or DNA in vitro.  
     
     
         28 . A method comprising contacting an oligonucleotide according to  claim 19  with RNA or DNA in vitro.  
     
     
         29 . A method comprising contacting an oligonucleotide according to  claim 20  with RNA or DNA in vitro.  
     
     
         30 . A method comprising contacting an oligonucleotide according to  claim 2  with RNA or DNA in a cellular assay.  
     
     
         31 . A method comprising contacting an oligonucleotide according to  claim 5  with RNA or DNA in a cellular assay.  
     
     
         32 . A method comprising contacting an oligonucleotide according to  claim 6  with RNA or DNA in a cellular assay.  
     
     
         33 . A method comprising contacting an oligonucleotide according to  claim 17  with RNA or DNA in a cellular assay.  
     
     
         34 . A method comprising contacting an oligonucleotide according to  claim 18  with RNA or DNA in a cellular assay.  
     
     
         35 . A method comprising contacting an oligonucleotide according to  claim 19  with RNA or DNA in a cellular assay.  
     
     
         36 . A method comprising contacting an oligonucleotide according to  claim 20  with RNA or DNA in a cellular assay.  
     
     
         37 . A method comprising contacting an oligonucleotide according to  claim 21  with RNA or DNA in vitro.  
     
     
         38 . A method comprising contacting an oligonucleotide according to  claim 21  with RNA or DNA in a cellular assay.  
     
     
         39 . A method comprising contacting an oligonucleotide according to  claim 22  with RNA or DNA in vitro.  
     
     
         40 . A method comprising contacting an oligonucleotide according to  claim 22  with RNA or DNA in a cellular assay.  
     
     
         41 . A method comprising: 
 selecting a parameter associated with interaction of an oligonucleotide, a target nucleic acid and human RNase H1 polypeptide,    selecting a cell containing human RNase H1 polypeptide and a target nucleic acid,    selecting first and second oligonucleotides each having a sequence substantially commpementary to said target nucleic acid,    contacting said cell with said first oligonucleotide and measuring said parameter,    contacting said cell with said second oligonucleotide and measuring said parameter,    comparing measurements, and    using said comparison to select one of said first or said second oligonucleotides for modulating said target in the presence of said human RNase H1 polypeptide in said cell.    
     
     
         42 . The method of  claim 41  wherein said parameter is one of site preference for cleavage, sequence preference for cleavage or processivity of cleavage.  
     
     
         43 . The method of  claim 41  wherein said measurement is a measurement of at least one of K d , K max , K m  or K cat .  
     
     
         44 . An oligonucleotide for modulating a target nucleic acid in the presence of human RNase H1 polypeptide wherein said oligonucleotide is identified using the process of  claim 41 .  
     
     
         45 . An optimized oligonucleotide for modulating a target nucleic acid wherein said optimized oligonucleotide is identified using the process of  claim 41 .  
     
     
         46 . The method of  claim 41  further comprising: 
 selecting a further oligonucleotide,    contacting said cell with said further oligonucleotide and measuring said parameter,    comparing the measurement of this further oligonucleotide to that of said selected first or said second oligonucleotides and selecting one of said first, said second or said further oligonucleotide.