Oligonucleotides having A-DNA form and B-DNA form conformational geometry
Modified oligonucleotides containing both A-form conformation geometry and B-from conformation geometry nucleotides are disclosed. The B-form geometry allows the oligonucleotide to serve as substrates for RNase H when bound to a target nucleic acid strand. The A-form geometry imparts properties to the oligonucleotide that modulate binding affinity and nuclease resistance. By utilizing C2′ endo sugars or O4′ endo sugars, the B-form characteristics are imparted to a portion of the oligonucleotide. The A-form characteristics are imparted via use of either 2′-O-modified nucleotides that have 3′ endo geometries or use of end caps having particular nuclease stability or by use of both of these in conjunction with each other.
1. An oligonucleotide comprising a plurality of nucleotides, wherein:
a first portion of said plurality of nucleotides have B-form conformational geometry and are joined together in a continuous sequence, at least two of said nucleotides of said first portion being ribonucleotides; and
a further portion of said plurality of nucleotides are ribonucleotide that have A-form conformation geometry and are joined together in at least one continuous sequence.
2. The oligonucleotide of claim 1 wherein each nucleotide of said first portion, independently, is 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′-C≡CH ribonucleotide.
3. The oligonucleotide of claim 1 wherein each of said nucleotides of said first portion are joined together in said continuous sequence by phosphate, phosphorothioate, phosphorodithioate or boranophosphate linkages.
4. The oligonucleotide of claim 1 wherein each nucleotide of said further portion, independently, is a 2′-fluoro nucleotide or a nucleotide having a 2′-substituent having the formula I or II:
wherein
E is C 1 -C 10 alkyl, N(Q 1 )(Q 2 ) or N═C(Q 1 )(Q 2 );
each Q 1 and Q 2 is, independently, H, C 1 -C 10 alkyl, dialkylaminoalkyl, a nitrogen protecting group, a tethered or untethered conjugate group, a linker to a solid support, or Q 1 and Q 2 , together, are joined in a nitrogen protecting group or a ring structure that can include at least one additional heteroatom selected from N and O;
R 3 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)Z, C(═O)N(H)Z or OC(═O)N(H)Z;
Z is H or C 1 -C 8 alkyl;
L 1 , L 2 and L 3 form 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 heteroatoms selected from oxygen, nitrogen and sulfur and wherein said ring system is aliphatic, unsaturated aliphatic, aromatic, or saturated or unsaturated heterocyclic;
Y 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(Q 1 )(Q 2 ), O(Q 1 ), halo, S(Q 1 ), or CN;
each q 1 is, independently, from 2 to 10;
each q 2 is, independently, 0 or 1;
m is 0, 1 or 2;
p is from 1 to 10; and
q 3 is from 1 to 10 with the proviso that when p is 0, q 3 is greater than 1.
5. The oligonucleotide of claim 1 wherein each of said nucleotides of said further portion, independently, is 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 .
6. The oligonucleotide of claim 1 wherein all of said nucleotides of said further portion are joined together in a continuous sequence by 3′-5′ phosphodiester, 2′-5′ phosphodiester, phosphorothioate, Sp phosphorothioate, Rp phosphorothioate, phosphorodithioate, 3′-deoxy-3′-amino phosphoroamidate, 3′-methylenephosphonate, methylene(methylimino), dimethylhydrazino, amide 3, amide 4 or boranophosphate linkages.
7. The oligonucleotide of claim 1 wherein at least two of said nucleotides of said further portion are joined together in a continuous sequence that is positioned 3′ to said continuous sequence of said first portion of said plurality of nucleotides.
8. The oligonucleotide of claim 1 wherein at least two of said nucleotides of said further portion are joined together in a continuous sequence that is positioned 5′ to said continuous sequence of said first portion.
9. The oligonucleotide of claim 1 wherein at least two of said nucleotides of said further portion are joined together in a continuous sequence that is positioned 3′ to said continuous sequence of said first portion and at least two of said further portion are joined together in a continuous sequence that is positioned 5′ to said continuous sequence of said first portion.
10. The oligonucleotide of claim 1 wherein each nucleotide of said first portion, independently, is 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′=CH 2 ribonucleotide, a 2′-CH 3 ribonucleotide, a 2′-C 2 H 5 ribonucleotide, a 2′-CH═CH 2 ribonucleotide or a 2′-C≡CH ribonucleotide.
11. The oligonucleotide of claim 1 wherein each nucleotide of said first portion, independently, is 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 or a 2′-CH 3 ribonucleotide.
12. The oligonucleotide of claim 1 wherein each nucleotide of said first portion, independently, is a 2′-SCH 3 ribonucleotide, a 2′-NH 2 ribonucleotide or a 2′-CH 3 ribonucleotide.
13. The oligonucleotide of claim 1 wherein each nucleotide of said first portion is a 2′-SCH 3 ribonucleotide.
14. The oligonucleotide of claim 1 wherein each nucleotide of said first portion, independently, is 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′-CH 3 ribonucleotide, a 2′-CH═CH 2 ribonucleotide or a 2′-C≡CH ribonucleotide; and
each nucleotide of said further portion, independently, is 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 .
15. The oligonucleotide of claim 1 wherein said further portion comprises at least two nucleotides joined together in a continuous sequence that is positioned at the 3′ terminus end of said oligonucleotide.
16. The oligonucleotide of claim 1 wherein said further portion comprises at least two nucleotides joined together in a continuous sequence that is positioned at the 5′ terminus of said oligonucleotide.
17. The oligonucleotide of claim 1 wherein said further portion comprises at least two nucleotides joined together in a continuous sequence that is positions at the 3′ terminus of said oligonucleotide; and
at least two nucleotides joined together in a continuous sequence that is positions at the 5′ terminus of said oligonucleotide.
18. The oligonucleotide of claim 15 wherein said at least two nucleotides joined together comprise nucleotides joined together by a 2′-5′ phosphodiester linkage, a 3′-methylenephosphonate linkage, a Sp phosphorothioate linkage, a methylene(methylimino) linkage, a dimethyhydrazino linkage, a 3′-deoxy-3′-amino phosphoroamidate linkage, an amide 3 linkage or an amide 4 linkage.
19. The oligonucleotide of claim 18 wherein said two nucleotides are joined together by a 2′-5′ phosphodiester linkage, a 3′-methylenephosphonate linkage, a Sp phosphorothioate linkage or a methylene(methylimino) linkage.
20. The oligonucleotide of claim 16 wherein said at least two nucleotides joined together comprise nucleotides joined together by a 2′-5′ phosphodiester linkage, a 3′-methylenephosphonate linkage, a Sp phosphorothioate linkage, a methylene(methylimino) linkage, a dimethyhydrazino linkage, a 3′-deoxy-3′-amino phosphoroamidate linkage, an amide 3 linkage or an amide 4 linkage.
21. The oligonucleotide of claim 20 wherein said two nucleotides are joined together by a 2′-5′ phosphodiester linkage, a 3′-methylenephosphonate linkage, a Sp phosphorothioate linkage or a methylene(methylimino) linkage.
22. The oligonucleotide of claim 17 wherein said at least two nucleotides joined together and positioned at said 3′ terminus comprise nucleotides joined together by a 2′-5′ phosphodiester linkage, a 3′-methylenephosphonate linkage, a Sp phosphorothioate linkage, a methylene(methylimino) linkage, a dimethyhydrazino linkage, a 3′-deoxy-3′-amino phosphoroamidate linkage, an amide 3 linkage or an amide 4 linkage; and
wherein said at least two nucleotides joined together and positioned at said 5′ terminus comprise nucleotides joined together by a 2′-5′ phosphodiester linkage, a 3′-methylenephosphonate linkage, a Sp phosphorothioate linkage, a methylene(methylimino) linkage, a dimethyhydrazino linkage, a 3′-deoxy-3′-amino phosphoroamidate linkage, an amide 3 linkage or an amide 4 linkage.
23. The oligonucleotide of claim 22 wherein said two nucleotides joined together at said 3′ terminus and said two nucleotides joined together at said 5′ terminus are, independently, joined together by 2′-5′ phosphodiester linkages, 3′-methylenephosphonate linkages, Sp phosphorothioate linkages or methylene(methylimino) linkages.
24. The oligonucleotide of claim 15 wherein at least one of said two nucleotides joined together is a 2′-alkylamino substituted nucleotide.
25. The oligonucleotide of claim 16 wherein at least one of said two nucleotides joined together is a 2′-alkylamino substituted nucleotide.
26. The oligonucleotide of claim 17 wherein at least one of said two nucleotides joined together at said 3′ terminus is a 2′-alkylamino substituted nucleotide, and
wherein at least one of said two nucleotides joined together at said 5′ terminus is a 2′-alkylamino substituted nucleotide.