Enzymatic nucleic acids containing 5'-and/or 3'-cap structures

• US 5,998,203 A
• Filed: 04/16/1996
• Issued: 12/07/1999
• Est. Priority Date: 04/16/1996
• Status: Expired due to Fees

• Current Assignee: Ribozyme Pharmaceuticals, Inc. (Alnylam Pharmaceuticals Incorporated)
• Original Assignee: Ribozyme Pharmaceuticals, Inc. (Alnylam Pharmaceuticals Incorporated)
• Inventors: Beigelman, Leonid, Matulic-Adamic, Jasenka, Jarvis, Thale, Karpeisky, Alexander, Usman, Nassim, DiRenzo, Anthony, Wincott, Francine
• Primary Examiner(s): Degen, Nancy
• Assistant Examiner(s): LARSON, THOMAS GEORGE
• Application Number:
  US08/632,882
• Time in Patent Office: 1,330 Days
• Field of Search: 435/6, 435/91.31, 435/199, 435/240.1, 435/240.2, 435/366, 435/375, 435/325, 514/44, 536/23.1, 536/23.2, 536/24.3, 536/24.5, 536/25.3
• US Class Current: 435/325
• CPC Class Codes: C07H 19/06 Pyrimidine radicals
• CPC Class Codes: C07H 19/16 Purine radicals
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 2310/121 Hammerhead

Abstract

An enzymatic nucleic acid molecule comprising a 5'"'"'- and/or a 3'"'"'-cap structure, wherein said structure is not a 5'"'"'-5'"'"'-linked inverted nucleotide or a 3'"'"'-3'"'"'-linked inverted nucleotide.

Claims

1. An enzymatic nucleic acid molecule comprising a 5'"'"'-cap structure, a 3'"'"'-cap structure, or both a 5'"'"'- and a 3'"'"'-cap structure, wherein said 5'"'"'-cap structure is selected from the group consisting of 4'"'"',5'"'"'-methylene nucleotide;

1-(β

-D-erythrofuranosyl)nucleotide;

4'"'"'-thio nucleotide, carbocyclic nucleotide;

5'"'"'-amino-alkyl phosphate;

1,3-diamino-2-propyl phosphate, 3-aminopropyl phosphate;

6-aminohexyl phosphate;

12-aminododecyl phosphate;

hydroxypropyl phosphate;

1,5-anhydrohexitol nucleotide;

L-nucleotide;

alpha-nucleotide;

threo-pentofuranosyl nucleotide;

acyclic 3'"'"',4'"'"'-seco nucleotide;

3,4-dihydroxybutyl nucleotide;

3,5-dihydroxypentyl nucleotide, 5'"'"'-5'"'"'-inverted abasic moiety;

1,4-butanediol phosphate;

5'"'"'-amino; and

bridging or non bridging methylphosphonate and 5'"'"'-mercapto moieties, and wherein said 3'"'"'-cap structure is selected from the group consisting of 4'"'"'-thio nucleoside, carbocyclic nucleotide;

1,5-anhydrohexitol nucleotide;

L-nucleotide;

alpha-nucleotide;

modified base nucleotide;

threo-pentofuranosyl nucleotide;

acyclic 3'"'"',4'"'"'-seco nucleotide;

3,4-dihydroxybutyl nucleotide;

3,5-dihydroxypentyl nucleotide;

3'"'"'-3'"'"'-inverted abasic moiety;

3'"'"'-2'"'"'-inverted nucleotide moiety;

3'"'"'-2'"'"'-inverted abasic moiety;

1,4-butanediol;

hexylphosphate;

aminohexyl phosphate;

3'"'"'-phosphate; and

a bridging methylphosphonate moiety.

2. The enzymatic nucleic acid molecule of claim 1, wherein said enzymatic nucleic acid molecule is in a hammerhead motif.

3. The enzymatic nucleic acid molecule of claim 1, wherein said enzymatic nucleic acid molecule is in a hairpin, hepatitis delta virus, group I intron, VS RNA or RNase P RNA motif.

4. The enzymatic nucleic acid molecule of claim 1, wherein said enzymatic nucleic acid molecule comprises said 5'"'"'-cap structure.

5. The enzymatic nucleic acid molecule of claim 1, wherein said enzymatic nucleic acid molecule comprises said 3'"'"'-cap structure.

6. The enzymatic nucleic acid molecule of claim 1, wherein said enzymatic nucleic acid molecule comprises said 5'"'"'-cap structure and said 3'"'"'-cap structure.

7. The enzymatic nucleic acid molecule of claim 1, wherein said 5'"'"'- and 3'"'"'-cap structures are different.

8. The enzymatic nucleic acid molecule of claim 1, wherein said enzymatic nucleic acid molecule comprises at least one ribonucleotide residue.

9. The enzymatic nucleic acid molecule of claim 2, wherein said enzymatic nucleic acid molecule comprises:

at least five ribose residues;

at least three 5'"'"' terminal nucleotides are substituted with phosphorothioate linkages;

position No. 4 of said enzymatic nucleic acid molecule is substituted with a 2'"'"'-C-allyl modification; and

wherein said enzymatic nucleic acid molecule comprises at least ten 2'"'"'-O-methyl modifications.

10. The enzymatic nucleic acid molecule of claim 2, wherein said enzymatic nucleic acid molecule comprises:

at least five ribose residues;

a 2'"'"'-amino modification at position No. 4, at position No. 7, or at both position No. 4 and position No. 7; and

wherein said enzymatic nucleic acid molecule comprises at least ten 2'"'"'-O-methyl modifications.

11. The enzymatic nucleic acid molecule of claim 2, wherein said enzymatic nucleic acid molecule comprises:

at least five ribose residues;

abasic substitution at position No. 4, at position No. 7, or at both position No. 4 and position No. 7; and

wherein said enzymatic nucleic acid molecule comprises at least ten 2'"'"'-O-methyl modifications.

12. The enzymatic nucleic acid molecule of claim 2, wherein said enzymatic nucleic acid molecule comprises:

at least five ribose residues;

6-methyl uridine substitutions at position No. 4, at position No. 7, or at both position No. 4 and position No. 7; and

wherein said enzymatic nucleic acid molecule comprises at least ten 2'"'"'-O-methyl modifications.

13. The enzymatic nucleic acid molecule of claim 2, wherein said enzymatic nucleic acid molecule comprises at least five ribose residues;

pyridin-4-one substitution at position No. 4, at position No. 7, or at both position No. 4 and position No. 7; and

wherein said enzymatic nucleic acid molecule comprises at least ten 2'"'"'-O-methyl modifications.

14. The enzymatic nucleic acid molecule of claim 2, wherein said enzymatic nucleic acid molecule comprises a stem II region of length greater than or equal to two base-pairs.

15. The enzymatic nucleic acid molecule of claim 3, wherein said hairpin enzymatic nucleic acid molecule comprises a stem IV region of length greater than or equal to two base-pairs.

16. The enzymatic nucleic acid molecule of claim 3, wherein said hairpin enzymatic nucleic acid molecule comprises a stem II region of length between three and seven base-pairs.

17. The enzymatic nucleic acid of claim 1, wherein said 3'"'"'-cap structure is a 3'"'"'-3'"'"' linked inverted ribose.

18. The enzymatic nucleic acid of claim 1, wherein said 3'"'"'-cap structure is a 3'"'"'-2'"'"' linked inverted ribose.

19. The enzymatic nucleic acid of claim 1, wherein said 3'"'"'-cap structure is a 3'"'"'-2'"'"' linked inverted nucleotide.

20. The enzymatic nucleic acid of claim 1, wherein said 5'"'"'-cap structure is a 5'"'"'-5'"'"' linked inverted ribose.

21. The enzymatic nucleic acid of claim 1, wherein said 5'"'"'-cap structure is a 5'"'"'-amino group.

22. The enzymatic nucleic acid of claim 1, wherein said 5'"'"'-cap structure is a 4'"'"'-thio nucleotide.

23. The enzymatic nucleic acid of claim 1, wherein said 5'"'"'-cap structure is a 1,3-diamino-2-propyl phosphate group.

24. The enzymatic nucleic acid of claim 1, wherein said 5'"'"'-cap structure is a L-nucleotide.

25. The enzymatic nucleic acid of claim 1, wherein said 5'"'"'-cap structure is an alpha-nucleotide.

26. The enzymatic nucleic acid of claim 1, wherein said 5'"'"'-cap structure is a threo-pentafuranosyl group.

27. The enzymatic nucleic acid of claim 1, wherein said 5'"'"'-cap structure is a 3,4-dihydroxybutyl nucleotide.

28. The enzymatic nucleic acid of claim 1, wherein said 5'"'"'-cap structure is a 3,5-dihydroxypentyl nucleotide.

29. The enzymatic nucleic acid of claim 1, wherein said 5'"'"'-cap structure is a 1-(β

-D-erythrofuranosyl)nucleotide.

30. The enzymatic nucleic acid of claim 1, wherein said 3'"'"'-cap structure is a 4'"'"'-thio group.

31. The enzymatic nucleic acid of claim 1, wherein said 3'"'"'-cap structure is a L-nucleotide.

32. The enzymatic nucleic acid of claim 1, wherein said 3'"'"'-cap structure is an alpha-nucleotide.

33. The enzymatic nucleic acid of claim 1, wherein said 3'"'"'-cap structure is a 3,4-dihydroxybutyl nucleotide.

34. The enzymatic nucleic acid of claim 1, wherein said 3'"'"'-cap structure is a 3,5-dihydroxypentyl nucleotide.

35. The enzymatic nucleic acid of claim 1, wherein said 5'"'"'-cap structure is a 5'"'"'-amino group and said 3'"'"'-cap structure is a 3'"'"'-3'"'"'-inverted abasic nucleotide.

36. The enzymatic nucleic acid of claim 1, wherein said 3'"'"'-cap structure is a 3'"'"'-3'"'"'-inverted abasic nucleotide.

37. The enzymatic nucleic acid of claim 1, wherein said 5'"'"'-cap structure is a 5'"'"'-amino group and said 3'"'"'-cap structure is a 3'"'"'-2'"'"'-inverted abasic nucleotide.

38. The enzymatic nucleic acid of claim 1, wherein said 3'"'"'-cap structure is a 3'"'"'-2'"'"'-inverted abasic nucleotide.

39. A mammalian cell including the enzymatic nucleic acid molecule of claim 1.

40. The mammalian cell of claim 39, wherein said mammalian cell is a human cell.

41. A method for producing an enzymatic nucleic acid molecule having activity to cleave an RNA or a single-stranded DNA molecule, comprising the step of forming said enzymatic nucleic acid molecule with a 5'"'"'-cap structure, a 3'"'"'-cap structure, or both a 5'"'"'- and a 3'"'"'-cap structure,

wherein said 5'"'"'-cap structure is selected from the group consisting of 4'"'"',4'"'"'-methylene nucleotide;

1-(β

-D-erythrofuranosyl)nucleotide;

4'"'"'-thio nucleotide, carbocyclic nucleotide;

5'"'"'-amino-alkyl phosphate;

1,3-diamino-2-propyl phosphate, 3-aminopropyl phosphate;

6-aminohexyl phosphate;

12-aminododecyl phosphate;

hydroxypropyl phosphate;

1,5-anhydrohexitol nucleotide;

L-nucleotide;

alpha-nucleotide;

threo-pentofuranosyl nucleotide;

acyclic 3'"'"',4'"'"'-seco nucleotide;

3,4-dihydroxybutyl nucleotide;

3,5-dihydroxypentyl nucleotide, 5'"'"'-5'"'"'-inverted abasic moiety;

1,4-butanediol phosphate;

5'"'"'-amino; and

bridging or non bridging methylphosphonate and 5'"'"'-mercapto moieties, and wherein said 3'"'"'-cap structure is selected from the group consisting of 4'"'"'-thio nucleoside, carbocyclic nucleotide;

1,5-anhydrohexitol nucleotide;

L-nucleotide;

alpha-nucleotide;

modified base nucleotide;

threo-pentofuranosyl nucleotide;

acyclic 3'"'"',4'"'"'-seco nucleotide;

3,4-dihydroxybutyl nucleotide;

3,5-dihydroxypentyl nucleotide;

3'"'"'-3'"'"'-inverted abasic moiety;

3'"'"'-2'"'"'-inverted nucleotide moiety;

3'"'"'-2'"'"'-inverted abasic moiety;

1,4-butanediol;

hexylphosphate;

aminohexyl phosphate;

3'"'"'-phosphate; and

a bridging methylphosphonate moiety.