Gapped oligonucleotides

• US 7,015,315 B1
• Filed: 06/06/1995
• Issued: 03/21/2006
• Est. Priority Date: 12/24/1991
• Status: Expired due to Term

• Current Assignee: Ionis Pharmaceuticals, Inc.
• Original Assignee: ISIS Pharmaceuticals Incorporated
• Inventors: Monia, Brett P., Cook, Phillip Dan
• Primary Examiner(s): Low, Christopher S. F.
• Application Number:
  US08/465,866
• Time in Patent Office: 3,941 Days
• Field of Search: 435/6, 435/375, 435/377, 435/877, 514/44, 536/23.1, 536/24.3, 536/24.31, 536/24.32, 536/24.33, 536/24.5, 536/25.1
• US Class Current: 536/23.1
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: A61P 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07H 19/04 Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
• CPC Class Codes: C07H 19/06 Pyrimidine radicals
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/16 Purine radicals
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• 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: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C07H 23/00 Compounds containing boron, silicon, or a metal, e.g. chelates, vitamin B12 esters with inorganic acids C07H11/00; metal salts, see parent compounds
• CPC Class Codes: C07J 43/003 not condensed
• CPC Class Codes: C07K 14/003 Peptide-nucleic acids (PNAs)
• 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 15/1135 against oncogenes or tumor suppressor genes
• CPC Class Codes: C12N 15/1137 against enzymes viral enzymes C12N15/1131; receptors C12N15/1138
• CPC Class Codes: C12N 2310/3125 Methylphosphonates
• CPC Class Codes: C12N 2310/315 Phosphorothioates
• CPC Class Codes: C12N 2310/32 : of the sugar
• CPC Class Codes: C12N 2310/321 : 2'-O-R Modification
• CPC Class Codes: C12N 2310/322 : 2'-R Modification
• CPC Class Codes: C12N 2310/33 : of the base
• CPC Class Codes: C12N 2310/333 : Modified A
• CPC Class Codes: C12N 2310/336 : Modified G
• CPC Class Codes: C12N 2310/3521 : Methyl
• CPC Class Codes: C12N 2310/3523 : Allyl
• CPC Class Codes: C12N 2310/3527 : Other alkyl chain
• CPC Class Codes: C12N 2310/3531 : Hydrogen
• CPC Class Codes: C12N 2310/3533 : Halogen
• CPC Class Codes: C12N 9/0069 : acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
• CPC Class Codes: C12Q 1/68 : involving nucleic acids
• CPC Class Codes: C12Q 1/6813 : Hybridisation assays
• CPC Class Codes: C12Q 1/6832 : Enhancement of hybridisation reaction
• CPC Class Codes: C12Q 2521/319 : Exonuclease
• CPC Class Codes: C12Q 2525/101 : incorporating non-naturally occurring nucleotides, e.g. inosine
• CPC Class Codes: C12Q 2525/125 : incorporating agents resulting in resistance to degradation
• CPC Class Codes: C12Y 113/11012 : Linoleate 13S-lipoxygenase (1.13.11.12)

Abstract

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

Claims

1. An oligonucleotide which is specifically hybridizable with single-stranded DNA or RNA and which comprises a sequence of nucleotide units, wherein said sequence is divided into:

a first region having 2′

-O-alkyl substituted nucleotide units in which said 2′

-O-alkyl substituent has as least 2 carbon atoms, and

a second region composed of nucleotide units having 2′

-deoxy sugar moieties, said nucleotide units of at least one of said first or second regions being connected by phosphorothioate linkages.

2. The oligonucleotide of claim 1 wherein said nucleotide units of said first and second regions are connected by phosphorothioate linkages.

3. The oligonucleotide of claim 1 wherein said nucleotide units of said first region are connected by phosphodiester linkages and said nucleotide units of said second region are connected by phosphorothioate linkages.

4. The oligonucleotide of claim 1 wherein said nucleotide units of said first region are connected by phosphorothioate linkages and said nucleotide units of said second region are connected by phosphodiester linkages.

5. The oligonucleotide of claim 1 wherein said second region has at least three nucleotide units.

6. The oligonucleotide of claim 1 wherein said second region has at least five nucleotide units.

7. The oligonucleotide of claim 1 having 5 to 50 nucleotide units.

8. The oligonucleotide of claim 1 having a third region, said third region having 2′

-O-alkyl substituted nucleotide units, wherein said second region is positioned between said first and third regions.

9. The oligonucleotide of claim 8 wherein said nucleotide units of said first, second and third regions are connected by phosphorothioate linkages.

10. The oligonucleotide of claim 8 wherein said nucleotide units of said first and third regions are connected by phosphodiester linkages and said nucleotide units of said second region are connected by phosphorothioate linkages.

11. The oligonucleotide of claim 8 wherein said nucleotide units of said first and third regions are connected by phosphorothioate linkages and said nucleotide units of said second region are connected by phosphodiester linkages.

12. The oligonucleotide of claim 8 wherein said second region has at least three nucleotide units.

13. The oligonucleotide of claim 8 wherein said second region has at least five nucleotide units.

14. The oligonucleotide of claim 8 having 5 to 50 nucleotide units.

15. The oligonucleotide of claim 8 wherein said nucleotide units of said first region are connected by phosphodiester linkages and said nucleotide units of said second and third regions are connected by phosphorothioate linkages.

16. The oligonucleotide of claim 1 wherein at least one nucleotide unit of said first region is a 2′

-O-alkyl-O-alkyl nucleotide unit.

17. An oligonucleotide which is specifically hybridizable with single-stranded DNA or RNA and which comprises a sequence of nucleotide units, wherein said sequence is divided into a first region having 2′

-O-alkyl substituted nucleotide units and a second region composed of nucleotide units having 2′

-deoxy sugar moieties, wherein said nucleotide units of said first region are connected by phosphodiester linkages and said nucleotide units of said second region are connected by phosphorothioate linkages.

18. The oligonucleotide of claim 17 wherein said second region has at least three nucleotide units.

19. The oligonucleotide of claim 17 wherein said second region has at least five nucleotide units.

20. The oligonucleotide of claim 17 having 5 to 50 nucleotide units.

21. The oligonucleotide of claim 17 having a third region, said third region having 2′

-O-alkyl substituted nucleotide units, wherein said second region is positioned between said first and third regions.

22. The oligonucleotide of claim 17 wherein at least one nucleotide unit of said first region is a 2′

-O-alkyl-O-alkyl nucleotide unit.

23. The oligonucleotide of claim 21 wherein said nucleotide units of said third region are connected by phosphorothioate linkages.

24. The oligonucleotide of claim 21 wherein said nucleotide units of said third region are connected by phosphodiester linkages.

25. The oligonucleotide of claim 21 wherein said second region has at least three nucleotide units.

26. The oligonucleotide of claim 21 wherein said second region has at least five nucleotide units.

27. The oligonucleotide of claim 21 having 5 to 50 nucleotide units.

28. An oligonucleotide which is specifically hybridizable with single-stranded DNA or RNA and which comprises a sequence of nucleotide units, wherein said sequence is divided into:

a first region having 2′

-O-alkyl substituted nucleotide units in which said 2′

-O-alkyl substituent has as least 2 carbon atoms, said nucleotide units of said first region being connected either only by phosphodiester linkages or only by phosphorothioate linkages; and

a second region composed of nucleotide units having 2′

-deoxy sugar moieties, said nucleotide units of at least one of said first or second regions being connected by phosphorothioate linkages.

29. An oligonucleotide which is specifically hybridizable with single-stranded DNA or RNA and which comprises a sequence of nucleotide units, wherein said sequence is divided into:

a first region having 2′

-O-alkyl substituted nucleotide units in which said 2′

-O-alkyl substituent has as least 2 carbon atoms; and

a second region composed of nucleotide units having 2′

-deoxy sugar moieties; and

a third region having 2′

-O-alkyl substituted nucleotide units;

wherein

said second region is positioned between said first and third regions,

said nucleotide units of at least one of said first or second regions is connected by phosphorothioate linkages; and

said nucleotide units of said first region or said third region are connected either only by phosphodiester linkages or only by phosphorothioate linkages.

30. An oligonucleotide which is specifically hybridizable with single-stranded DNA or RNA and which comprises a sequence of nucleotide units, wherein said sequence is divided into a first region having 2′

-O-alkyl substituted nucleotide units and a second region composed of nucleotide units having 2′

-deoxy sugar moieties, wherein said nucleotide units of said first region are connected only by phosphodiester linkages and said nucleotide units of said second region are connected by phosphorothioate linkages.