Gapped 2' modified oligonucleotides

• US 5,623,065 A
• Filed: 06/21/1994
• Issued: 04/22/1997
• Est. Priority Date: 08/13/1990
• Status: Expired due to Term

• Current Assignee: ISIS Pharmaceuticals Incorporated
• Original Assignee: ISIS Pharmaceuticals Incorporated
• Inventors: Monia, Brett P., Cook, Phillip D.
• Primary Examiner(s): Low, Christopher S. F.
• Application Number:
  US08/244,993
• Time in Patent Office: 1,036 Days
• Field of Search: 514/44, 536/23.1, 536/23.2, 536/23.5, 536/23.51, 536/23.52, 536/23.53, 536/25.1, 536/25.2, 435/91.1, 435/91.2, 435/91.4, 435/91.5, 935/9, 935/6, 935/10
• 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: 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 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: 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 that 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 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.

Claims

1. A compound comprising a plurality of units linked by covalent linkages in a sequence that is hybridizable to a complementary nucleic acid encoding a ras gene product, 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;

said sequence of linked units is divided into at least two segments, wherein;

a first of said segments 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 non-phosphorous linkages,

said carbocyclic nucleosides linked by charged and neutral phosphorous linkages,

said carbocyclic nucleosides linked 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,

said β

-nucleosides linked by non-phosphorous linkages; and

a second of said segments including said nucleobases linked by non-phosphorous linkages and nucleobases that are attached to phosphate linkages via a non-sugar tethering moiety;

said sequence of linked nucleosides has at least one of SEQ ID NOS;

1-6; and

said compound has increased binding affinity to said complementary nucleic acid, inhibits translation of said complementary nucleic acid, and promotes RNase H cleavage in vitro.

2. The compound of claim 1 wherein said non-phosphate linkage is a peptide linkage.

3. A compound of claim 1 including a plurality of said first segments.

4. A compound of claim 1 including a plurality of said second regions.

5. A compound of claim 3 including a plurality of said first segments.

6. A compound comprising a plurality of nucleosides linked by covalent linkages in a sequence that is hybridizable to a complementary nucleic acid encoding a ras gene product, wherein:

said nucleosides are selected from α

-nucleosides, β

-nucleosides, 4'"'"'-thionucleosides and carbocyclic nucleosides;

said linkages are selected from charged phosphorous linkages, neutral phosphorous linkages or non-phosphorous linkages;

said sequence of linked nucleosides contains at least two nucleoside segments, wherein;

a first of said segments 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 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 non-phosphorous linkages,

said β

-nucleosides linked by charged and neutral 2'"'"'-5'"'"' linkages, and

said β

-nucleosides linked by non-phosphorous linkages;

a second of said segments consists of 2'"'"'-deoxy-erythro-pentofuranosyl β

nucleosides linked by charged 3'"'"'-5'"'"' phosphorous linkages having a negative charge at physiological pH;

said sequence of linked nucleosides has at least one of SEQ ID NOS;

1-6; and

said compound has increased binding affinity to said complementary nucleic acid, inhibits translation of said complementary nucleic acid, and promotes RNase H cleavage in vitro.

7. A compound of claim 6 wherein said second nucleoside segments is positioned between said first nucleoside segment and a third nucleoside segment, said third nucleoside segment 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.

8. A compound of claim 6 wherein said charged phosphorous linkages are selected from phosphodiester, phosphorothioate, phosphorodithioate, phosphoroselenate or phosphorodiselenate linkages.

9. A compound of claim 6 wherein said charged phosphorous linkages are phosphodiester or phosphorothioate.

10. A compound of claim 6 wherein said neutral phosphorous linkages are selected from alkyl and aryl phosphonates, alkyl and aryl phosphoroamidites, alkyl and aryl phosphotriesters, hydrogen phosphonate and boranophosphate linkages.

11. A compound of claim 6 wherein said non-phosphorous linkages are selected from 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.

12. A compound of claim 6 wherein said first nucleoside segment includes at least two α

-nucleoside linked by a charged or neutral 3'"'"'-5'"'"' phosphorous linkages.

13. A compound of claim 6 wherein said second segment includes at least 3 of said 2'"'"'-deoxy-erythro-pentofuranosyl β

-nucleosides.

14. A compound comprising a plurality of units linked by covalent linkages in a sequence that is hybridizable to a complementary nucleic acid encoding a ras gene product, 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 3'"'"'-5'"'"' phosphorous, neutral 3'"'"'-5'"'"' phosphorous, charged 2'"'"'-5'"'"' phosphorous neutral 2'"'"'-5'"'"' phosphorous or non-phosphorous linkages;

said sequence of linked units is divided into at least two segments, wherein;

a first of said segments includes linked moieties selected from;

said nucleobases linked by non-phosphorous linkages,

said nucleobases linked to phosphate linkages via non-sugar tethering groups,

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 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 non-phosphorous linkages,

said β

-nucleosides linked by charged and neutral 2'"'"'-5'"'"' linkages, and

said β

-nucleosides linked by non-phosphorous linkages;

a second of said segments includes 2'"'"'-deoxy-erythro-pentofuranosyl β

nucleosides linked by charged 3'"'"'-5'"'"' phosphorous linkages having a negative charge at physiological pH;

said sequence of linked nucleosides has at least one of SEQ ID NOS;

1-6; and

said compound has increased binding affinity to said complementary nucleic acid, inhibits translation of said complementary nucleic acid, and promotes RNase H cleavage in vitro.

15. The compound of claim 14 wherein said first segment includes at least two nucleobases linked by a non-phosphate linkage.

16. The compound of claim 15 wherein said non-phosphate linkage is a peptide linkage.

17. The compound of claim 15 wherein said second segment is positioned between said first segment and a third segment, said third segment including linked moieties selected from:

said nucleobases linked by non-phosphorous linkages,

said nucleobases linked to phosphate linkages via a non-sugar tethering moiety,

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.

18. A compound of claim 15 wherein said nucleobases are selected from adenine, guanine, cytosine, uracil, thymine, xanthine, hypoxanthine, 2-aminoadenine, alkyl adenine, 5-halo uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudo uracil), 4-thiouracil, 8-substituted adenine and guanine, or 5-trifluoromethyl uracil and cytosine.

19. A method of modifying in vitro a sequence-specific RNA, comprising contacting a test solution containing RNase H and said RNA with a compound of claim 14 under conditions that effect hybridization of said RNA and said compound and cleavage of said RNA.