Gapped 2' modified oligonucleotides

• US 6,146,829 A
• Filed: 08/31/1998
• Issued: 11/14/2000
• Est. Priority Date: 12/24/1991
• Status: Expired due to Fees

• Current Assignee: ISIS Pharmaceuticals Incorporated (Ionis Pharmaceuticals, Inc.)
• Original Assignee: ISIS Pharmaceuticals Incorporated (Ionis Pharmaceuticals, Inc.)
• Inventors: Monia, Brett P., Cook, Phillip Dan
• Primary Examiner(s): Schwartzman, Robert A.
• Assistant Examiner(s): LARSON, THOMAS GEORGE
• Application Number:
  US09/144,611
• Time in Patent Office: 806 Days
• Field of Search: 435/6, 435/91.5, 435/91.51, 435/91.53, 435/199, 536/23.1, 536/24.3, 536/24.5
• US Class Current: 435/6.11
• 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 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: C07K 14/003 Peptide-nucleic acids (PNAs)
• CPC Class Codes: C07K 14/82 Translation products from oncogenes
• CPC Class Codes: C12N 15/1135 against oncogenes or tumor suppressor genes
• 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 2310/3521 Methyl
• 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: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• 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

Abstract

Oligonucleotides and other macromolecules are provided that have increased nuclease resistance, substituent groups for increasing binding affinity to complementary strand, and sub-sequences 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 method of modifying in vitro a sequence-specific ribonucleic acid, comprising contacting a test solution containing RNase HI and said sequence-specific ribonucleic acid with an oligonucleotide having a sequence of nucleotides under conditions that effect hybridization of said sequence-specific ribonucleic acid and said oligonucleotide, where at least one of said nucleotides is functionalized to increase nuclease resistance of the oligonucleotide, where a plurality of the nucleotides have a substituent group located thereon to increase binding affinity of the oligonucleotide to a complementary strand of sequence-specific ribonucleic acid, and where a plurality of the nucleotides have 2'"'"'-deoxy-erythro-pentofiranosyl sugar moieties.

2. The method of claim 1 wherein said substituent group for increasing binding affinity comprises a 2'"'"'-substituent group.

3. The method of claim 1 wherein said substituent group for increasing binding affinity comprises a 2'"'"'-substituent group that is fluoro, C1-C9 alkoxy, C1-C9 aminoalkoxy, allyloxy, imidazolealkoxy or poly(ethylene glycol).

4. The method of claim 1 wherein each of said nucleotides is a phosphorothioate or phosphorodithioate nucleotide.

5. The method of claim 1 wherein the 3'"'"' terminal nucleotide of said oligonucleotide includes a nuclease resistance modifying group on at least one of the 2'"'"' or the 3'"'"' positions of said nucleotide.

6. The method of claim 1 wherein:

a plurality of said nucleotides bear substituent groups that increases binding affinity of said oligonucleotide to said sequence-specific ribonucleic acid, said substituent-bearing nucleotides being divided into a first nucleotide unit sub-sequence and a second nucleotide unit sub-sequence; and

said plurality of 2'"'"'-deoxy-erythro-pentofuranosyl nucleotides is positioned in said sequence of nucleotides between said first nucleotide unit sub-sequence and said second nucleotide unit sub-sequence.

7. The method of claim 1 wherein:

a plurality of said nucleotides bear substituent groups that increase binding affinity of said oligonucleotide to said complementary strand of nucleic acid; and

at least a portion of said substituent-bearing nucleotides are consecutively located at one of the 3'"'"' terminus or the 5'"'"' terminus of said oligonucleotide.

8. The method of claim 1 wherein at least five of said nucleotides have 2'"'"'-deoxy-erythro-pentofuran-osyl sugar moieties, said at least five 2'"'"'-deoxy-erythro-pentofuranosyl nucleotides being consecutively located in said sequence of nucleotides.

9. The method of claim 1 wherein from one to about eight of said nucleotides bear a substituent group that increases the binding affinity of said oligonucleotide to said complementary strand, said substituent-bearing nucleotides being consecutively located in said sequence of nucleotides.

10. The method of claim 1 wherein:

from one to about eight of said nucleotides bear a substituent group for increasing the binding affinity of said oligonucleotide to said complementary strand, said substituent-bearing nucleotides being consecutively located in said sequence of nucleotides; and

at least five of said nucleotides have 2'"'"'-deoxy-erythro-pentofuranosyl sugar moieties, said at least five 2'"'"'-deoxy-erythro-pentofuranosyl nucleotides being consecutively located in said sequence of nucleotides.

11. A method of modifying in vitro a sequence-specific ribonucleic acid, comprising contacting a test solution containing a RNase H and said sequence-specific ribonucleic acid with a compound comprising a plurality of units linked by covalent linkages in a sequence, wherein said contacting occurs under conditions that effect hybridization of said sequence-specific ribonucleic acid and said compound, and:

said units are selected from nucleosides and nucleobases;

said nucleosides are selected from α

-nucleosides, β

-nucleosides including 2'"'"'-deoxy-erythro-pentofuranosyl 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; and

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

a first of said regions includes said nucleobases linked by non-phosphorous linkages and nucleobases that are attached to phosphate linkages via non-sugar tethering groups, and 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; and

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

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

12. The method of claim 11 wherein said first region includes at least two nucleobases linked by a non-phosphate linkage.

13. The method of claim 12 wherein said non-phosphate linkage is a peptide linkage.

14. The method of claim 11 wherein said second region is positioned between said first region and a third region, said third region including said nucleobases linked by non-phosphorous linkages and nucleobases that are attached to phosphate linkages via a non-sugar tethering moiety, and 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.

15. A method of claim 11 wherein said nucleobases are selected from adenine, guanine, cytosine, uracil, thymine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl adenine, 2-propyl and other alkyl adenine, 5-halo uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, amino, thiol, thiolalkyl, hydroxyl and other 8 substituted adenine and guanine, or 5-trifluoromethyl uracil and cytosine.