DNA-Armed ribozymes and minizymes

• US 6,365,730 B1
• Filed: 12/08/1992
• Issued: 04/02/2002
• Est. Priority Date: 06/19/1990
• Status: Expired due to Fees

• Current Assignee: Gene Shears Pty Limited
• Original Assignee: Gene Shears Pty Limited
• Inventors: Jennings, Philip Anthony, Hendry, Philip, McCall, Maxine June
• Primary Examiner(s): WANG, ANDREW J
• Application Number:
  US07/986,776
• Time in Patent Office: 3,402 Days
• Field of Search: 435/6, 435/91.31, 435/91.21, 435/91.1, 435/172.3, 435/320.1, 435/375, 536/23.2, 536/24.5, 514/44
• US Class Current: 536/24.5
• 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: 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 2310/111 spanning the whole gene, or a large part of it
• CPC Class Codes: C12N 2310/121 Hammerhead
• CPC Class Codes: C12N 2310/3183 Diol linkers, e.g. glycols or propanediols
• CPC Class Codes: C12N 2310/322 2'-R Modification
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113

Abstract

The invention describes catalytic nucleic acid based compounds capable of cleaving nucleic acid polymers both in vivo and in vitro. Two embodiments of this invention are compounds with a short stem that does not base pair, a minizyme, and compounds with DNA hybridizing arms and RNA catalytic domain and stem, DNA-armed ribozymes. The compounds of this invention, while nucleotide based may be substituted or modified in the sugar, phosphate, or base. Methods of use and methods of treatment are also described.

Claims

1. A compound having the structure:

wherein each X represents a ribonucleotide or a deoxyribonucleotide, which is the same or different and may be modified or substituted in its sugar, phosphate or base;

wherein each x represents a nucleotide which may be modified or substituted in its sugar, phosphate or base;

wherein each of A, C, U, and G represents a different ribonucleotide which may be modified in its sugar, phosphate or base;

wherein at least one nucleotide is modified or substituted in its sugar;

wherein each of (x)_n and (x)_n′

, represents an oligonucleotide having a predetermined sequence sufficiently complementary to a predefined RNA target sequence to allow hybridization to the RNA target sequence and each of n and n′

represents an integer which defines the number of nucleotides in the oligonucleotide with the proviso that the sum of n+n′

is sufficient to allow the compound to hybridize with the RNA target sequence;

wherein each * represents base pairing between the nucleotides located on either side thereof;

wherein each solid line represents a chemical linkage providing covalent bonds between the nucleotides located on either side thereof;

wherein a represents an integer which defines a number of nucleotides with the proviso that a may be 0 or 1 and if 0, the A located 5′

of (X)_a is bonded to the X located 3′

of (X)_a;

wherein each of m and m′

represents 0 or an integer which is greater than or equal to 1; and

wherein (X)_b represents an oligonucleotide and b represents an integer which is greater than or equal to 2.

2. The compound of claim 1, wherein each of m and m′

are one and b is four.

3. The compound of claim 1, wherein each of m and m′

are seven and b is four.

4. The compound of claim 1, wherein (x)_n or (x)_n′

represents an oligodeoxyribonucleotide.

5. The compound of claim 1, wherein (x)_n and (x)_n′

represent oligodeoxyribonucleotides.

6. The compound of claim 1, wherein each X represents a ribonucleotide.

7. The compound of claim 1, wherein each X represents a deoxyribonucleotide.

8. The compound of claim 1 having the structure:

wherein each X represents a ribonucleotide or a deoxyribonucleotide, which is the same or different and may be modified or substituted in its sugar, phosphate or base;

wherein each x represents a nucleotide which may be modified or substituted in its sugar, phosphate or base;

wherein each of A, C, U, and G represents a different ribonucleotide which may be modified in its sugar, phosphate or base;

wherein c represents a cytosine nucleotide which may be modified or substituted in its sugar, phosphate or base;

wherein at least one nucleotide is modified or substituted in its sugar;

wherein each of (x)_n and (x)_n′

represents an oligonucleotide having a predetermined sequence sufficiently complementary to a predefined RNA target sequence to allow hybridization to the RNA target sequence and each of n and n′

represents an integer which defines the number of nucleotides in the oligonucleotide with the proviso that the sum of n+n′

is sufficient to allow the compound to hybridize with the RNA target sequence;

wherein each * represents base pairing between the nucleotides located on either side thereof;

wherein each solid line represents a chemical linkage providing covalent bonds between the nucleotides located on either side thereof;

wherein a represents an integer which defines a number of nucleotides with the proviso that a may be 0 or 1 and if 0, the A located 5′

of (X)_a is bonded to the X located 3′

of (X)_a;

wherein each of m and m′

represents 0 or an integer which is greater than or equal to 1; and

wherein (X)_b represents an oligonucleotide and b represents an integer which is greater than or equal to 2.

9. The compound of claim 8, wherein each of m and m′

are one and b is four.

10. The compound of claim 8, wherein each of m and m′

are seven and b is four.

11. The compound of claim 8, wherein (x)_n or (x)_n′

represents an oligodeoxyribonucleotide.

12. The compound of claim 8, wherein (x)_n and (x)_n′

represent oligodeoxyribonucleotides.

13. The compound of claim 8, wherein each X represents a ribonucleotide.

14. The compound of claim 8, wherein each X represents a deoxyribonucleotide.

22. The compound of claim 1, wherein each at least one nucleotide in the compound is a 2′

-methoxyribonucleotide.

23. The compound of claim 1, wherein each at least one nucleotide in the compound is a a-anomer of the nucleotide.

24. The compound of claim 1, wherein the 3′

end of the oligonucleotide (x)_n and/or the 5′

end of the oligonucleotide (x)_n′

contain a blocking group which is resistant to nuclease attack.

25. The compound of claim 24, wherein the blocking group is selected from the group consisting of optionally substituted alkyl, optionally substituted phenyl, optionally substituted alkanoyl, phosphorothioate, phosphoroamidate or methyl phosphonate nucleotide analogues which are resistant to nuclease attack, spermine, and spermidine.

15. A compound having the structure:

wherein each X represents a ribonucleotide or a deoxyribonucleotide, which is the same or different and may be modified or substituted in its sugar, phosphate or base;

wherein each x represents a nucleotide which may be modified or substituted in its sugar, phosphate or base;

wherein at least one nucleotide represented by X or x is modified or substituted in its sugar;

wherein each of A, C, U, and G represents a different ribonucleotide;

wherein each of (x)_n and (x)_n′

represents an oligonucleotide having a predetermined sequence sufficiently complementary to a predefined RNA target sequence and each of n and n′

represents an integer which defines the number of nucleotides in the oligonucleotide with the proviso that the sum of n+n′

is sufficient to allow the compound to hybridize with the RNA target sequence;

wherein each * represents base pairing between the nucleotides located on either side thereof;

wherein each solid line represents a chemical linkage providing covalent bonds between the nucleotides located on either side thereof;

wherein a represents an integer which defines a number of nucleotides with the proviso that a may be 0 or 1 and if 0, the A located 5′

of (X)_a is bonded to the X located 3′

of (X)_a;

wherein each of m and m′

represents 0 or an integer which is greater than or equal to 1; and

wherein (X)_b represents an oligonucleotide and b represents an integer which is greater than or equal to 2.

16. The compound of claim 15, wherein each of m and m′

are one and b is four.

17. The compound of claim 15, wherein each of m and m′

are seven and b is four.

18. The compound of claim 15, wherein (x)_n or (x)_n′

represents an oligodeoxyribonucleotide.

19. The compound of claim 15 having the structure:

wherein each X represents a ribonucleotide or a deoxyribonucleotide, which is the same or different and may be modified or substituted in its sugar, phosphate or base;

wherein each x represents a nucleotide which may be modified or substituted in its sugar, phosphate or base;

wherein c represents a cytosine nucleotide which may be modified or substituted in its sugar, phosphate or base;

wherein at least one nucleotide represented by X or x is modified or substituted in its sugar;

wherein each of A, C, U, and G represents a different ribonucleotide;

wherein each of (x)_n and (x)_n′

represents an oligonucleotide having a predetermined sequence sufficiently complementary to a predefined RNA target sequence to allow hybridization to the RNA target sequence and each of n and n′

represents an integer which defines the number of nucleotides in the oligonucleotide with the proviso that the sum of n+n′

is sufficient to allow the compound to hybridize with the RNA target sequence;

wherein each * represents base pairing between the nucleotides located on either side thereof;

wherein each solid line represents a chemical linkage providing covalent bonds between the nucleotides located on either side thereof;

wherein a represents an integer which defines a number of nucleotides with the proviso that a may be 0 or 1 and if 0, the A located 5′

of (X)_a is bonded to the X located 3′

of (X)_a;

wherein each of m and m′

represents 0 or an integer which is greater than or equal to 1; and

wherein (X)_b represents an oligonucleotide and b represents an integer which is greater than or equal to 2.

20. The compound of claim 19, wherein each of m and m′

are one and b is four.

21. The compound of claim 19, wherein (x)_n or (x)_n′

represents an oligodeoxyribonucleotide.

26. The compound of claim 15, wherein each at least one nucleotide in the compound is a 2′

-methoxyribonucleotide.

27. The compound of claim 15, wherein each at least one nucleotide in the compound is a a-anomer of the nucleotide.

28. The compound of claim 15, wherein the 3′

end of the oligonucleotide (x)_n and/or the 5′

end of the oligonucleotide (x)_n′

contain a blocking group which is resistant to nuclease attack.

29. The compound of claim 28, wherein the blocking group is selected from the group consisting of optionally substituted alkyl, optionally substituted phenyl, optionally substituted alkanoyl, phosphorothioate, phosphoroamidate or methyl phosphonate nucleotide analogues which are resistant to nuclease attack, spermine, and spermidine.