Complex formation between dsDNA and pyrrole imidazole polyamides

• US 6,143,901 A
• Filed: 04/21/1997
• Issued: 11/07/2000
• Est. Priority Date: 07/31/1996
• Status: Expired due to Term

• Current Assignee: California Institute of Technology
• Original Assignee: Genesoft, Inc. (Oscient Pharmaceuticals Corporation)
• Inventors: Dervan, Peter B.
• Primary Examiner(s): Higel, Floyd D.
• Application Number:
  US08/837,524
• Time in Patent Office: 1,296 Days
• Field of Search: 548/312.4, 435/75
• US Class Current: 548/312.4
• 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 47/645 Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
• CPC Class Codes: C07D 207/34 with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
• CPC Class Codes: C07D 233/90 Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
• CPC Class Codes: C07D 403/14 containing three or more hetero rings
• CPC Class Codes: C07K 7/02 Linear peptides containing at least one abnormal peptide link

Abstract

Methods and compositions are provided for forming complexes between dsDNA and oligomers of heterocycles, aliphatic amino acids, particularly omega-amino acids, and a polar end group. By appropriate choice of target sequences and composition of the oligomers, complexes are obtained with low dissociation constants. The formation of complexes can be used for identification of specific dsDNA sequences, for inhibiting gene transcription, and as a therapeutic for inhibiting proliferation of undesired cells or expression of undesired genes.

Claims

1. A method for forming a specific complex between a target sequence of double-stranded DNA and an oligomer of N-methyl pyrrole (Py) and N-methyl imidazole (Im) comprising the steps of:

identifying a target sequence of double-stranded DNA;

contacting the target sequence of double-stranded DNA with a transcription inhibiting amount of a first oligomer containing from 6 to 30 N-heterocycles chosen from the group consisting of N-methyl pyrrole (Py) carboxamide and N-methyl imidazole (Im) carboxamide, wherein the N-heterocycles are connected by linking groups;

forming a complex of the first oligomer and the target sequence of double-stranded DNA, wherein pairs of the carboxamides form pairwise hydrogen bonds with nucleotide base pairs in the minor groove of the double stranded DNA, wherein a Im/Py carboxamide pair forms hydrogen bonds with a G/C nucleotide base pair, a Py/Im carboxamide pair forms hydrogen bonds with a C/G nucleotide base pair, a Py/Py carboxamide pair forms hydrogen bonds with an A/T nucleotide base pair or a T/A nucleotide base pair, wherein the formed complex has a dissociation constant of no more than about one nanomolar.

6. A method according to claim 1, wherein at least one of said second oligomers contains at least 2 unpaired N-heterocycles.

7. A method according to claim 6, wherein each of said second oligomers contains at least 3 unpaired N-heterocycles.

8. A method according to claim 1, wherein said first oligomer contains at least 8 N-heterocycles.

9. A method according to claim 1, wherein said oligomer contains an internal β

-alanine dividing a chain of 6 N-heterocycles and separated from said γ

-aminobutryic acid by at least 2 heterocycles.

2. A method according to claim 2, wherein at least one of said linking groups is an amido group.

3. The method of claim 2, containing the step of forming a complex between the first oligomer, the target sequence of double-stranded DNA and a second oligomer containing from six to 30 N-heterocycles chosen from the group consisting of N-methyl pyrrole (Py) carboxamide and N-methyl imidazole (Im) carboxamide.

4. A method according to claim 3, wherein there are not more than 2 consecutive Ims.

5. The method of claim 3 wherein the first and the second oligomers each have one β

-alanine separating units of at least 2 N-heterocycles.

10. A method of detecting target dsDNA in a sample employing from 1 to 2 oligomers of N-heterocycles selected from the group consisting of N-nethyl pyrrole (Py) and N-methyl imidazole (Im), wherein said N-heterocycles and other members of said oligomers are selected to provide a K_d ≦

1nM, wherein said oligomer is defined as having at least 6 said heterocycles, where the order of heterocycles in relation to said target dsDNA is defined as Im/Py in juxtaposition to G/C, Py/Im in juxtaposition to C/G, and Py/Py in juxtaposition to A/T and T/A, said oligomer containing at least two units of 3 consecutive heterocycles forming complementary pairs with itself as a first oligomer or another oligomer as second oligomers, where when said oligomer forms said complementary pairs with itself, said oligomer contains an internal γ

-aminobutyric acid, and when two oligomers form said complementary pairs, said oligomers contain an internal β

-alanine, said internal β

-alanine being in juxtaposition to A/T and T/A and forming a complementary pair with itself, said heterocycles being linked by one or more groups for forming hydrogen bonds to available nitrogen or oxygen atoms, or nitrogen and oxygen atoms of said dsDNA, at least one oligomer joined to a moiety for detection of complex formation between said target dsDNA and said oligomers, said method comprising the steps of;

combining said oligomers and said sample under complex forming conditions; and

detecting the presence of said target dsDNA in said sample as a complex with said oligomers by means of said moiety.

11. A method according to claim 10, wherein said moiety is an enzyme, a fluorescer, a chemiluminescer, a solid surface, a hapten which binds to a receptor, or a radioactive isotope.

12. A method according to claim 10, wherein said method further comprises:

separating any complex from any other dsDNA in said sample before detecting said complex.

34. The method of claim 10 or 14, wherein a second γ

-aminobutyric acid joins the termini to define a ring of said first oligomer.

13. A method for isolating target dsDNA from a mixture of dsDNA employing employing from 1 to 2 oligomers of N-heterocycles selected from the group consisting of N-methyl pyrrole (Py) and N-methyl imidazole (Im), wherein said N-heterocycles and other members of said oligomers are selected to provide a K_d ≦

1 nM, wherein said oligomer is defined as having at least 6 said heterocycles, where the order of heterocycles in relation to said target dsDNA is defined as Im/Py in juxtaposition to G/C, Py/Im in juxtaposition to C/G, and Py/Py in juxtaposition to A/T and T/A, said oligomer containing at least two units of 3 consecutive heterocycles forming complementary pairs with itself as a first oligomer or another oligomer as second oligomers, where when said oligomer forms said complementary pairs with itself, said oligomer containing an internal γ

-aminobutyric acid, and when two oligomers form said complementary pairs, said oligomers contain an internal β

-alanine, said internal β

-alanine being in juxtaposition to A/T and T/A and forming a complementary pair with itself, said heterocycles being linked by one or more groups for forming hydrogen bonds to available nitrogen or oxygen atoms, or nitrogen and oxygen atoms of said dsDNA, at least one oligomer joined to a moiety for separation of complexes between said target dsDNA and said oligomers, said method comprising the steps of;

combining said oligomers and said sample under complex forming conditions; and

;

separating complexes which form by means of said moiety.

14. A method according to claim 13, wherein said moiety is a hapten and said oligomers and mixture are combined with a receptor for said hapten bound to a solid surface.

15. A composition containing from 1 to 2 oligomers of N-heterocycles selected from the group consisting of N-methyl pyrrole (Py) and N-methyl imidazole (Im), wherein said N-heterocycles and other members of said oligomers are selected to provide a K_d ≦

1 nM, wherein said oligomer is defined as having at least 6 said heterocycles, where the order of heterocycles in relation to said target dsDNA is defined as Py in juxtaposition to A, G and T, and Im in juxtaposition to C, said oligomer containing at least two units of 3 consecutive heterocycles forming complementary pairs with itself as a first oligomer or another oligomer as second oligomers, where when said oligomer forms said complementary pairs with itself, said oligomer contains an internal γ

-aminobutyric acid, and when two oligomers form said complementary pairs, said oligomers contain an internal β

-alanine, said internal β

-alanine being in juxtaposition to A and T and forming a complementary pair with itself, said heterocycles being linked by one or more groups for forming hydrogen bonds to available nitrogen or oxygen, or nitrogen and oxygen atoms of said dsDNA.

16. A composition according to claim 15, wherein at least one of said linking groups is an amido group.

17. A composition according to claim 15, wherein said composition contains one oligomer.

18. A composition according to claim 15, wherein at least one of said 1 to 2 oligomers contains at least 7 N-heterocycles.

19. A composition according to claim 15, wherein at least one of said 1 to 2 oligomers contains a β

-alanine internal to six consecutive N-heterocycles and separated from said γ

-aminobutyric acid by at least 2 heterocycles.

35. The composition of claim 15, wherein a second γ

-aminobutyric acid joins the termini to define a ring of said oligomers.

20. A method of forming a specific complex with a target sequence with of double stranded DNA comprising the steps of:

a. identifying the target sequence of double stranded DNA;

b. contacting the target sequence with a transcription inhibiting amount of at least one oligomer that contains about 6 to about 30 heterocycles; and

c. forming at least two complementary pairs of heterocycles in apposition to at least two nucleotide base pairs of the target sequence,

wherein the specific complex formed has a binding affinity of at least about 10⁸ M^-1.

21. The method of claim 20 wherein the heterocycles are chosen from the group consisting of pyrrole, triazole, furan, thiophen, oxazole, thiazole, cyclopentadiene, pyridine, pyrimidine, triazine, and methyl-, ethyl- and propyl- derivatives thereof.

22. The method of claim 21 wherein the heterocycles are chosen from the group consisting of pyrrole, imidazole, and N-methyl-, N-ethyl- and N-propyl- derivatives thereof.

23. The method of claim 21 wherein the heterocycles are chosen from the group consisting of N-methyl-pyrrole and N-methyl-imidazole.

24. The method of claim 20 wherein said oligomer contains at least one aliphatic amino acid having from 2 to 6 carbon atoms.

25. The method of claim 23 wherein at least one aliphatic amino acid is interposed between heterocycles.

26. The method of claim 23 wherein at least one aliphatic amino acid is at the amino terminal of the oligomer of heterocycles.

27. The method of claim 23 wherein at least one aliphatic amino acid is at the carboxyl terminal of the oligomer of heterocycles.

28. The method of claim 24 wherein at least one aliphatic amino acid is beta-alanine.

29. The method of claim 24 wherein at least one aliphatic amino acid is gamma-aminobutyric acid.

30. The method of claim 20 wherein the specific complex formed has a binding affinity of at least about 10⁹ M^-1.

31. The method of claim 23 wherein the oligomer is terminated by an alkyl chain containing a polar group located about 2 to about 4 carbon atoms from the bond linking the alkyl chain to the rest of the oligomer.

32. The method of claim 20 wherein the transcription inhibiting amount is about 0.1 nanomolar to about 1 millimolar.

33. The method of claim 20 wherein the transcription inhibiting amount is about 10 nanomolar to about 1 micromolar.