PNA-DNA-PNA chimeric macromolecules

• US 5,700,922 A
• Filed: 11/24/1993
• Issued: 12/23/1997
• Est. Priority Date: 12/24/1991
• Status: Expired due to Term

• Current Assignee: ISIS Pharmaceuticals Incorporated
• Original Assignee: ISIS Pharmaceuticals Incorporated
• Inventors: Cook, Phillip Dan
• Primary Examiner(s): Low, Christopher S. F.
• Application Number:
  US08/158,352
• Time in Patent Office: 1,490 Days
• Field of Search: 514/44, 435/91.1, 435/91.5, 536/23.5, 536/24.1, 536/24.2, 536/24.33, 536/25.1, 536/25.2, 536/25.3, 536/25.31, 536/24.5, 536/24.31, 536/24.32
• 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: A61P 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 33/00 Antiparasitic agents
• 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 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 9/005 containing within the molecule the substructure with m, n > 0 and m+n > 0, A, B, D, E being heteroatoms; X being a bond or a chain, e.g. muramylpeptides
• 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

Abstract

Macromolecules are provided that have increased nuclease resistance, increasing binding affinity to a complementary strand, and that activate RNase H enzyme. The macromolecules have the structure PNA-DNA-PNA where the DNA portion is composed of subunits of 2'"'"'-deoxy-erythro-pentofuranosyl nucleotides and the PNA portions are composed of subunits of peptide nucleic acids. Such 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 therapeutics.

Claims

1. A macromolecule of the structure:

space="preserve" listing-type="equation">PNA-DNA-PNA

wherein;

said DNA of said PNA-DNA-PNA comprises at least one 2'"'"'-deoxynucleotide; and

each of said PNAs of said PNA-DNA-PNA comprise at least one peptide nucleic acid subunit having formula (I);

##STR6## wherein n is at least 2,

each of L¹ and Lⁿ is independently selected from the group consisting of hydrogen, hydroxy, (C₁ -C₄)alkanoyl, nucleobases, aromatic moieties, DNA intercalators, nucleobase-binding groups, heterocyclic moieties, and reporter ligands, at least one of L¹ to Lⁿ being a nucleobase, a DNA intercalator, or a nucleobase-binding group;

each of R³ and R⁴ is independently selected from the group consisting of hydrogen, (C₁ -C₄)alkyl, hydroxy- or alkoxy- or alkylthio-substituted (C₁ -C₄)alkyl, hydroxy, alkoxy, alkylthio and amino;

each of C¹ to Cⁿ is (CR⁶ R⁷)_y where R⁶ is hydrogen and R⁷ is selected from the group consisting of the side chains of naturally occurring alpha amino acids, or R⁶ and R⁷ are independently selected from the group consisting of hydrogen, (C₂ -C₆)alkyl, aryl, aralkyl, heteroaryl, hydroxy, (C₁ -C₆)alkoxy, (C₁ -C₆)alkylthio, NR³ R⁴ and SR⁵, where R³ and R⁴ are as defined above, and R⁵ is hydrogen, (C₁ -C₆)alkyl, hydroxy-, alkoxy-, or alkylthio-substituted (C₁ -C₆)alkyl, or R⁶ and R⁷, together, are alicyclic or heterocyclic;

each of D¹ to Dⁿ is (CR⁶ R⁷)_z where R⁶ and R⁷ are as defined above;

each of y and z is zero or an integer from 1 to 10, the sum y+z being greater than 2 but not more than 10;

each of G¹ to G^n-1 is --NR³ CO--, --NR³ CS--, --NR³ SO-- --NR³ SO₂ --, --CONR³ --, --CSNR³ --, --SONR³ -- or --SO₂ NR₃ -- where R³ is as defined above;

each pair of A¹ to Aⁿ and B¹ to Bⁿ are selected such that;

(a) A is a group of formula (IIa), (IIb) or (IIc) and B is N or R³ N⁺ ;

or

(b) A is a group of formula (IId) and B is CH;

##STR7## where;

X is O, S, Se, NR³, CH₂ or C(CH₃)₂ ;

Y is a single bond, O, S or NR⁴ ;

each of p and q is zero or an integer from 1 to 5, the sum p+q being not more than 10;

each of r and s is zero or an integer from 1 to 5, the sum r+s being not more than 10;

each R¹ and R² is independently selected from the group consisting of hydrogen, (C₁ -C₄)alkyl which may be hydroxy- or alkoxy- or alkylthio-substituted, hydroxy, alkoxy, alkylthio, amino and halogen;

Q is --CO₂ H, --CONR'"'"'R", --SO₃ H or --SO₂ NR'"'"'R" or an activated derivative of --CO₂ H or --SO₃ H; and

I is --NR'"'"'"R"" or --NR'"'"'"C(O)R"";

where;

R'"'"', R", R'"'"'", and R"" are independently selected from the group consisting of hydrogen, alkyl, amino protecting groups, reporter ligands, intercalators, chelators, peptides, carbohydrates, lipids, steroids, nucleosides, nucleotides, nucleotide diphosphates, nucleotide triphosphates, oligonucleotides, oligonucleosides and soluble and non-soluble polymers, provided that at least one R'"'"' is said DNA of said PNA-DNA-PNA and at least one R"" is said DNA of said PNA-DNA-PNA.

14. A macromolecule of claim 1 wherein said PNA-DNA-PNA macromolecule hybridizes to a strand of nucleic acid.

15. A macromolecule of claim 14 wherein said strand of nucleic acid is a RNA strand.

16. A macromolecule of claim 1 wherein:

said DNA of said PNA-DNA-PNA includes at least three 2'"'"'-deoxynucleotides linked together in a sequence; and

each PNA of said PNA-DNA-PNA includes at least two peptide nucleic acid subunits.

17. A macromolecule of claim 1 wherein said 2'"'"'-deoxynucleotide is a phosphodiester, a phosphorothioate or a phosphorodithioate 2'"'"'-deoxynucleotide.

18. A macromolecule of claim 1 wherein said DNA of said PNA-DNA-PNA includes at least three 2'"'"'-deoxynucleotides linked together in a sequence by phosphodiester, phosphorothioate or phosphorodithioate linkages.

19. A macromolecule of claim 1 wherein each of said PNAs of said PNA-DNA-PNA comprises a compound having a formula selected from the group consisting of IIIa, IIIb and IIIc:

##STR11## wherein;

each L is independently selected from the group consisting of hydrogen, phenyl, heterocyclic moieties, and nucleobases;

each R^7'"'"' is independently selected from the group consisting of hydrogen, and the side chains of naturally occurring alpha amino acids;

n is an integer from 1 to 60;

each of k, l, and m is independently zero or an integer from 1 to 5;

p is zero or 1;

R^h is OH or NR'"'"'R"; and

Rⁱ is --R"" or --C(O)R"".

20. A macromolecule of claim 19 where each of said PNAs of said PNA-DNA-PNA comprise a compound having a formula selected from the group consisting of (IIIa), (IIIb), and (IIIc), wherein each L is independently selected from the group consisting of the nucleobases thymine (T), adenine (A), cytosine (C), guanine (G) and uracil (U), k and m are zero or 1, and n is an integer from 1 to 30.

21. A compound of claim 20 wherein:

said DNA of said PNA-DNA-PNA includes at least three of said 2'"'"'-deoxynucleotides linked together in a sequence;

each PNA of said PNA-DNA-PNA includes at least two peptide nucleic acid subunits; and

said 2'"'"'-deoxynucleotides are joined via phosphodiester, phosphorothioate or phosphorodithioate linkages.

22. A macromolecule of claim 1 wherein each of said PNAs of said PNA-DNA-PNA is covalently bound to said DNA of said PNA-DNA-PNA with an amide, amine or ester linkage.

2. A macromolecule of the structure:

space="preserve" listing-type="equation">PNA-DNA or DNA-PNA

wherein;

said DNA of said PNA-DNA or DNA-PNA comprises at least one 2'"'"'-deoxynucleotide; and

said PNA of said PNA-DNA or DNA-PNA comprises at least one peptide nucleic acid subunit having formula (I);

##STR8## wherein;

n is at least 2,

each of L¹ to Lⁿ is independently selected from the group consisting of hydrogen, hydroxy, (C₁ -C₄)alkanoyl, nucleobases, aromatic moieties, DNA intercalators, nucleobase-binding groups, heterocyclic moieties, and reporter ligands, at least one of L¹ to Lⁿ being a nucleobase, a DNA intercalator, or a nucleobase-binding group;

each of R³ and R⁴ is independently selected from the group consisting of hydrogen, (C₁ -C₄)alkyl, hydroxy- or alkoxy- or alkylthio-substituted (C₁ -C₄)alkyl, hydroxy, alkoxy, alkylthio and amino;

each of C¹ to Cⁿ is (CR⁶ R⁷)_y where R⁶ is hydrogen and R⁷ is selected from the group consisting of the side chains of naturally occurring alpha amino acids, or R⁶ and R⁷ are independently selected from the group consisting of hydrogen, (C₂ -C₆)alkyl, aryl, aralkyl, heteroaryl, hydroxy, (C₁ -C₆)alkoxy, (C₁ -C₆)alkylthio, NR³ R⁴ and SR⁵, where R³ and R⁴ are as defined above, and R⁵ is hydrogen, (C₁ -C₆)alkyl, hydroxy-, alkoxy-, or alkylthio-substituted (C₁ -C₆)alkyl, or R⁶ and R⁷, together, are alicyclic or heterocyclic;

each of D¹ to Dⁿ is (CR⁶ R⁷)_z where R⁶ and R⁷ are as defined above;

each of y and z is zero or an integer from 1 to 10, the sum y+z being greater than 2 but not more than 10;

each of G¹ to G^n-1 is --NR³ CO--, --NR³ CS--, --NR³ SO-- --NR³ SO₂ --, --CONR³ --, --CSNR³ --, --SONR³ -- where R³ is as defined above;

each pair of A¹ to Aⁿ and B¹ to Bⁿ are selected such that;

(a) A is a group of formula (IIa), (IIb) or (IIc) and B is N or R³ N⁺ ;

or

(b) A is a group of formula (IId) and B is CH;

##STR9## where X is O, S, Se, NR³, CH₂ or C(CH₃)₂ ;

Y is a single bond, O, S or NR⁴ ;

each of p and q is zero or an integer from 1 to 5, the sum p+q being not more than 10;

each of r and s is zero or an integer from 1 to 5, the sum r+s being not more than 10;

each R¹ and R² is independently selected from the group consisting of hydrogen, (C₁ -C₄)alkyl which may be hydroxy- or alkoxy- or alkylthio-substituted, hydroxy, alkoxy, alkylthio, amino and halogen;

Q is --CO₂ H, --CONR'"'"'R", --SO₃ H or --SO₂ NR'"'"'R" or an activated derivative of --CO₂ H or --SO₃ H; and

I is --NR'"'"'"R"" or --NR'"'"'"C(O)R"";

where;

R'"'"', R", R'"'"'", and R"" are independently selected from the group consisting of hydrogen, alkyl, amino protecting groups, reporter ligands, intercalators, chelators, peptides, carbohydrates, lipids, steroids, nucleosides, nucleotides, nucleotide diphosphates, nucleotide triphosphates, oligonucleotides, oligonucleosides and soluble and non-soluble polymers, provided that R'"'"' is said DNA of said PNA-DNA or DNA-PNA or R"" is said DNA of said PNA-DNA or DNA-PNA.

3. The macromolecule of claim 2 having the structure PNA-DNA.

4. The macromolecule of claim 2 having the structure DNA-PNA.

5. A macromolecule of claim 2 wherein said PNA-DNA or DNA-PNA macromolecule hybridizes to a strand of nucleic acid.

6. A macromolecule of claim 5 wherein said strand of nucleic acid is a RNA strand.

7. A macromolecule of claim 2 wherein:

said DNA of said PNA-DNA or DNA-PNA includes at least three 2'"'"'-deoxynucleotides linked together in a sequence; and

said PNA of said PNA-DNA or DNA-PNA includes at least two peptide nucleic acid subunits.

8. A macromolecule of claim 2 wherein said 2'"'"'-deoxynucleotide is a phosphodiester, a phosphorothioate or a phosphorodithioate 2'"'"'-deoxynucleotide.

9. A macromolecule of claim 2 wherein said DNA of said PNA-DNA or DNA-PNA includes at least three 2'"'"'-deoxynucleotides linked together in a sequence by phosphodiester, phosphorothioate or phosphorodithioate linkages.

10. A macromolecule of claim 2 wherein said PNA of said PNA-DNA or DNA-PNA comprises a compound having a formula selected from the group consisting of IIIa, IIIb and IIIc:

##STR10## wherein;

each L is independently selected from the group consisting of hydrogen, phenyl, heterocyclic moieties, nucleobases;

each R^7'"'"' is independently selected from the group consisting of hydrogen, and the side chains of naturally occurring alpha amino acids;

n is an integer from 1 to 60;

each of k, l, and m is independently zero or an integer from 1 to 5;

p is zero or 1;

R^h is OH or NR'"'"'R"; and

Rⁱ is --R"" or --C(O)R"".

11. A macromolecule of claim 10 where said PNA of said PNA-DNA or DNA-PNA comprises a compound having a formula selected from the group consisting of (IIIa), (IIIb), and (IIIc), wherein each L is independently selected from the group consisting of the nucleobases thymine (T), adenine (A), cytosine (C), guanine (G) and uracil (U), k and m are zero or 1, and n is an integer from 1 to 30.

12. A compound of claim 11 wherein:

said DNA of said PNA-DNA or DNA-PNA includes at least three of said 2'"'"'-deoxynucleotides linked together in a sequence;

said PNA of said PNA-DNA or DNA-PNA includes at least two peptide nucleic acid subunits; and

said 2'"'"'-deoxynucleotides are joined via phosphodiester, phosphorothioate or phosphorodithioate linkages.

13. A macromolecule of claim 2 wherein said PNA of said PNA-DNA or DNA-PNA is covalently bound to said DNA of said PNA-DNA or DNA-PNA with an amide, amine or ester linkage.

23. A macromolecule of the structure:

PNA-(amide link)-DNA-(amide link)-PNA;

wherein;

said DNA of said structure comprises at least one 2'"'"'-deoxynucleotide;

each of said PNAs of said structure comprise at least one peptide nucleic acid subunit; and

each of said amide links of said structure includes an amide linkage of the structure;

space="preserve" listing-type="equation">--C(═

O)--NH-- or --NH--C(═

O)--.