Complex formation between DSDNA and oligomer of cyclic heterocycles
AssigneeCalifornia Institute of Technology
InventorPeter B. Dervan
Life of this patent
Estimating the term…
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.
Claims61
A method for forming a specific complex between target dsDNA and an oligomer selected to bind at a minor groove site of said target dsDNA at a Ka of at least 109 M−1,
- said oligomer comprising organic cyclic groups of from 5 to 6 ring atoms, where at least 60% of the cyclic groups are heterocycles having from 1 to 3 ring atoms that are heteroatoms independently selected from the group consisting of nitrogen, oxygen and sulfur, and wherein at least 60% of the heterocycles have at least one nitrogen ring atom, wherein said oligomer is defined as comprising at least 6 of said nitrogen containing heterocycles disposed in a non-branching configuration relative to one another, where at least one of said heterocycles is specific for A, G, C or T, said oligomer comprising an internal molecule forming a hairpin turn, and
- said heterocycles being linked by one or more groups for forming hydrogen bonds to available nitrogen and oxygen atoms of said dsDNA, said method comprising:
- bringing together under complex forming conditions, said oligomer with dsDNA, whereby complexes form between said oligomer at said target dsDNA.
A method for forming a specific complex between target dsDNA and a first and a second oligomer at a minor groove site, where said first and second oligomers are selected to provide binding at a Ka of at least 109 M−1,
- said first and second oligomers comprising organic cyclic groups of from 5 to 6 ring atoms, where at least 60% of the cyclic groups are heterocycles having from 1 to 3 ring atoms that are heteroatoms independently selected from the group consisting of nitrogen, oxygen and sulfur, and wherein at least 60% of the heterocycles have at least one nitrogen ring atom, wherein said first and second oligomers are defined as having at least 6 of said nitrogen containing heterocycles disposed in a non-branching configuration relative to one another, where at least one of said heterocycles is specific for A, G, C or T,
- 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, said method comprising:
- bringing together under complex forming conditions, said first and second oligomers with dsDNA, whereby complexes form between said first and second oligomers and said target dsDNA.
A method for forming a specific complex between target dsDNA in the minor groove and from 1 to 2 oligomers, where the 1 to 2 oligomers are selected to provide binding to said minor groove site, said 1 to 2 oligomers comprised of nitrogen containing heterocycles consisting of N-methyl pyrrole (Py) and N-methyl imidazole (Im), wherein said nitrogen containing heterocycles and other members of said 1 to 2 oligomers are selected to provide a Ka of greater than about 109 M−1, wherein each of said oligomers is comprised of at least 6 said nitrogen containing heterocycles disposed in a non-branching configuration relative to one another,
- where the order of nitrogen containing heterocycles in relation to said target dsDNA is defined as Im/Py in juxtaposition to G/C, Py/Im in juxtaposition to CM, and Py/Py in juxtaposition to A/T and T/A, said nitrogen containing heterocycles being linked by one or more linking groups for forming hydrogen bonds to available nitrogen or oxygen, or nitrogen and oxygen atoms of said dsDNA, said method comprising:
- bringing together under complex forming conditions, said 1 to 2 oligomers with dsDNA, whereby complexes form between said 1 to 2 oligomers and any target dsDNA.
A method for forming a specific complex between target dsDNA in the minor groove and a pair of oligomers of N-heterocycles consisting of N-methyl pyrrole (Py) and N-methyl imidazole (Im), wherein said N-heterocycles are selected to provide binding in said minor groove with a Ka of at least 109 M−1, wherein said oligomer is comprised of 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 and a complementary pair of heterocycles refers to the complementary pair of nucleotides,
- said oligomers comprise an internal β-alanine, said internal β-alanine being in juxtaposition to A and T and forming a complementary pair with itself, each oligomer terminating in a glycine or β-alanine amino acid joined to an alkyl chain of from 2 to 4 carbon atoms comprising a polar group, said heterocycles being linked by chains of oligomers 2 atoms comprising NH groups for forming hydrogen bonds to available nitrogen and oxygen atoms of said dsDNA,
- with the proviso that hydrogen atoms away from the surface of said minor groove may be substituted with substituents of a total of not greater than 30 carbon atoms, said method comprising:
- bringing together under complex forming conditions, said oligomers with dsDNA, whereby complexes form between said oligomers and any target dsDNA.
A method for forming a specific complex between target dsDNA in the minor groove and an oligomer of nitrogen containing heterocycles selected from the group consisting of N-methyl pyrrole (Py) and N-methyl imidazole (Im), wherein said nitrogen containing heterocycles are selected to provide binding in said minor groove with a Ka of at least 109 M−1, wherein said oligomer is comprised of at least 6 said nitrogen containing heterocycles disposed in a non-branching configuration relative to one another, where the order of nitrogen containing 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 nitrogen containing 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,
- with the proviso that hydrogen atoms away from the surface of said minor groove may be substituted with substituents of a total of not greater than 30 carbon atoms, said method comprising:
- bringing together under complex forming conditions, said oligomer with dsDNA, whereby complexes form between said oligomer and any target dsDNA.
A method for forming a specific complex between target dsDNA in the minor groove and a pair of oligomers of nitrogen containing heterocycles selected from the group consisting of N-methyl pyrrole (Py) and N-methyl imidazole (Im), wherein said nitrogen containing heterocycles are selected to provide binding in said minor groove with a Ka of at least 109 M−1, wherein said oligomer is comprised of at least 6 said nitrogen containing heterocycles disposed in a non-branching configuration relative to one another, where the order of nitrogen containing 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 nitrogen containing 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,
- with the proviso that hydrogen atoms away from the surface of said minor groove may be substituted with substituents of a total of not greater than 30 carbon atoms, said method comprising:
- bringing together under complex forming conditions, said oligomers with dsDNA, whereby complexes form between said oligomers and any target dsDNA.
A method for detecting the presence of target dsDNA in a sample, employing a composition comprising an oligomer selected to provide binding to a minor groove site of said target dsDNA at a Ka of at least 109 M−1,
- said oligomer comprising organic cyclic groups of from 5 to 6 ring atoms, where at least 60% of the cyclic groups are heterocycles having from 1 to 3 ring atoms that are heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur, and wherein at least 60% of the heterocycles have at least one nitrogen ring atom, wherein said oligomer is defined as having at least 6 of said nitrogen containing heterocycles disposed in a non-branching configuration relative to one another, where at least one of said heterocycles is specific for A, G, C or T, said oligomer comprising an internal molecule forming a hairpin turn,
- said heterocycles being linked by one or more groups for forming hydrogen bonds to available nitrogen and oxygen atoms of said dsDNA, with the proviso that hydrogen atoms away from the surface of said minor groove may be substituted with substituents of a total of not greater than 100 carbon atoms, and
- a moiety for detecting said complex, said method comprising:
- combining said composition and said sample under complex forming conditions;
- and detecting the presence of said target dsDNA in said sample as a complex with said oligomer by means of said moiety.
A method of detecting target dsDNA in a sample employing a composition comprising a first and a second oligomer, where the first and second oligomer are selected to provide binding to a minor groove site of said target dsDNA, said first and second oligomers comprised of nitrogen containing heterocycles selected from the group consisting of N-methyl pyrrole (Py) and N-methyl imidazole (Im), wherein said nitrogen containing heterocycles and other members of said oligomers are selected to provide a Ka of greater than about 109 M−1, wherein each said oligomer is comprised of at least 6 said nitrogen containing heterocycles disposed in a non-branching configuration relative to one another,
- where the order of nitrogen containing 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 nitrogen containing 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, with the proviso that hydrogen atoms away from the surface of said minor groove may be substituted with substituents of a total of not greater than 100 carbon atoms,
- at least one oligomer joined to a moiety for detection of complex formation between said target dsDNA and said oligomers, said method comprising:
- combining said composition 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.
A method for isolating target dsDNA from a mixture of dsDNA employing a composition comprising an oligomer selected to provide binding to said target dsDNA at a Kd≦1 nM, said oligomer including organic cyclic groups of from 5 to 6 ring atoms, where at least 60% of the rings are heterocycles having from 1 to 3 ring atoms that are heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur, and wherein at least 60% of the heterocycles have at least one nitrogen ring atom, wherein said oligomer is defined as having at least 6 of said nitrogen containing heterocycles, where at least one of said heterocycles is specific for A, G, C or T, and a moiety for separating said complex, 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, said method comprising:
- combining said composition with said mixture of dsDNA under complex forming conditions;
- and
- separating complexes which form by means of said moiety.
A method for isolating target dsDNA from a mixture of dsDNA employing a composition comprising a first oligomer and a second oligomer, where said oligomers are selected to provide binding to said target dsDNA at a Ka of at least 109 M−1,
- said oligomers comprising organic cyclic groups of from 5 to 6 ring atoms, where at least 60% of the rings are heterocycles having from 1 to 3 ring atoms that are heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur, and wherein at least 60% of the heterocycles have at least one nitrogen ring atom, wherein each said oligomer is defined as having at least 6 of said nitrogen containing heterocycles, where at least one of said heterocycles is specific for A, G, C or T,
- 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, and
- a moiety for detecting said complex, at least one oligomer joined to a moiety for separation of complexes between said target dsDNA and said oligomers, said method comprising:
- combining said composition and said sample under complex forming conditions;
- and separating complexes which form by means of said moiety.
A composition comprising:
- oligomers comprising nitrogen containing heterocycles selected to provide binding to a minor groove site of said target dsDNA, wherein said nitrogen containing heterocycles are selected from a group consisting of N-methyl pyrrole (Py) and N-methyl imidazole (Im), wherein said nitrogen containing heterocycles and other members of said oligomers are selected to provide a Ka of greater than about 109 M−1, wherein said oligomer is comprised of at least 6 said nitrogen containing heterocycles,
- where the order of nitrogen containing heterocycles in relation to said target dsDNA is defined as Im/Py in juxtaposition to G/C, Py/In in juxtaposition to C/G, and Py/Py in juxtaposition to A/T and T/A,
- said oligomer comprising at least two units of consecutive nitrogen containing heterocycles,
- said oligomer chosen from the group consisting of an oligomer that forms said complementary pairs with itself and an oligomer that forms complementary pairs with another oligomer,
- where said oligomer that forms said complementary pairs with itself comprises an internal γ-aminobutyric acid, and said oligomer that forms complementary pairs with another oligomer comprises 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 nitrogen containing 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.
A method for isolating target dsDNA from an extended dsDNA comprising said target dsDNA, said method comprising:
- bringing together said larger fragment of dsDNA and a pair of oligomers of nitrogen containing heterocycles selected from the group consisting of N-methyl pyrrole (Py) and N-methyl imidazole (Im), wherein said nitrogen containing heterocycles are selected to provide binding in said minor groove with a Ka of at least 109 M−1, wherein said oligomer is comprised of at least 6 said nitrogen containing heterocycles, where the order of nitrogen containing 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 oligomers comprise an internal β-alanine, said internal β-alanine being in juxtaposition to A and T and forming a complementary pair with itself, said nitrogen containing 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,
- with the proviso that hydrogen atoms away from the surface of said minor groove may be substituted with substituents of a total of not greater than 30 carbon atoms, and at least one terminal end of an oligomer is a functionality capable of cleaving dsDNA,
- whereby a complex is formed between said oligomers and said extended dsDNA;
- and
- cleaving said extended dsDNA by means of said functionality.
A minor-groove binding polyamide reagent which binds to duplex DNA in a sequence specific manner, said polyamide reagent comprising an oligomer of nitrogen containing heterocycles selected from the group consisting of N-methyl pyrrole (Py) and N-methyl imidazole (Im), wherein said nitrogen containing heterocycles are selected to provide binding in said minor groove with a Ka of at least 109 M−1, wherein said oligomer is composed of at least 6 said nitrogen containing heterocycles, where the order of nitrogen containing 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 comprising at least two units of 2 consecutive nitrogen containing heterocycles forming complementary pairs with itself, said oligomer comprises an internal γ-aminobutyric acid between said two units, and a unit of 6 nitrogen containing heterocycles comprises an internal β-alanine, said internal β-alanine being in juxtaposition to A and T and forming a complementary pair with itself, and has from about one to three chemical moieties appended thereto, wherein, the chemical moieties confer upon the polyamide reagent properties which render the polyamide reagent useful for a purpose selected from the group consisting of purifying duplex DNA in a sequence specific manner, and detecting duplex DNA in a sequence specific manner.
A minor groove binding polyamide reagent which binds to duplex DNA in specific manner, said polyamide reagent comprising a pair of oligomers of nitrogen containing heterocycles selected from the group consisting of N-methyl pyrrole (Py) and N-methyl imidazole (Im), wherein said nitrogen containing heterocycles are selected to provide binding in said minor groove with a Ka of at least 109 M−1, wherein each said oligomer is comprised of at least 6 said nitrogen containing heterocycles, where the order of nitrogen containing 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 oligomers comprise an internal β-alanine, said internal β-alanine being in juxtaposition to A and T and forming a complementary pair with itself, said nitrogen containing 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, and has from about one to three chemical moieties appended thereto, wherein, the chemical moieties confer upon the polyamide reagent properties which render the polyamide reagent useful for a purpose selected from the group consisting of purifying duplex DNA in a sequence specific manner, and detecting duplex DNA in a sequence specific manner.
A method of detecting target dsDNA in a sample employing a composition comprising a first and a second oligomer, where first and second the oligomers are selected to provide binding to a minor groove site of said target dsDNA, said first and second oligomers comprised of N-heterocycles 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 Ka of greater than about 109 M−1, wherein said each oligomer is comprised of 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 and a complementary pair of heterocycles refers to the complementary pair of nucleotides,
- said first and second oligomer each comprising at least two units of consecutive heterocycles forming complementary pairs with the other oligomer,
- said oligomers comprising an internal β-alanine, said internal β-alanine being in juxtaposition to A/T and T/A and forming a complementary pair with itself,
- each said oligomer terminating in a glycine or β-alanine amino acid joined to an alkyl chain of from 2 to 4 carbon atoms comprising a polar group, said heterocycles being linked by chains of 2 atoms comprising NH groups for forming hydrogen bonds to available nitrogen and oxygen atoms of said dsDNA, with the proviso that hydrogen atoms away from the surface of said minor groove may be substituted with substituents of a total of not greater than 100 carbon atoms,
- at least one oligomer joined to a moiety for detection of complex formation between said target dsDNA and said oligomers, said method comprising:
- combining said composition 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.
A minor-groove binding polyamide reagent which binds to duplex DNA in specific manner, said polyamid reagent comprising an oligomer of N-heterocycles selected from the group consisting of N-methyl pyrrole (Py) and N-methyl imidazole (Im), wherein said N-heterocycles are selected to provide binding in said minor groove with a Ka of at least 109 M−1, wherein said oligomer is composed of 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 comprising at least two units of 2 consecutive heterocycles forming complementary pairs with itself, said oligomer comprises an internal γ-aminobutyric acid between said two units, and a unit of 6 N-heterocycles comprises an internal β-alanine, said internal β-alanine being in juxtaposition to A and T and forming a complementary pair with itself, and has from about one to three chemical moieties appended thereto, wherein, the chemical moieties confer upon the polyamide reagent properties which render the polyamide reagent useful for a purpose selected from the group consisting of purifying duplex DNA in a sequence specific manner, and detecting duplex DNA in a sequence specific manner.
A minor-groove binding polyamide reagent which binds to duplex DNA in specific manner, said polyamide reagent comprising a pair of oligomers of N-heterocycle selected from the group consisting of N-methyl pyrrole (Py) and N-methyl imidazole (Im), wherein said N-heterocycles are selected to provide binding in said minor groove with a Ka of at least 109 M−1, wherein each said oligomer is comprised of 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 CIG, and Py/Py in juxtaposition to A/T and T/A
- said oligomers comprise 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 atoms, or nitrogen and oxygen atoms of aid dsDNA, and has from about one to three chemical moieties appended thereto, wherein, the chemical moieties confer upon the polyamide reagent properties which render the polyamide reagent useful for a purpose selected from the group consisting of purifying duplex DNA in a sequence specific manner, and detecting duplex DNA in a sequence specific manner.
Dependencies are inferred from the claim text.