Organic compounds -- part of the class 532-570 series – Organic compounds – Heterocyclic carbon compounds containing a hetero ring...
Reexamination Certificate
1999-08-11
2002-10-29
Lambkin, Deborah C. (Department: 1613)
Organic compounds -- part of the class 532-570 series
Organic compounds
Heterocyclic carbon compounds containing a hetero ring...
C548S312400, C548S312700, C548S313100, C548S314700, C548S334500, C548S557000
Reexamination Certificate
active
06472537
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to polyamides which bind to predetermined sequences in the minor groove of double stranded DNA.
2. Description of the Related Art
The design of synthetic ligands that read the information stored in the DNA double helix has been a long standing goal of chemistry. Cell-permeable small molecules which target predetermined DNA sequences are useful for the regulation of gene-expression. Oligodeoxynucleotides that recognize the major groove of double-helical DNA via triple-helix formation bind to a broad range of sequences with high affinity and specificity. Although oligonucleotides and their analogs have been shown to interfere with gene expression, the triple helix approach is limited to purine tracks and suffers from poor cellular uptake. The. development of pairing rules for minor groove binding polyamides derived from N-methylpyrrole (Py) and N-methylimidazole (Im) amino acids provides another code to control sequence specificity. An Im/Py pair distinguishes G·C from C·G and both of these from A·T or T·A base pairs. Wade, W. S., Mrksich, M. & Dervan, P. B. describes the design of peptides that bind in the minor groove of DNA at 5′-(A,T)G(A,T)C(A,T)-3′ sequences by a dimeric side-by-side motif.
J. Am. Chem. Soc
. 114, 8783-8794 (1992); Mrksich, M. et al. describes antiparallel side-by-side motif for sequence specific-recognition in the minor groove of DNA by the designed peptide 1-methylimidazole-2-carboxamidenetropsin.
Proc. Natl. Acad. Sci. USA
89, 7586-7590 (1992); Trauger, J. W., Baird, E. E. Dervan, P. B. describes the recognition of DNA by designed ligands at subnanomolar concentrations.
Nature
382, 559-561 (1996). A Py/Py pair specifies A·T from G·C but does not distinguish A·T from T·A. Pelton, J. G. & Wemmer, D. E. describes the structural characterization of a 2-1 distamycin A-d(CGCAAATTTGGC) complex by two-dimensional NMR.
Proc. Natl. Acad. Sci. USA
86, 5723-5727 (1989); White, S., Baird, E. E. & Dervan, P. B. Describes the effects of the A·T/T·A degeneracy of pyrrole-imidazole polyamide recognition in the minor groove of DNA.
Biochemistry
35, 6147-6152 (1996); White, S., Baird, E. E. & Dervan, P. B. describes the pairing rules for recognition in the minor groove of DNA by pyrrole-imidazole polyamides.
Chem. & Biol
. 4, 569-578 (1997); White, S., Baird, E. E. & Dervan, P. B. describes the 5′-3′ N-C orientation preference for polyamide binding in the minor groove. In order to break this degeneracy, a new aromatic amino acid, 3-hydroxy-N-methylpyrrole (Hp) incorporated into a polyamide and paired opposite Py, has been found to discriminate A·T from T·A. The replacement of a single hydrogen atom on the pyrrole with a hydroxy group in a Hp/Py pair regulates affinity and specificity of a polyamide by an order of magnitude. Utilizing Hp together with Py and Im in polyamides to form four aromatic amino acid pairs (Im/Py, Py/Im, Hp/Py, and Py/Hp) provides a code to distinguish all four Watson-Crick base pairs in the minor groove of DNA.
SUMMARY OF THE INVENTION
The invention encompasses improved polyamides for binding to the minor groove of double stranded (“duplex”) DNA. The polyamides are in the form of a hairpin comprising two groups of at least three consecutive carboxamide residues, the two groups covalently linked by an aliphatic amino acid residue, preferably y-aminobutyric acid or 2,4 diaminobutyric acid, the consecutive carboxamide residues of the first group pairing in an antiparallel manner with the consecutive carboxamide residues of the second group in the minor groove of double stranded DNA. The improvement relates to the inclusion of a binding pair of Hp/Py carboxamides in the polyamide to bind to a T·A base pair in the minor groove of double stranded DNA or Py/Hp carboxamide binding pair in the polyamide to bind to an A·T base pair in the minor groove of double stranded DNA. The improved polyamides have at least three consecutive carboxamide pairs for binding to at least three DNA base pairs in the minor groove of a duplex DNA sequence that has at least one A·T or T·A DNA base pair, the improvement comprising selecting a Hp/Py carboxamide pair to correspond to a T·A base pair in the minor groove or a Py/Hp carboxamide pair to bind to an A·T DNA base pair in the minor groove. Preferably the binding of the carboxamide pairs to the DNA base pairs modulates the expression of a gene.
In one preferred embodiment, the polyamide includes at least four consecutive carboxamide pairs for binding to at least four base pairs in a duplex DNA sequence. In another preferred embodiment, the polyamide includes at least five consecutive carboxamide pairs for binding to at least five base pairs in a duplex DNA sequence. In yet another preferred embodiment, the polyamide includes at least six consecutive carboxamide pairs for binding to at least six base pairs in a duplex DNA sequence. In one preferred embodiment, the improved polyamides have four carboxamide binding pairs that will distinguish A·T, TEA, C·G and G·C base pairs in the minor groove of a duplex DNA sequence. The duplex DNA sequence can be a regulatory sequence, such as a promoter sequence or an enhancer sequence, or a gene sequence, such as a coding sequence or a non-coding sequence. Preferably, the duplex DNA sequence is a promoter sequence.
The preparation and the use of polyamides for binding in the minor groove of double stranded DNA are extensively described in the art. This invention is an improvement of the existing technology that uses 3-hydroxy-N-methylpyrrole to provide carboxamide binding pairs for DNA binding polyamides.
The invention encompasses polyamides having &ggr;-aminobutyric acid or a substituted &ggr;-aminobutyric acid to form a hairpin with a member of each carboxamide pairing on each side of it. Preferably the substituted &ggr;-aminobutyric acid is a chiral substituted &ggr;-aminobutyric acid such as (R)-2,4-diaminobutyric acid. In addition, the polyamides may contain an aliphatic amino acid residue, preferably a &bgr;-alanine residue, in place of a non-Hp carboxamide. The &bgr;-alanine residue is represented in formulas as &bgr;. The &bgr;-alanine residue becomes a member of a carboxamide binding pair. The invention further includes the substitution as a &bgr;·&bgr; binding pair for non-Hp containing binding pair. Thus, binding pairs in addition to the Hp/Py and Py/Hp are Im/&bgr;, &bgr;/Im, Py/&bgr;, &bgr;/Py, and &bgr;/&bgr;.
The polyamides of the invention can have additional moieties attached covalently to the polyamide. Preferably the additional moieties are attached as substituents at the amino terminus of the polyamide, the carboxy terminus of the polyamide, or at a chiral (R)-2,4-diaminobutyric acid residue. Suitable additional moieties include a detectable labeling group such as a dye, biotin or a hapten. Other suitable additional moieties are DNA reactive moieties that provide for sequence specific cleavage of the duplex DNA.
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Baird Eldon E.
Dervan Peter B.
California Institute of Technology
Foley & Lardner
Lambkin Deborah C.
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