IMPROVED POLYAMIDES FOR BINDING IN THE MINOR GROOVE OF DOUBLE STRANDED DNA

Abstract: The invention encompasses improved polyamides for binding to specific nucleotide sequences in the minor groove of double stranded DNA. The 3-hydroxy-N-methylpyrrole/N-methylpyrrole carboxamide pair specifically recognizes the T.A base pair, while the N-methylpyrrole/3-hydroxy-N-methylpyrrole pair recognizes A.T nucleotide pairs. Similarly, an N-methylimidizole/N-methylpyrrole carboxamide pair specifically recognizes the G.C nucleotide pair, and the N-methylpyrrole/N-methylimidizole carboxamide pair recognizes the C.G nucleotide pair.

Description: IMPROVED POLY AMIDES FOR BINDING IN THE MINOR GROOVE OF DOUBLE STRANDED DNA The U.S. Government has certain rights in this invention pursuant to Grant Nos. GM 26453, 27681 and 47530 awarded by the National Institute of Health. CROSS REFERENCE TO RELATED APPLICATIONS This application is a continuation-in-part of PCT US97/03332 filed February 20, 1997, Serial No. 08/853,522 filed May 8, 1997 and PCT/US 97/12722 filed July 21, 1997 which are continuation-in-part applications of Serial No. 08/837,524, filed April 21, 1997, Serial No. 08/607,078, filed February 26, 1996, provisional application Serial No. 60/042,022, filed April 16, 1997 and provisional application Serial No. 60/043,444, filed April 8, 1997. BACKGROUND OF THE INVENTION Field of the Invention This invention relates to polyamides which bind to predetermined sequences in the minor groove of double stranded DNA. 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 l-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 γ-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, T*A, 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 γ-aminobutyric acid or a substituted γ- aminobutyric acid to form a hairpin with a member of each carboxamide pairing on each side of it. Preferably the substituted γ-aminobutyric acid is a chiral substituted γ-aminobutyric acid such as (R)-2,4-diaminobutyric acid. In addition, the polyamides may contain an aliphatic amino acid residue, preferably a β-alanine residue, in place of a non-Hp carboxamide. The β- alanine residue is represented in formulas as β. The β-alanine residue becomes a member of a carboxamide binding pair. The invention further includes the substitution as a β«β binding pair for non-Hp containing binding pair. Thus, binding pairs in addition to the Hp/Py and Py/Hp are Im/β, β/Im, Py/β, β/Py, and β/β. 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. Brief Description of the Drawings Figure 1 illustrates the structure of polyamide lj, 2j_and 3. Figure 2 illustrates the pairing of polyamides to DNA base pairs. Figure 3 illustrates the DNase footprint titration of compounds 2 and 3. Figure 4 illustrates a list of the structures of representative Hp containing polyamides. Figure 5 illustrates the synthesis of a protected Hp monomer for solid phase synthesis. Figure 6 illustrates the solid phase synthesis of polyamide 2. Figure 7 illustrates the 1H-NMR characterization of polyamide 2. Figure 8 illustrates the Mass spectral characterization of polyamide 2. Figure.9 illustrates 1H-NMR characterization of synthesis purity. Figure 10 illustrates DNasel footprint titration experiment. Figure 11 illustrates the synthesis of bifunctional conjugate of polyamide 2. Figure 12 illustrates affinity cleaving evidence for oriented hairpin formation. Figure 13 illustrates increased sequence specificity of Hp/Py containing polyamides. Figure 14 illustrates 8-ring hairpin polyamides which target 5'-WGTNNW-3' sites. Figure 15 illustrates 8-ring hairpin polyamides which target 5'-WGANNW-3' sites. Figure 16 illustrates 8-ring hairpin polyamides which target 5'-WGGNNW-3' sites. Figure 17 illustrates 8-ring hairpin polyamides which target 5'-WGCNNW-3' sites. DETAILED DESCRIPTION OF THE INVENTION Within this application, unless otherwise stated, definitions of the terms and illustration of the techniques of this application may be found in any of several well-known references such as: Sambrook, J., et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989); Goeddel, D., ed., Gene Expression Technology, Methods in Enzymology, 185, Academic Press, San Diego, CA (1991); "Guide to Protein Purification" in Deutshcer, M.P., ed., Methods in Enzymology, Academic Press, San Diego, CA (1989); Innis, et al., PCR Protocols: A Guide to Methods and Applications, Academic Press, San Diego, CA (1990); Freshney, R.I., Culture of Animal Cells: A Manual of Basic Technique, 2 nd Ed., Alan Liss, Inc. New York, NY (1987); Murray, E.J., ed., Gene Transfer and Expression Protocols, pp. 109-128, The Humana Press Inc., Clifton, NJ and Lewin, B., Genes VI, Oxford University Press, New York (1997). For the purposes of this application, -promoter is a regulatory sequence of DNA that is involved in the binding of RNA polymerase to initiate transcription of a gene. A gene is a segment of DNA involved in producing a peptide, polypeptide or protein, including the coding region, non-coding regions preceding ("leader") and following ("trailer") the coding region, as well as intervening non-coding sequences ("introns") between individual coding segments ("exons"). Coding refers to the representation of amino acids, start and stop signals in a three base "triplet" code. Promoters are often upstream (" '5 to") the transcription initiation site of the corresponding gene. Other regulatory sequences of DNA in addition to promoters are known, including sequences involved with the binding of transcription factors, including response elements that are the DNA sequences bound by inducible factors. Enhancers comprise yet another group of regulatory sequences of DNA that can increase the utilization of promoters, and can function in either orientation (5 '-3' or 3 '-5') and in any location (upstream or downstream) relative to the promoter. Preferably, the regulatory sequence has a positive activity, i.e., binding of an endogeneous ligand (e.g. a transcription factor) to the regulatory sequence increases transcription, thereby resulting in increased expression of the corresponding target gene. In such a case, interference with transcription by binding a polyamide to a regulatory sequence would reduce or abolish expression of a gene. The promoter may also include or be adjacent to a regulatory sequence known in the art as a silencer. A silencer sequence generally has a negative regulatory effect on expression of the gene. In such a case, expression of a gene may be increased directly by using a polyamide to prevent binding of a factor to a silencer regulatory sequence or indirectly, by using a polyamide to block transcription of a factor to a silencer regulatory sequence. It is to be understood that the polyamides of this invention bind to double stranded DNA in a sequence specific manner. The function of a segment of DNA of a given sequence, such as 5'-TATAAA-3', depends on its position relative to other functional regions in the DNA sequence. In this case, if the sequence 5'-TATAAA-3' on the coding strand of DNA is positioned about 30 base pairs upstream of the transcription start site, the sequence forms part of the promoter region (Lewin, Genes VI, pp. 831-835). On the other hand, if the sequence 5'- TATAAA-3' is downstream of the transcription start site in a coding region and in proper register with the reading frame, the sequence encodes the tyrosyl and lysyl amino acid residues (Lewin, Genes VI, pp. 213-215). While not being held to one hypothesis, it is believed that the binding of the polyamides of this invention modulate gene expression by altering the binding of DNA binding proteins, such as RNA polymerase, transcription factors, TBF, TFIIIB and other proteins. The effect on gene expression of polyamide binding to a segment of double stranded DNA is believed to be related to the function, e.g., promoter, of that segment of DNA. It is to be understood by one skilled in the art that the improved polyamides of the present invention may bind to any of the above-described DNA sequences or any other sequence having a desired effect upon expression of a gene. In addition, U.S. Patent No. 5,578,444 describes numerous promoter targeting sequences from which base pair sequences for targeting an improved polyamide of the present invention may be identified. It is generally understood by those skilled in the art that the basic structure of DNA in a living cell includes both major and a minor groove. For the purposes of describing the present invention, the minor groove is the narrow groove of DNA as illustrated in common molecular biology references such as Lewin, B., Genes VI, Oxford University Press, New York (1997). To affect gene expression in a cell, which may include causing an increase or a decrease in gene expression, a effective quantity of one or more polyamide is contacted with the cell and internalized by the cell. The cell may be contacted in vivo or in vitro. Effective extracellular concentrations of polyamides that can modulate gene expression range from about 10 nanomolar to about 1 micromolar. Gottesfeld, J.M., et al., Nature 387 202-205 (1997). To determine effective amounts and concentrations of polyamides in vitro, a suitable number of cells is plated on tissue culture plates and various quantities of one or more polyamide are added to separate wells. Gene expression following exposure to a polyamide can be monitored in the cells or medium by detecting the amount of the protein gene product present as determined by various techniques utilizing specific antibodies, including ELISA and western blot. Alternatively, gene expression following exposure to a polyamide can be monitored by detecting the amount of messenger RNA present as determined by various techniques, including northern blot and RT-PCR. Similarly, to determine effective amounts and concentrations of polyamides for in vivo administration, a sample of body tissue or fluid, such as plasma, blood, urine, cerebrospinal fluid, saliva, or biopsy of skin, muscle, liver, brain or other appropriate tissue source is analyzed. Gene expression following exposure to a polyamide can be monitored by detecting the amount of the protein gene product present as determined by various techniques utilizing specific antibodies, including ELISA and western blot. Alternatively, gene expression following exposure to a polyamide can be monitored by the detecting the amount of messenger RNA present as determined by various techniques, including northern blot and RT-PCR. The polyamides of this invention may be formulated into diagnostic and therapeutic compositions for in vivo or in vitro use. Representative methods of formulation may be found in Remington: The Science and Practice of Pharmacy, 19th ed., Mack Publishing Co., Easton, PA (1995). For in vivo use, the polyamides may be incorporated into a physiologically acceptable pharmaceutical composition that is administered to a patient in need of treatment or an animal for medical or research purposes. The polyamide composition comprises pharmaceutically acceptable carriers, excipients, adjuvants, stabilizers, and vehicles. The composition may be in solid, liquid, gel, or aerosol form. The polyamide composition of the present invention may be administered in various dosage forms orally, parentally, by inhalation spray, rectally, or topically. The term parenteral as used herein includes, subcutaneous, intravenous, intramuscular, intrasternal, infusion techniques or intraperitoneally. The selection of the precise concentration, composition, and delivery regimen is influenced by, inter alia, the specific pharmacological properties of the particular selected compound, the intended use, the nature and severity of the condition being treated or diagnosed, the age, weight, gender, physical condition and mental acuity of the intended recipient as well as the route of administration. Such considerations are within the purview of the skilled artisan. Thus, the dosage regimen may vary widely, but can be determined routinely using standard methods. Polyamides of the present invention are also useful for detecting the presence of double stranded DNA of a specific sequence for diagnostic or preparative purposes. The sample containing the double stranded DNA can be contacted by polyamide linked to a solid substrate, thereby isolating DNA comprising a desired sequence. Alternatively, polyamides linked to a suitable detectable marker, such as biotin, a hapten, a radioisotope or a dye molecule, can be contacted by a sample containing double stranded DNA. The design of bifunctional sequence specific DNA binding molecules requires the integration of two separate entities: recognition and functional activity. Polyamides that specifically bind with subnanomolar affinity to the minor groove of a predetermined sequence of double stranded DNA are linked to a functional molecule, providing the corresponding bifunctional conjugates useful in molecular biology, genomic sequencing, and human medicine. Polyamides of this invention can be conjugated to a variety of functional molecules, which can be independently chosen from but is not limited to arylboronic acids, biotins, polyhistidines comprised from about 2 to 8 amino acids, haptens to which an antibody binds, solid phase supports, oligodeoxynucleotides, N-ethylnitrosourea, fluorescein, bromoacetamide, iodoacetamide, DL-α-lipoic acid, acridine, captothesin, pyrene, mitomycin, texas red, anthracene, anthrinilic acid, avidin, DAPI, isosulfan blue, malachite green, psoralen, ethyl red, 4-(psoraen-8-yloxy)-bύtyrate, tartaric acid, (+)-α-tocopheral, psoralen, EDTA, methidium, acridine, Ni(II) » Gly-Gly-His, TO, Dansyl, pyrene, N-bromoacetamide, and gold particles. Such bifunctional polyamides are useful for DNA affinity capture, covalent DNA modification, oxidative DNA cleavage, DNA photocleavage. Such bifunctional polyamides are useful for DNA detection by providing a polyamide linked to a detectable label. Detailed instructions for synthesis of such bifunctional polyamides can be found in copending U.S. provisional application 60/043,444, the teachings of which are incorporated by reference. DNA complexed to a labeled polyamide can then be determined using the appropriate detection system as is well known to one skilled in the art. For example, DNA associated with a polyamide linked to biotin can be detected by a streptavidin / alkaline phosphatase system. The present invention also describes a diagnostic system, preferably in kit form, for assaying for the presence of the double stranded DNA sequence bound by the polyamide of this invention in a body sample, such brain tissue, cell suspensions or tissue sections, or body fluid samples such as CSF, blood, plasma or serum, where it is desirable to detect the presence, and preferably the amount, of the double stranded DNA sequence bound by the polyamide in the sample according to the diagnostic methods described herein. The diagnostic system includes, in an amount sufficient to perform at least one assay, a specific polyamide as a separately packaged reagent. Instructions for use of the packaged reagent(s) are also typically included. As used herein, the term "package" refers to a solid matrix or material such as glass, plastic (e.g., polyethylene, polypropylene or polycarbonate), paper, foil and the like capable of holding within fixed limits a polyamide of the present invention. Thus, for example, a package can be a glass vial used to contain milligram quantities of a contemplated polyamide or it can be a microliter plate well to which microgram quantities of a contemplated polypamide have been operatively affixed, i.e., linked so as to be capable of being bound by the target DNA sequence. "Instructions for use" typically include a tangible expression describing the reagent concentration or at least one assay method parameter such as the relative amounts of reagent and sample to be admixed, maintenance time periods for reagent or sample admixtures, temperature, buffer conditions and the like. A diagnostic system of the present invention preferably also includes a detectable label and a detecting or indicating means capable of signaling the binding of the contemplated polyamide of the present invention to the target DNA sequence. As noted above, numerous detectable labels, such as biotin, ~ and detecting or indicating means, such as enzyme-linked (direct or indirect) streptavidin, are well known in the art. Figure 1 shows representative structures of polyamides. ImlmPyPy-γ-ImPyPyPy-β-Dp (1), ImlmPyPy-γ-ImHpPyPy-β-Dp (2), and ImlmHpPy-γ-ImPyPyPy-β-Dp (3). (Hp = 3- hydroxy-N-methylpyrrole, Im = N-methylimidazole, Py = N-methylpyrrole, β = β-alanine, γ = γ-aminobutyric acid, Dp = Dimethylaminopropylamide). Polyamides were synthesized by solid phase methods using Boc-protected 3-methoxypyrrole. imidazole, and pyrrole aromatic amino acids, cleaved from the support by aminolysis, deprotected with sodium thiophenoxide, and purified by reversed phase HPLC. Baird, E. E. & Dervan, P. B. describes the solid phase synthesis of polyamides containing imidazole and pyrrole amino acids. J. Am. Chem. Soc. 118, 6141-6146 (1996); also see PCT US 97/003332. The identity and purity of the polyamides were verified by Η NMR, analytical HPLC, and matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS-monoisotopic): 1 1223.6 (1223.6 calculated), 2 1239.6 (1239.6 calculated); 3 1239.6 (1239.6 calculated). Figure 2 illustrates binding models for polyamides 1-3 in complex with 5'-TGGTCA-3' and 5'-TGGACA-3' (A«T and T«A in fourth position highlighted). Filled and unfilled circles represent imidazole and pyrrole rings respectively; circles containing an H represent 3- hydroxypyrrole, the curved line connecting the polyamide subunits represents γ-aminobutyric acid, the diamond represents β-alanine, and the + represents the positively charged dimethylaminopropylamide tail group. Figure 3 shows quantitative DNase I footprint titration experiments with polyamides 2 and 3 on the 3' 32 P labeled 250-bp pJK6 EcoRI/PvuII restriction fragment. Lane 1, intact DNA; lanes 2-11 DNase I digestion products in the presence of 100, 50, 20, 10, 5, 2, 1, 0.5, 0.2, 0.1 nM polyamide, respectively; lane 12, DNase I digestion products in the absence of polyamide; lane 13, adenine-specific chemical sequencing. Iverson, B. L. & Dervan, P. B. describes an adenine-specific DNA chemical sequencing reaction. Methods Enzymol. 15, 7823-7830 (1987). All reactions were done in a total volume of 400 μL. A polyamide stock solution or H 2 O was added to an assay buffer containing radiolabeled restriction fragment, with the final solution conditions of 10 mM Tris-HCl, 10 mM KC1, 10 mM MgCl 2 , 5 mM CaCl 2 , pH 7.0. Solutions were allowed to equilibrate for 4-12 h at 22 °C before initiation of footprinting reactions. Footprinting reactions, separation of cleavage products, and data analysis were carried out as described. White, S., Baird, E. E. & Dervan, P. B. Effects of the A»T/T»A degeneracy of pyrrole-imidazole polyamide recognition in the minor groove of DNA. Biochemistry 35, 6147- 6152 (1996). Figure 4 shows the structure and equilibrium dissociation constant for numerous compounds of the present invention. Polyamides are shown in complex with their respective match site. Filled and unfilled circles represent imidazole (Im) and pyrrole (Py) rings, respectively; circles containing an H represent 3-hydroxypyrrole (Hp), the curved line connecting the polyamide subunits represents γ-aminobutyric acid (γ), the diamond represents β-alanine (β), and the + represents the positively charged dimethylaminopropylamide tail group (Dp). The equilibrium dissociation constants are the average values obtained from three DNase I footprint titration experiments. The standard deviation for each set is less than 15% of the reported number. Assays were carried out in the presence of 10 mM Tris » HCl, 10 mM KC1, 10 mM MgCl 2 , and 5 mM CaCl 2 at pH 7.0 and 22°C. Figure 5 shows the synthetic scheme for 3-O-methyl-N-Boc protected pyrrole-2- carboxylate. The hydroxypyrrole monoester can be prepared in 0.5 kg quantity using published procedures on enlarged scale. Figure 6 shows the solid phase synthetic scheme for ImlmPyPy-γ-ImHpPyPy-β-Dp starting from commercially available Boc-β-Pam-Resin: (i) 80% TFA/DCM, 0.4 M PhSH; (ii) Boc-Py-OBt, DIEA, DMF; (iii) 80% TFA/DCM, 0.4 M PhSH; (iv) Boc-Py-OBt, DIEA, DMF; (v) 80% TFA/DCM, 0.4 M PhSH; (vi) Boc-3-OMe-Py-OH, HBTU, DMF, DIEA; (vii) 80% TFA/DCM, 0.4 M PhSH; (viii) Boc-Im-OH, DCC, HOBt; (ix) 80% TFA/DCM, 0.4 M PhSH; (x) Boc-γ-aminobutyric acid, DIEA, DMF; (xi) 80% TFA/DCM, 0.4 M PhSH; (xii) Boc-Py- OBt, DIEA, DMF; (xiii) 80% TFA/DCM, 0.4 M PhSH; (xiv) Boc-Py-OBt, DMF, DIEA; (xv) 80% TFA/DCM, 0.4 M PhSH; (vxi) Boc-Im-OH, DCC, HOBt (xvii) 80% TFA DCM, 0.4 M PhSH; (xviii) imidazole-2-carboxylic acid, HBTU, DIEA; (xviv) dimethylaminopropylamine, 55 °C, 18h. Purification by reversed phase HPLC provides ImlmPyPy-γ-ImOpPyPy-β-Dp. (Op = 3-methoxypyrrole). Treatment of the 3-methyoxypyrrole polyamide with thiophenol, NaH, DMF, at 100 °C for 120 min provides polyamide 2 after reverse phase HPLC purification. Figure 7 shows the aromatic region from 7-11 ppm for the 1H-NMR spectrum determined at 300 MHz for ImlmPyPy-γ-ImOpPyPy-β-Dp and ImlmPyPy-γ-ImHpPyPy-β-Dp. This region of the spectrum may be used to determine compound identity and purity. Figure 8 shows-the MALDI-TOF mass spectrum determined in positive ion mode with a monoisotopic detector for the polyamides for ImlmPyPy-γ-ImOpPyPy-β-Dp and ImlmPyPy-γ- ImHpPyPy-β-Dp. This spectrum may be used to determine compound identity and purity. Figure 9 shows the methyl group region from 3.5-4.0 ppm for the 1H-NMR spectrum determined at 300 MHz for ImPyPy-γ-OpPyPy-β-Dp and ImPyPy-γ-HpPyPy-β-Dp. This region of the spectrum may be used to directly follow the progress for conversion of 3-methoxypyrrole to 3-hydroxypyrrole. Fig. 10 shows quantitative DNase I footprint titration experiments with the polyamides ImPyPy-γ-PyHpPy-β-Dp and ImHpPy-γ-PyPyPy-β-Dp on the 3'- 32 P labeled 370-bp pDEHl EcøRI/RvwII restriction fragment. Intact lane, labeled restriction fragment no polyamide or DNase I added; lanes 1-10, DNase I digestion products in the presence of 10 μM, 5 μM, 2 μM, 1 μM, 500 nM, 200 nM, 100 tiM, 50 nM, 20 nM, 10 nM ImPyPy-γ-PyPyPy-β-Dp, respectively or 1 μM, 500 nM, 200 nM, 100 nM, 50 nM, 20 nM, 10 nM, 5 nM, 2 nM, 1 nM ImHpPy-γ- PyPyPy-β-Dp, respectively; DNase I lane, DNase I digestion products in the absence of polyamide; A lane, adenine-specific chemical sequencing. Iverson, B. L. & Dervan, P. B. describes an adenine-specific DNA chemical sequencing reaction. Methods Enzymol. 15, 7823- 7830 (1987). All reactions were done in a total volume of 40 μL. A polyamide stock solution or H 2 O was added to an assay buffer containing radiolabeled restriction fragment, with the final solution conditions of 10 mM Tris-HCl. 10 mM KC1, 10 mM MgCl 2 , 5 mM CaCl 2 , pH 7.0. Solutions were allowed to equilibrate for 4-12 h at 22 °C before initiation of footprinting reactions. Footprinting reactions, separation of cleavage products, and data analysis were carried out as described. White, S., Baird, Ε. Ε. & Dervan, P. describe the pairing rules for recognition in the minor groove of DNA by pyrrole-imidazole polyamides. Chemistry & Biology 4, 569-578 (1997). Figure 1 1 shows the synthesis of a bifunctional polyamide which incorporates the Hp/Py pair. Treatment of a sample of ImlmPyPy-γ-ImHpPyPy-β-Pam-resin (see Figure 6) with 3,3'- diamino-N-methyldipropylamine, 55°C, 18 h followed by reverse phase HPLC purification provides the Op polyamide with a free primary amine group which can be coupled to an activated carboxylic acid derivative. Treatment with (i) ΕDTA-dianhydride, DMSO/ΝMP, DIΕA, 55 °C; (ii) 0.1M ΝaOH, followed by reverse phase HPLC purification provides the Op- Py-Im-polyamide-ΕDTA conjugate. Treatment of the 3-methyoxypyrrole polyamide with thiophenol, NaH, DMF, at 100 °C for 120 min provides polyamide 2 after reverse phase HPLC purification. Figure 12 shows the determination of the binding orientation of hairpin polyamides ImlmPyPy-γ-ImHpPyPy-β-Dp-EDTA'Fe(II) 2-E « Fe(II) and ImlmHpPy-γ-ImPyPyPy-β-Dp- EDTA'Fe(II) 3-E » Fe(II) by affinity cleaving footprint titration. Top and bottom left: Affinity cleavage experiments on a 3' P labeled 250-bp pJK6 EcoRI/ Pvu II restriction fragment. The 5'-TGGACA-3' and 5'-TGGTCA-3' sites are shown on the right side of the autoradiogram. Top left: lane 1, adenine-specific chemical sequencing reaction; lanes 2-6, 6.5 μM, 1.0 μM, 100 nM, 10 nM, 1 nM polyamide 2-Ε » Fe(II); lane 7, intact restriction fragment, no polyamide added. Bottom left: lane 1, A reaction; lanes 2-6, 8.5 μM, 1.0 μM, 100 nM, 10 nM, 1 nM polyamide 3-E»Fe(II); lane 7, intact DNA. All reactions were carried out in a total volume of 40 μL. A stock solution of polyamide or H 2 0 was added to a solution containing 20 kcpm labeled restriction fragment, affording final solution conditions of 25 mM Tris- Acetate, 20 mM NaCl, 100 μM/ bp calf thymus DNA, at pH 7.0. Solutions were allowed to equilibrate for a minimum of 4 h at 22°K before initiation of reactions. Affinity cleavage reactions were carried out as described White, S., Baird, E.E. & Dervan, P.B. Effects of the A*T/T»A degeneracy of pyrrole-imidazole polyamide recognition in the minor groove of DNA. Biochemistry 35, 6147- 6152 (1996). Top and bottom right: Affinity cleavage patterns of 2-E«Fe(II) and 3-E»Fe(II) at 100 nM bound to 5'-TGGACA-3' and 5'-TGGTCA-3'. Bar heights are proportional to the relative cleavage intensities at each base pair. Shaded and nonshaded circles denote imidazole and pyrrole carboxamides, respectively. Nonshaded diamonds represent the β-alanine moiety. A curved line represents the γ-aminobutyric acid, and the + represents the positively charged dimethylaminopropylamide tail group. The boxed Fe denotes the EDTA'Fe(II) cleavage moiety. Figure 13 shows quantitative DNase I footprint titration experiments with the polyamides ImPyPyPyPy-γ-ImPyPyPyPy-β-Dp and ImHpPyPyPy-γ-ImHpPyPyPy-β-Dp on the 3' 32 P labeled 252-bp pJK7 EcoRI/ Pvu II restriction fragment. For ImPyPyPyPy-γ- ImPyPyPyPy-β-Dp gel (left): lane 1, DNase I digestion products in the absence of polyamide; lanes 2-18, DNase I digestion products in the presence of 1.0 μM, 500 nM, 200, 100, 65, 40, 25, 15, 10, 6.5, 4.0, 2.5, 1.5, 1.0, 0.5, 0.2, 0.1 nM polyamide; lane 19, DNase I digestion products in the absence of polyamide; lane 20, intact restriction fragment; lane 21, guanine-specific chemical sequencing reaction; lane 22, adenine-specific chemical sequencing reaction. For ImHpPyPyPy-γ-ImHpPyPyPy-β-Dp gel (right): lane 1, intact DNA; lane 2, DNase I digestion products in the absence of polyamide; lanes 3-19, 1.0 μM, 500 nM, 200, 100, 50, 20, 10, 5, 2, 1, 0.5, 0.2. 0.1, 0.05, 0.01, 0.005, 0.001 nM polyamide; lane 20, DNase I digestion products in the absence of polyamide; lane 21, A reaction. All reactions were done in a total volume of 400 μL. A polyamide stock solution or H 2 0 was added to an assay buffer containing radiolabeled restriction fragment, with the final solution conditions of 10 mM Tris-HCl, 10 mM KCl, 10 mM MgCl 2 , 5 mM CaCl 2 , pH 7.0. Solutions were allowed to equilibrate for 4-12 h at 22°C before initiation of footprinting reactions. Footprinting reactions, separation of cleavage products, and data analysis were carried as described. White, S., Baird, E.E. & Dervan, P.B. Effects of the A-T/T-A degeneracy of pyrrole-imidazole polyamide recognition in the minor groove of DNA. Biochemistry 35, 6147-6152 (1996). Fig. 14 shows the 8-ring Hp-Py-Im-polyamide hairpins described by the pairing code of the present invention. The eight ring haiφin template is shown at the top. A polyamide having the formula X 1 X 2 X 3 X 4 -γ-X 5 X 6 X 7 X 8 wherein γ is the -NH-CH 2 -CH 2 -CH 2 -CONH- haiφin linkage derived from γ-aminobutyric acid or a chiral haiφin linkage derived from R-2,4- diaminobutyric acid; X 4 /X 5 , X 3 /X 6 , X 2 /X7 > and XJX 8 represent carboxamide binding pairs which bind the DNA base pairs. The minor groove sequence to be bound is represented as 5'- WGTNNW-3', where the 5'-GTNN-3' core sequence is defined as position a, b, c, and d (W = A or T, N = A, G, C, or T). A linear sequence of aromatic amino acids fills the haiφin template in order to satisfy the ring pairing requirements to correspond to the DNA base pairs in the minor groove to be bound. The ring pairing code as applied is listed in Table 2. The 16 unique haiφin polyamides which target 16 5'-WGTNNW-3' sequences are drawn as binding models where filled and unfilled circles represent imidazole and pyrrole rings respectively; circles containing an H represent 3-hydroxypyrrole, and the curved line connecting the polyamide subunits represents γ-aminobutyric acid. Fig. 15 shows the 8-ring Hp-Py-Im-polyamide haiφins described by the pairing code of the present invention. The eight ring haiφin template is shown at the top. A polyamide having the formula X,X 2 X 3 X 4 -γ-X 5 X 6 X 7 X 8 wherein γ is the -NH-CH 2 -CH 2 -CH 2 -CONH- haiφin linkage derived from γ-aminobutyric acid or a chiral haiφin linkage derived from R-2,4- diaminobutyric acid; X /X 5 , X 3 /X 6 , X . /Xγ, and XJX 8 represent carboxamide binding pairs which bind the DNA base pairs. The minor groove sequence to be bound is represented as 5'- WGANNW-3', where the 5'-GANN-3' core sequence is defined as position a, b, c, and d (W = A or T, N = A, G, C, or T). A linear sequence of aromatic amino acids fills the haiφin template in order to satisfy the ring pairing requirements to correspond to the DNA base pairs in the minor groove to be bound. The ring pairing code as applied is listed in Table 2. The 16 unique haiφin polyamides which target 16 5'-WGANNW-3' sequences are drawn as binding models where filled and unfilled circles represent imidazole and pyrrole rings respectively; circles containing an H represent 3-hydroxypyrrole, and the curved line connecting the polyamide subunits represents γ-aminobutyric acid. Fig. 16 shows the 8-ring Hp-Py-Im-polyamide haiφins described by the pairing code of the present invention. The eight ring haiφin template is shown at the top. A polyamide having the formula X,X 2 X 3 X -γ-X 5 X 6 X 7 X 8 wherein γ is the -NH-CH 2 -CH 2 -CH 2 -CONH- haiφin linkage derived from γ-aminobutyric acid or a chiral haiφin linkage derived from R-2,4- diaminobutyric acid; X-j/Xs, X 3 /X 6 , X 2 /X7 > and Xι/X 8 represent carboxamide binding pairs which bind the DNA base pairs. The minor groove sequence to be bound is represented as 5'- WGGNNW-3', where the 5'-GGNN-3' core sequence is defined as position a, b, c, and d (W = A or T, N = A, G, C, or T). A linear sequence of aromatic amino acids fills the haiφin template in order to satisfy the ring pairing requirements to correspond to the DNA base pairs in the minor groove to be bound. The ring pairing code as applied is listed in Table 2. The 16 unique haiφin polyamides which target 16 5'-WGGNNW-3' sequences are drawn as binding models where filled and unfilled circles represent imidazole and pyrrole rings respectively; circles containing an H represent 3-hydroxypyrrole, and the curved line connecting the polyamide subunits represents γ-aminobutyric acid. Fig. 17 shows the 8-ring Hp-Py-Im-polyamide haiφins described by the pairing code of the present invention. The eight ring haiφin template is shown at the top. A polyamide having the formula X 1 X 2 X 3 X 4 -γ-X 5 X 6 X 7 X 8 wherein γ is the -NH-CH 2 -CH 2 -CH 2 -CONH- haiφin linkage derived from γ-aminobutyric acid or a chiral haiφin linkage derived from R-2,4- diaminobutyric acid; X 4 /X 5 , X 3 /X 6 , X 2 /X 7 , and X]/X 8 represent carboxamide binding pairs which bind the DNA base pairs. The minor groove sequence to be bound is represented as 5'- WGCNNW-3', where the 5'-GCNN-3' core sequence is defined as position a, b, c, and d (W = A or T, N = A, G, C, or T). A linear sequence of aromatic amino acids fills the haiφin template in order to satisfy the ring pairing requirements to correspond to the DNA base pairs in the minor groove to be bound. The ring pairing code as applied is listed in Table 2. The 16 unique haiφin polyamides which target 16 5'-WGCNNW-3' sequences are drawn as binding models where filled and unfilled circles represent imidazole and pyrrole rings respectively; circles containing an H represent 3-hydroxypyrrole, and the curved line connecting the polyamide subunits represents γ-aminobutyric acid. Four-ring polyamide subunits, covalently coupled to form eight-ring haiφin structures, bind specifically to 6-bp target sequences at subnanomolar concentrations. Trauger, J.W., Baird, E. E. & Dervan, P.B. describe the recognition of DNA by designed ligands at subnanomolar concentrations. Nature 382, 559-561 (1996); Swalley, S. E., Baird, E. E. & Dervan, P. B. describe the discrimination of 5'-GGGG-3', 5'-GCGC-3', and 5'-GGCC'3' sequences in the minor groove of DNA by eight-ring haiφin polyamides. J. Am. Chem. Soc. 119, 6953-6961 (1997). The DNA-binding affinities of three eight-ring haiφin polyamides shown in Figure 1 as compound 1, 2, and 3 containing pairings of Im Py, Py/Im opposite G»C, C*G and either Py/Py, Hp/Py, or Py/Hp at a common single point opposite T«A and A«T has been determined. Equilibrium dissociation constants KJ) for ImlmPyPy-γ-ImPyPyPy-β-Dp 1, ImlmPyPy-γ-ImHpPyPy-β-Dp 2, ImlmHpPy-γ-ImPyPyPy-β-Dp 3 of Figure 1 are shown in Table 1. Brenowitz, M., Senear, D. F., Shea, M. A. & Ackers, G. K. describe a quantitative DNase footprint titration method for studying protein-DNA interactions. Methods Enzymol. 130, 132-181 (1986); The I j values were determined by quantitative DNase I footprint titration experiments: on a 3' 32 P-labeled 250-bp DNA fragment containing the target sites, 5'- TGGACA-3' and 5'-TGGTCA-3' which differ by a single A»T base pair in the fourth position. The DNase footprint gels are shown in Figure 3. K Λ = 0.077 nM K d = 0. \ 5 nM K d = 15 nM K ά = 0.83 nM 5"-T G G [*- " J C A-3 ' 5 ' -T G G [SI C A-3 ' •#®CK #® K 3 Hp/Py +XKX>0# +X X 0 ^ 77 3'-A C C [AJ G T-5 ' 3 '-A C C T G T-5' K d = 0.48 nM K d =37 nM *The reported dissociation constants are the average values obtained from three DNase I footprint titration experiments. The standard deviation for each data set is less than 15% of the reported number. Assays were carried out in the presence of 10 mM Tris'HCl, 10 M KCl, 10 mM MgCl 2 , and 5 mM CaCl 2 at pH 7.0 and 22 °C. tRing pairing opposite T«A and A»T in the fourth position. ^Calculated as K d (5'-TGGACA-3')/K d (5'-TGGTC A-3'). Based on the pairing rules for polyamide-DNA complexes both of these sequences are a match for control polyamide 1 which places a Py/Py pairing opposite A*T and T»A at both sites. It was determined that in polyamide 1 (Py/Py) binds to 5'- TGGTCA-3' and 5'-TGGACA-3' within a factor of 2 (K^ = 0.077 or 0.15 nM respectively). In contrast, polyamide 2 (Py/Hp) binds to 5'-TGGT_CA-3' and 5'-TGGACA-3' with dissociation constants which differ by a factor of 18 (Kj = 15 nM and 0.83 nM respectively). By reversing the pairing in polyamide 3 (Hp/Py) the dissociation constants differ again in the opposite direction by a factor of 77 (K D = 0.48 nM and 37 nM respectively. Control experiments performed on separate DNA fragments; reveal that neither a 5'-TGGGCA-3' or a 5'-TGGCCA- 3' site is bound by polyamide 2 or 3 at concentrations < 100 nM, indicating that the Hp/Py and Py/Hp ring pairings do not bind opposite G»C or C«G. The A»T vs. T«A discrimination is achieved preferably when the two neighboring base pairs are G*C and C«G (GTC vs. GAC). The specificity of polyamides 2 and 3 for sites which differ by a single A«T/T»A base pair results from small chemical changes. Replacing the Py/Py pair in 1 with a Py/Hp pairing as in 2, a single substitution of C3-OH for C3-H, destabilizes interaction with 5'-TGGT_CA-3' by 191-fold, a free energy difference of 3.1 kcal mol '1 . Interaction of 2 with 5'-TGGACA-3' is destabilized only 6-fold relative to 1, a free energy difference of 1.1 kcal mol "1 . Similarly, replacing the Py/Py pair in 1 with Hp/Py as in 3 destabilizes interaction with 5'-TGGACA-3' by 252-fold, a free energy difference of 3.2 kcal mol "1 . Interaction of 3 with 5'TGGTCA-3' is destabilized only 6-fold relative to 1, a free energy difference of 1.0 kcal mol '1 . The polyamides of this invention provide for coded targeting of predetermined DNA sequences with affinity and specificity comparable to sequence-specific DNA binding proteins. Hp, Im, and Py polyamides complete the minor groove recognition code using three aromatic amino acids which combine to form four ring pairings (Im/Py, Py/Im, Hp/Py, and Py/Hp) which complement the four Watson-Crick base pairs, as shown in TABLE 2. There are a possible 240 four base pair sequences which contain at least 1 A»T or T»A base pair and therefore can advantageously use an Hp/Py, or Py/Hp carboxamide binding. Polyamides binding to any of these sequences can be designed in accordance with the code of TABLE 2. TABLE 2 Pairing code for minor groove recognition* Pair G « C C » G T » A A » T Im/Py Py Im Hp/Py Py/Hp * favored (+), disfavored (-) For certain G » C rich sequences the affinity of polyamide»DNA complexes may be enhanced by substitution of an Im/β pair for Im/Py at G»C and β/Im for Py/Im at C*G. At A»T and T » A base pairs, either a Py/β, β/Py, and β/β may be used. The alternate aliphatic/aromatic amino acid pairing code is described in Table 3. TABLE 3 Aliphatic /Aromatic substitution for ring pairings* Pair Substitution Im Py Im/β Py/Im β/Im Hp/Py Py/β, β/Py, Hp/β, β/β Py/Hp Py/β, β/Py, β/Hp, β/β U. S. Patent 5,578,444 describes numerous promoter region targeting sequences from which base pair sequences for targeting a polyamide can be identified. PCT U.S. 97/003332 describes methods for synthesis of polyamides which are suitable for preparing polyamides of this invention. The use of β-alanine in place of a pyrrole amino acid in the synthetic methods provides aromatic/aliphatic pairing (Im/β, β/Im, Py/β, and β/Py) and aliphatic/aliphatic pairing (β/β) substitution. The use of γ-aminobutyric acid, or a substituted γ-aminobutyric acid such as (R)-2,4 diaminobutyric acid, provides for preferred haiφin turns. The following examples illustrate the synthesis of polyamides of the present invention. Example 1 : PREPARATION OF A PROTECTED Hp MONOMER FOR SOLID PHASE SYNTHESIS. Distamycin and its analogs have previously been considered targets of traditional multistep synthetic chemistry. Arcamone, F., Orezzi, P. G., Barbieri, W., Nicolella, V. & Penco, S. describe a solution phase synthesis of distamycin Gazz. Chim. Ital. 1967, 97, 1097. The repeating amide of distamycin is formed from an aromatic carboxylic acid and an aromatic amine. The aromatic acid is often unstable to decarboxylation, and the aromatic amines have been found to be air and light sensitive. Lown, J. W. & Krowicki, K. describe a solution phase synthesis of Distamycin J. Org. Chem. 1985, 50, 3774. The variable coupling yields, long reaction times (often >24 h), numerous side products, and reactive intermediates (acid chlorides and trichloro ketones) characteristic of the traditional solution phase coupling reactions make the synthesis of the aromatic carboxamides problematic. B. Merrifield describes the solid phase synthesis of a tetrapeptide J. Am. Chem. Soc. 1963, 85, 2149. In order to implement an efficient solid phase methodology for the synthesis of the pyrrole- imidazole polyamides, the following components were developed: (1) a synthesis which provides large quantities of appropriately protected monomer or dimer building blocks in high purity, (2) optimized protocols for forming an amide in high yield from a support-bound aromatic amine and an aromatic carboxylic acid, (3) methods for monitoring reactions on the solid support, and (4) a stable resin linkage agent that can be cleaved in high yield upon completion of the synthesis. Baird, E. E. & Dervan, P. B. describes the solid phase synthesis of polyamides containing imidazole and pyrrole amino acids. J. Am. Chem. Soc. 118, 6141-6146 (1996); also see PCT US 97/003332. In order to prepare polyamides which contain the 3-hydroxypyrrole monomer, a synthesis has been developed which allows the appropriately protected Boc-Op acid monomer to be prepared on 50 g scale. l NMR and 13 C NMR spectra were recorded on a General Electric-QE 300 NMR spectrometer in CD 3 OD or DMSO-- 6 , with chemical shifts reported in parts per million relative to residual CHD 2 OD or DMSO-c 5, respectively. IR spectra were recorded on a Perkin-Elmer FTIR spectrometer. High-resolution mass spectra were recorded using fast atom bombardment (FABMS) techniques at the Mass Spectrometry Laboratory at the University of California, Riverside. Reactions were executed under an inert argon atmosphere. Reagent grade chemicals were used as received unless otherwise noted. Still, W. C, Kahn, M. & Mitra, A. describe flash column chromatography J. Org. Chem. 1978, 40, 2923-2925. Flash chromatography was carried out using EM science Kieselgel 60 (230-400) mesh. Thin-layer chromatography was performed on EM Reagents silica gel plates (0.5 mm thickness). All compounds were visualized with short-wave ultraviolet light. Table 4 : Intermediates for preparation of Boc-protected 3-methoxypyrrole NAME STRUCTURE Ethyl 4-carboxy-3-hydfoxy-l- methylpyrrole-2-carboxylate. Ethyl 4-[(Benzyloxycarbonyl)amino]-3- hydroxy-l-methylpyrrole-2-carboxylate Ethyl 4-[(Benzyloxycarbonyl)amino]-3- methoxy-l-methylpyrrole-2-carboxylate Ethyl 4-[(tert-Butyloxycarbonyl)amino]-3- methoxy-l-methylpyrrole-2-carboxylate 4-[(tert-Butyloxycarbonyl)amino]-3-methoxy -1 -methylpy rrole-2-carboxylic acid Ethyl 4-[(benzyloxycarbonyl)amino]-3-hydroxy- 1 -methylpyrrole-2-carboxylate Ethyl-4- carboxy-3-hydroxy-l-methylpyrrole-2-carboxylate (60 g, 281.7 mmol) was dissolved in 282 mL acetonitrile. TEA (28.53 g, 282 mmol) was added, followed by diphenylphosphorylazide (77.61 g, 282 mmol). The mixture was refluxed for 5 hours, followed by addition of benzyl alcohol (270 ml) and reflux continued overnight. The solution was cooled and volitiles removed in vacuo. The residue was absorbed onto silca and chromatagraphed, 4: 1 hexanes : ethyl acetate, to give a white solid (21.58 g, 24%) H NMR (DMSO-d6) δ 8.73 (s, 1H), 8.31 (s, 1H), 7.31 (m, 5H), 6.96 (s, 1H), 5.08 (s, 2H), 4.21 (q, 2H, J = 7.1 Hz), 3.66 (s, 3H), 1.25 (t, 3H, J = 7.1 Hz); MS m/e 319.163 (M+H 319.122 calcd. for C16H18 2O5). Ethyl 4-[(tert-butoxycarbonyl)amino]-3-methoxy-l-methylpyrrole-2- carboxylate. Ethyl 4-[(benzyloxycarbonyl)amino]-3-hydroxy-l-methylpyrrole-2-carboxylate (13.4 g, 42.3 mmol) was dissolved in 110 mL acetone. Anhydrous K2CO3 (11.67 g, 84.5 mmol) was added, followed by methyliodide (5.96 g, 42.3 mmol) and dimethylaminopyridine (0.5 g, 4.23 mmol) and the mixture stirred overnight. The solid K2CO3 was removed by filtration and 200 ml water added. Volitiles were removed in vacuo and the solution made acidic with addition of IN H2SO4 . The aqueous layer was extracted with diethyl ether. Organic layers were combined, washed with 10% H2SO4, dried over MgSO4, and dried to give a white solid. The solid was used without further purification and dissolved in 38 ml DMF. DIEA (11 ml), Boc anhydride (9.23 g, 42.3 mmol), and 10 % Pd C (500 mg) were added and the solution stirred under hydrogen (1 arm) for 2.1 h. The slurry was filtered through celite which was washed with methanol. Water 250 ml was added and volitiles removed in vacuo. The aqueous layer was extracted with ether. Organic layers were combined, washed with water and brine, and dried over MgSO4. Solvent was removed in vacuo to give a white solid ( 8.94 g, 71%) H ΝMR (DMSO-d6) δ 8.43 (s, 1H), 7.03 (s, 1H), 4.19 (q, 2H, J = 7.1 Hz), 3.70 (s, 3H), 3.67 (s, 3H), 1.42 (s, 9H), 1.26 (t, 3H, J = 7.1); MS m/e 299.161 (M+H 299.153 calcd. for 4H22Ν2O5). Ethyl 4-[(benzyloxycarbonyl)amino]-3-hydroxy-l-methylpyrrole-2-carboxylate Ethyl 4- [(tert-butoxycarbonyl)amino]-3-methoxy-l-methylpyrrole-2-carboxylate (9.0 g, 30.2 mmol) was dissolved in 30 mL ethanol. NaOH (30 ml, 1 M, aq) was added and the solution stirred for 4 days. Water (200 ml) was added and ethanol removed in vacuo. The solution was extracted with diethyl ether, aqueous layer acidified to pH = 2-3, and extracted again with diethyl ether. Organic layers were dried over MgSO4, and solvent removed in vacuo to give a white solid (6.0 g, 20.5 mmol, 87% based on recovered SM) l U NMR (DMSO-d6) δ 12.14 (s, 1H), 8.37 (s, 1H), 6.98 (s, 1H), 3.69 (s, 3H), 3.66 (s, 3H), 1.42 (s, 9H); MS m/e 293.112 (M+H 293.104 calcd. for C12H18N2O5). EXAMPLE 2: SOLID PHASE SYNTHESIS OF 3-HYDROXYPYRROLE POLYAMIDES. Cycling protocols were optimized to afford high stepwise coupling yields (>99%). Deprotection by amino lysis affords up to 100 mg quantities of polyamide. Solid phase polyamide synthesis protocols were modified from the in situ neutralization Boc-chemistry protocols. Schnolzer, M., Alewood, P., Jones, A., Alewood, D., Kent, S.B.H. report rapid in situ neutralization for solid phase peptide synthesis Int. J. Peptide. Protein. Res. 1992, 40, 180. Coupling cycles are rapid, 72 min per residue for manual synthesis or 180 min per residue for machine-assisted synthesis, and require no special precautions beyond those used for ordinary solid phase peptide synthesis. Manual solid phase synthesis of a pyrrole-imidazole polyamide consists of a dichloromethane (DCM) wash, removal of the Boc group with trifluoroacetic acid (TFA)/DCM/thiophenol (PhSH), a DCM wash, a DMF wash, taking a resin sample for analysis, addition of activated monomer, addition of DIEA if necessary, coupling for 45 min, taking a resin sample for analysis, and a final DMF wash (Figure 5, Table I). In addition, the manual solid phase protocol for synthesis of pyrrole-imidazole polyamides has been adapted for use on a ABI 430A peptide synthesizer. The aromatic amine of the pyrrole and imidazole do not react in the quantitative ninhydrin test. Stepwise cleavage of a sample of resin and analysis by HPLC indicates that high stepwise yields (> 99%) are routinely achieved. Dicyclohexylcarbodiimide (DCC), Hydroxybenzotriazole (HOBt), 2-(lH-Benzotriazole- l-yl)-l,l,3,3-tetramethyluronium hexa-fluorophosphate (HBTU) and 0.2 mmol gram Boc-β- alanine-(-4-carboxamidomethyl)-benzyl-ester-copoly(styrene-divinylbenzene) resin (Boc-β- Pam-Resin) was purchased from Peptides International (0.2 mmol gram), NovaBiochem (0.6 mmol/gram), or Peninsula (0.6 mmol/gram). ( (R)-2-Fmoc-4-Boc-diaminobutyric acid, (S)-2- Fmoc-4-Boc-diaminobutyric acid, and (Λ)-2-amino-4-Boc-diaminobutyric acid were purchased from Bachem. N,N-diisopropylethylamine (DIEA), NN-dimethyl formamide (DMF), N- methylpyrrolidone (ΝMP), DMSO/ΝMP, Acetic anhydride (Ac 2 O), and 0.0002 M potassium cyanide/pyridine were purchased from Applied Biosystems. Dichloromethane (DCM) and triethylamine (TEA) were reagent grade from EM, thiophenol (PhSH), dimethylaminopropylamine (Dp), Sodium Hydride, (R)-α-methoxy-α- (trifuoromethyl)phenylacetic acid ((R)MPTA) and (S)-α-methoxy-α- (trifouromethyl)phenylacetic acid ((S)MPTA) were from Aldrich, trifluoroacetic acid (TFA) Biograde from Halocarbon, phenol from Fisher, and ninhydrin from Pierce. All reagents were used without further purification. Quik-Sep polypropylene disposable filters were purchased from Isolab Inc. Η ΝMR spectra were recorded on a General Electric-QE ΝMR spectrometer at 300 MHz with chemical shifts reported in parts per million relative to residual solvent. UV spectra were measured in water on a Hewlett-Packard Model 8452A diode array spectrophotometer. Optical rotations were recorded on a JASCO Dip 1000 Digital Polarimeter. Matrix-assisted, laser desoφtion/ionization time of flight mass spectrometry (MALDI-TOF) was performed at the Protein and Peptide Microanalytical Facility at the California Institute of Technology. HPLC analysis was performed on either a HP 1090M analytical HPLC or a Beckman Gold system using a RALΝEΝ Cι 8 , Microsorb MV, 5μm, 300 x 4.6 mm reversed phase column in 0.1% (wt/v) TFA with acetonitrile as eluent and a flow rate of 1.0 mL/min, gradient elution 1.25% acetonitrile/min. Preparatory reverse phase HPLC was performed on a Beckman HPLC with a Waters DeltaPak 25 x 100 mm, 100 μm C18 column equipped with a guard, 0.1% (wt v) TFA, 0.25% acetonitrile/min. 18MΩ water was obtained from a Millipore MilliQ water purification system, and all buffers were 0.2 μm filtered. Activation of Boc-3-methoxypyrrole acid. The amino acid (0.5 mmol) was dissolved in 2 mL DMF. HBTU (190 mg, 0.5 mmol) was added followed by DIEA (1 mL) and the resulting mixture was shaken for 5 min. Activation of Imidazole-2-carboxylic acid, γ-aminobutyric acid, Boc-glycine, and Boc-β- alanine. The appropriate amino acid or acid (2 mmol) was dissolved in 2 mL DMF. HBTU (720 mg, 1.9 mmol) was added followed by DIEA (1 mL) and the solution shaken for at least 5 min. Activation of Boc-Imidazole acid. Boc imidazole acid (257 mg, 1 mmol) and HOBt (135 mg, 1 mmol) were dissolved in 2 mL DMF, DCC (202 mg, 1 mmol) is then added and the solution allowed to stand for at least 5 min. Acetylation Mix. 2 mL DMF, DIEA (710 μL, 4.0 mmol), and acetic anhydride (380 μL, 4.0 mmol) were combined immediately before use. Manual Synthesis Protocol. Boc-β-alanine-Pam-Resin (1.25 g, 0.25 mmol) is placed in a 20 mL glass reaction vessel, shaken in DMF for 5 min and me reaction vessel drained. The resin was washed with DCM (2 x 30 s.) and the Boc group removed with 80% TFA/DCM/0.5M PhSH, 1 x 30s., 1 x 20 min The resin was washed with DCM (2 x 30 s.) followed by DMF (1 x 30 s.) A resin sample (5 - 10 mg) was taken for analysis. The vessel was drained completely and activated monomer added, followed by DIEA if necessary. The reaction vessel was shaken vigorously to make a slurry. The coupling was allowed to proceed for 90 min, and a resin sample taken. Acetic anhydride (1 mL) was added and the reaction shaken for 5 min. The reaction vessel was then washed with DMF, followed by DCM. Machine-Assisted Protocols. Machine-assisted synthesis was performed on a ABI 430A synthesizer on a 0.18 mmol scale (900 mg resin; 0.2 mmol/gram). Each cycle of amino acid addition involved: deprotection with approximately 80% TFA/DCM/0.4M PhSH for 3 minutes, draining the reaction vessel, and then deprotection for 17 minutes; 2 dichloromethane flow washes; an NMP flow wash; draining the reaction vessel; coupling for 1 hour with in situ neutralization, addition of dimethyl sulfoxide (DMSO)/NMP, coupling for 30 minutes, addition of DIEA, coupling for 30 minutes; draining the reaction vessel; washing with DCM, taking a resin sample for evaluation of the progress of the synthesis by HPLC analysis; capping with acetic anhydride/DIEA in DCM for 6 minutes; and washing with DCM. A double couple cycle is employed when coupling aliphatic amino acids to imidazole, all other couplings are performed with single couple cycles. The ABI 430A synthesizer was left in the standard hardware configuration for NMP- HOBt protocols. Reagent positions 1 and 7 were DIEA, reagent position 2 was TFA/0.5M thiophenol, reagent position 3 was 70% ethanolamine/methanol, reagent position 4 was acetic anhydride, reagent position 5 was DMSO/NMP, reagent position 6 was methanol, and reagent position 8 was DMF: New activator functions were written, one for direct transfer of the cartridge contents to the concentrator (switch list 21, 25, 26, 35, 37, 44), and a second for transfer of reagent position 8 directly to the cartridge (switch list 37, 39, 45, 46). Boc-Py-OBt ester (357 mg, 1 mmol) was dissolved in 2 ml DMF and filtered into a synthesis cartridge. Boc-Im acid monomer was activated (DCC/HOBt), filtered, and placed in a synthesis cartridge. Imidazole-2-carboxylic acid was added manually. At the initiation of the coupling cycle the synthesis was interrupted, the reaction vessel vented and the activated monomer added directly to the reaction vessel through the resin sampling loop via syringe. When manual addition was necessary an empty synthesis cartridge was used. Aliphatic amino acids (2 mmol) and HBTU (1.9 mmol) were placed in a synthesis cartridge. 3 ml of DMF was added using a calibrated delivery loop from reagent bottle 8, followed by calibrated delivery of 1 ml DIEA from reagent bottle 7, and a 3 minute mixing of the cartridge. The activator cycle was written to transfer activated monomer directly from the cartridge to the concentrator vessel, bypassing the activator vessel. After transfer, 1 ml of DIEA was measured into the cartridge using a calibrated delivery loop, and the DIEA solution combined with the activated monomer solution in the concentrator vessel. The activated ester in 2:1 DMF/DIEA was then transferred to the reaction vessel. All lines were emptied with argon before and after solution transfers. ImImOpPy-y-ImPyPyPy- -Dp. ImlmOpPy-γ-ImPyPyPy-β-Pam-Resin was synthesized in a stepwise fashion by machine-assisted solid phase methods from Boc-β-Pam-Resin (0.66 mmol/g). Baird, E. E. & Dervan, P. B. describes the solid phase synthesis of polyamides containing imidazole and pyrrole amino acids. J. Am. Chem. Soc. 118, 6141-6146 (1996); also see PCT US 97/003332. 3-hydroxypyrrole-Boc-amino acid (0.7 mmol) was incoφorated by placing the amino acid (0.5 mmol) and HBTU (0.5 mmol) in a machine synthesis cartridge. Upon automated delivery of DMF (2 mL) and DIEA (1 mL) activation occurs. A sample of ImlmOpPy-γ-ImPyPyPy-β-Pam-Resin (400 mg, 0.40 mmol/gram) was placed in a glass 20 mL peptide synthesis vessel and treated with neat dimethylaminopropylamine (2 mL) and heated (55 °C) with periodic agitation for 16 h. The reaction mixture was then filtered to remove resin, 0.1% (wt/v) TFA added (6 mL) and the resulting solution purified by reversed phase HPLC. ImlmOpPy-γ-ImPyPyPy-β-Dp is recovered upon lyophilization of the appropriate fractions as a white powder (97 mg, 49% recovery). UV (H 2 O) λ^ 246, 316 (66,000); Η NMR (DMSO-d 6 ) δ 10.24 (s, 1 H), 10.14 (s, 1 H), 9.99 (s, 1 H), 9.94 (s, 1 H), 9.88 (s, 1 H), 9.4 (br s, 1 H), 9.25 (s, 1 H), 9.11 (s, 1 H), 8.05 (m, 3 H), 7.60 (s, 1 H), 7.46 (s, 1 H), 7.41 (s, 1 H), 7.23 (d, 1), 7.21 (d, 1 H), 7.19 (d, 1 H), 7.13 (m, 2 H), 7.11 (m, 2 H), 7.02 (d, 1 H), 6.83 (m, 2 H), 3.96 (s, 6 H), 3.90 (s, 3 H), 3.81 (m, 6 H), 3.79 (s, 3 H), 3.75 (d, 9 H), 3.33 (q, 2 H, J= 5.4 Hz), 3.15 (q, 2 H, J= 5.5 Hz), 3.08 (q, 2 H, J= 6.0 Hz), 2.96 (quintet, 2 H, J= 5.6 Hz), 2.70 (d, 6 H, J= 4.5 Hz), 2.32 (m, 4 H), 1.71 (m, 4 H); MALDI-TOF-MS (monoisotopic), 1253.5 (1253.6 calc. for ImlmHpPy-y-ImPyPyPy. In order to remove the methoxy protecting group, a sample of ImlmOpPy-γ-ImPyPyPy-β-Dp (5 mg, 3.9 μmol) was treated with sodium thiophenoxide at 100 °C for 2 h. DMF (1000 μL) and thiophenol (500 μL) were placed in a (13 x 100 mm) disposable Pyrex screw cap culture tube. A 60 % dispersion of sodium hydride in mineral oil (100 mg) was slowly added. Upon completion of the addition of the sodium hydride, ImlmOpPy-γ-ImPyPyPy- β-Dp (5 mg) dissolved in DMF (500 μL) was added. The solution was agitated, and placed in a 100 °C heat block, and deprotected for 2 h. Upon completion of the reaction the culture tube was cooled to 0°C, and 7 ml of a 20 % (wt/v) solution of trifluoroacetic acid added. The aqueous layer is separated from the resulting biphasic solution and purified by reversed phase HPLC. ImlmHpPy-γ-ImPyPyPy-β-Dp is recovered as a white powder upon lyophilization of the appropriate fractions (3.8 mg, 77 % recovery). UV (H 2 O) λ^ x 246, 312 (66,000); Η NMR (DMSO-4,) δ 10.34 (s, 1 H), 10.24 (s, 1 H), 10.00 (s, 2 H), 9.93 (s, 1 H), 9.87 (s, 1 H), 9.83 (s, 1 H), 9.4 (br s, 1 H), 9.04 (s, 1 H), 8.03 (m, 3 H), 7.58 (s, 1 H), 7.44 (s, 1 H), 7.42 (s, 1 H), 7.23 (s, 1 H), 7.20 (m, 3 H), 7.12 (m, 2 H), 7.05 (d, 1 H), 7.02 (d, 1 H), 6.83 (s, 1 H), 6.79 (s, 1 H), 3.96 (s, 6 H), 3.90 (s, 3 H), 3.81 (s, 6 H), 3.79 (s, 3 H), 3.75 (d, 6 H), 3.33 (q, 2 H, J= 5.4 Hz), 3.14 (q, 2 H, J= 5.4 Hz), 3.08 (q, 2 H, J= 6.1 Hz), 2.99 (quintet, 2 H, J= 5.4 Hz), 2.69 (d, 6 H, J = 4.2 Hz), 2.31 (m, 4 H), 1.72 (m, 4 H); MALDI-TOF-MS (monoisotopic), 1239.6 (1239.6 calc. for C 57 H 71 N 2 Oπ). ImImPyPy- -ImOpPyPy- -Dp. ImlmPyPy-γ-ImOpPyPy-β-Pam-Resin was synthesized in a stepwise fashion by machine-assisted solid phase methods from Boc-β-Pam-Resin (0.66 mmol/g) as described for ImlmOpPy-γ-ImPyPyPy-β-Dp. A sample of ImlmPyPy-γ-ImOpPyPy- β-Pam-Resin (400 mg, 0.40 mmol/gram) was placed in a glass 20 mL peptide synthesis vessel and treated with neat dimethylaminopropylamine (2 mL) and heated (55 °C) with periodic agitation for 16 h. The reaction mixture was then filtered to remove resin, 0.1% (wt/v) TFA added (6 mL) and the resulting solution purified by reversed phase HPLC. ImlmPyPy-γ- ImOpPyPy-β-Dp is recovered upon lyophilization of the appropriate fractions as a white powder (101 mg, 50% recovery). UV (H 2 O) X™ * 246, 316 (66,000); MALDI-TOF-MS (monoisotopic), 1253.6 (1253.6 calc. for C 58 H 72 N 22 O n ). ImlmPyPy-y-ImHpPyPy. A sample of ImlmPyPy-γ-ImOpPyPy-β-Dp (5 mg, 3.9 μmol) was treated with sodium thiophenoxide and purified by reversed phase HPLC as described for I -mHpPy-γ-ImPyPyPy-β-Dp. ImlmPyPy-γ-ImHpPyPy-β-Dp is recovered upon lyophilization of the appropriate fractions as a white powder (3.2 mg, 66 % recovery). UV (H 2 O) λ- j ^ 246, 312 (66,000); MALDI-TOF-MS (monoisotopic), 1239.6 (1239.6 calc. for Cs^N^O,,). ImPyPy-y-OpPyPy- -Dp. ImPyPy-γ-OpPyPy-β-Pam-Resin was synthesized in a stepwise fashion by machine-assisted solid phase methods from Boc-β-Pam-Resin (0.66 mmol g). Baird, E. E. & Dervan, P. B. describes the solid phase synthesis of polyamides containing imidazole and pyrrole amino acids. J. Am. Chem. Soc. 118, 6141-6146 (1996); also see PCT US 97/003332. 3-hydroxypyrrole-Boc-amino acid (0.7 mmol) was incoφorated by placing the amino acid (0.5 mmol) and HBTU (0.5 mmol) in a machine synthesis cartridge. Upon automated delivery of DMF (2 mL) and DIEA (1 mL) activation occurs. A sample of ImPyPy-γ-OpPyPy-β-Pam-Resin (400 mg, 0.45 mmol gram) was placed in a glass 20 mL peptide synthesis vessel and treated with neat dimethylaminopropylamine (2 mL) and heated (55 °C) with periodic agitation for 16 h. The reaction mixture was then filtered to remove resin, 0.1% (wt v) TFA added (6 mL) and the resulting solution purified by reversed phase HPLC. ImPyPy-γ-OpPyPy-β-Dp is recovered upon lyophilization of the appropriate fractions as a white powder (45 mg, 25% recovery). UV (H 2 O) λ^ 246, 310 (50,000); Η NMR (DMSO-d 6 ) δ 10.45 (s, 1 H), 9.90 (s, 1 H), 9.82 (s, 1 H), 9.5 (br s, 1 H), 9.38 (s, 1 H), 9.04 (s, 1 H), 8.02 (m, 3 H), 7.37 (s, 1 H), 7.25 (m, 2 H), 7.15 (d, 1 H, J= 1.6 Hz), 7.1 1 (m, 2 H), 7.09 (d, 1 H), 7.03 (d, 1 H), 6.99 (d, 1 H), 6.87 (d, 1 H), 6.84 (d, 1 H), 3.96 (s, 3 H), 3.81 (s, 6 H), 3.77 (s, 6 H), 3.76 (s, 3 H), 3.74 (s, 1 H), 3.34 (q, 2 H, J= 5.6 Hz), 3.20 (q, 2 H, J= 5.8 Hz), 3.09 (q, 2 H, J = 6.1 Hz), 2.97 (quintet, 2 H, J- 5.3 Hz), 2.70 (d, 6 H, J= 3.9 Hz). 2.34 (m, 4 H), 1.73 (m, 4 H); MALDI-TOF-MS (monoisotopic), 1007.6 (1007.5 calc. for C 48 H 63 N 16 O 9 ). ImPyPy-y-HpPyPy. In order to remove the methoxy protecting group, a sample of ImPyPy-γ-OpPyPy-β-Dp (5 mg, 4.8 μmol) was treated with sodium thiophenoxide at 100 °C for 2 h. DMF (1000 μL) and thiophenol (500 μL) were placed in a (13 x 100 mm) disposable Pyrex screw cap culture tube. A 60 % dispersion of sodium hydride in mineral oil (100 mg) was slowly added. Upon completion of the addition of the sodium hydride, I lmPyPy-γ-ImOpPyPy- β-Dp (5 mg) dissolved in DMF (500 μL) was added. The solution was agitated, and placed in a 100 °C heat block, and deprotected for 2 h. Upon completion of the reaction the culture tube was cooled to 0°C, and 7 ml of a 20 % (wt/v) solution of trifluoroacetic acid added. The aqueous layer is separated from the resulting biphasic solution and purified by reversed phase HPLC. ImlmHpPy-γ-ImHpPyPy-β-Dp is recovered as a white powder upon lyophilization of the appropriate fractions (2.5 mg, 52 % recovery). UV (H 2 O) λ ma 246, 310 (50,000); Η NMR (OMSO-d 6 ) δ 10.44 (s, 1 H), 10.16 (s, 1 H), 9.90 (s, 1 H), 9.77 (s, 1 H), 9.5 (br s, 1 H), 9.00 (s, 1 H), 8.03 (m, 3 H), 7.37 (s, 1 H), 7.26 (m, 2 H), 7.14 (d, 1 H, J = 1.7 Hz), 7.12 (m, 2 H), 7.02 (d, 1 H), 6.93 (d, 1 H), 6.88 (d, 1 H), 6.82 (d, 1 H), 6.72 (d, 1 H), 3.96 (s, 3 H), 3.81 (s, 6 H), 3.77 (s, 3 H), 3.76 (s, 3 H), 3.74 (s, 1 H), 3.36 (q, 2 H, J = 5.4 Hz), 3.22 (q, 2 H, J = 5.9 Hz), 3.09 (q, 2 H, J= 5.5 Hz), 2.98 (quintet, 2 H, J= 5.3 Hz), 2.70 (d, 6 H, J= 4.3 Hz), 2.34 (m, 4 H), 1.78 (m, 4 H); MALDI-TOF-MS (monoisotopic), 994.2 (993.5 calc. for C 47 H 61 N 16 O 9 ). Table 5. Mass spectral characterization of Op and Hp polyamides, synthesized and purified as described for IrrilmOpPy-γ-ImPyPyPy-β-Dp and hnln HpPy-γ-ImPyPyPy-β-Dp. POLYAMIDE FORMULA (M+H)CALCD FOUND ImOpPy-γ-PyPyPy-β-Dp C 48 H 63 Nι 6 O 1007.5 1007.5 ImHpPy-γ-PyPyPy-β-Dp C 47 H 61 N 16 O 9 993.5 993.2 ImPyOp-γ-PyPyPy-β-Dp C 48 H 63 N 16 O 9 1007.5 1007.5 ImPyHp-γ-PyPyPy-β-Dp C 47 H 61 N[ 6 O 9 993.5 993.4 ImPyPy-γ-OpPyPy-β-Dp C 8 H 63 N 16 O 1007.5 1007.6 ImPyPy-γ-HpPyPy-β-Dp C 47 H 61 N 16 O 9 993.5 993.2 ImPyPy-γ-PyOpPy-β-Dp C 8 H 63 Nι 6 O 1007.5 1007.5 ImPyPy-γ-PyHpPy-β-Dp C 47 H 61 N 16 O 9 993.5 993.4 ImOpOp-γ-PyPyPy-β-Dp C 9 H 65 N 16 Oιo 1037.5 1037.5 ImHpHp-γ-PyPyPy-β-Dp C 47 H 6 ιN 16 O 1( ) 1009.5 1009.4 ImlmOpPy-γ-ImPyPyPy-β-Dp C 58 H 72 N 22 O ! i 1253.6 1253.5 ImlmHpPy-γ-ImPyPyPy-β-Dp C 57 H 71 N 22 O u 1239.6 1239.6 ImlmPyPy-γ-ImOpPyPy-β-Dp C 8 H 72 N 22 O t l 1253.6 1253.6 ImlmPyPy-γ-ImHpPyPy-β-Dp C 57 H 71 N 22 Oπ 1239.6 1239.6 ImOpPyPy-γ-ImOpPyPy- β-Dp C 6 oH 6 N 2 ιOι 2 1282.6 1282.6 ImHpPyPy-γ-ImHpPyPy-β-Dp C 5 H 72 N 2 ιOι 2 1254.6 1254.6 ImlmOpPy-γ-ImOpPyPy-β-Dp C 59 H 75 N 22 O 12 1283.6 1283.6 ImlmHpPy-γ-ImHpPyPy-β-Dp C 57 H 71 N 22 O 12 1255.6 1255.5 ImOpPyPy-γ-PyPyPyPy-β-Dp C 6u H 75 N 20 Oπ 1251.6 1251.5 ImPyPyPy-γ-PyPyOpPy-β-Dp C6oH 75 N2oOπ 1251.6 1251.5 ImlmPyPy-γ-ImPyOpPy-β-Dp C 58 H 72 N 22 θπ 1253.6 1253.7 ImOpPyPyPy-γ-ImOpPyPyPy-β-Dp C 72 H 88 N 25 Oι 1526.7 1526.6 ImHpPyPyPy-γ-ImHpPyPyPy- β-Dp C 70 H 84 N 25 O 14 1498.7 1498.0 ImlmPyPyPy-γ-ImOpOpPyPy-β-Dp C 71 H 87 N 26 O 14 1527.7 1527.7 EXAMPLE 3: DETERMINATION OF POLYAMIDE BINDING AFFINITY AND SEQUENCE SPECIFICITY. Representative- footprint titration experiments are shown in Figures 3 and 10. A 252-bp DNA fragment which is typically used for the footprint titration experiments provides 247 possible 6-bp binding sites for an eight-ring haiφin polyamide. Thus, in addition to providing DNA binding affinities, the footprint titration experiments also reveal DNA binding sequence- specificity. The DNA binding sequence specificity of polyamides which differ by a single Py Py, Hp/Py, or Py/Hp pair for sites which differ by a single A # T or T»A base pair are described in Tables 1, 6, and 7. (Quantitative DNase I Footprint Titrations All reactions were executed in a total volume of 400 μL (Brenowitz, M. et al., 1986). A polyamide stock solution or H 2 O (for reference lanes) was added to an assay buffer containing 3'- P radiolabeled restriction fragment (20,000 cpm), affording final solution conditions of 10 mM TrisΗCl, 10 mM KCl, 10 mM MgCl 2 , 5 mM CaCl 2 , pH 7.0, and either (i) a suitable concentration range of polyamide, or (ii) no polyamide (for reference lanes). The solutions were allowed to equilibrate for 24 hours at 22°C. Footprinting reactions were initiated by the addition of 10 μL of a stock solution of DNase I (at the appropriate concentration to give -55% intact DNA) containing 1 mM dithiothreitol and allowed to proceed for 7 minutes at 22°C. The reactions were stopped by the addition of 50 μL of a solution containing 2.25 M NaCl, 150 mM EDTA, 23 μM base pair calf thymus DNA, and 0.6 mg/ml glycogen, and ethanol precipitated. The reactions were resuspended in 1 x TBE/ 80% formamide loading buffer, denatured by heating at 85°C for 15 minutes, and placed on ice. The reaction products were separated by electrophoresis on an 8% polyacrylamide gel (5% crosslinking, 7 M urea) in 1 x TBE at 2000 V for 1.5 h. Gels were dried on a slab dryer and then exposed to a storage phosphor screen at 22°C. Photostimuable storage phosphor imaging plates (Kodak Storage Phosphor Screen SO230 obtained from Molecular Dynamics) were pressed flat against dried gel samples and exposed in the dark at 22°C for 12-24 hours. A Molecular Dynamics 400S Phosphorlmager was used to obtain all data from the storage screens (Johnston et al., 1990). The data were analyzed by performing volume integration of the target site and reference blocks using the ImageQuant v. 3.3 software running on a Compaq Pentium 80. Quantitative DNase I Footprint Titration Data Analysis was performed by taking a background-corrected volume integration of rectangles encompassing the footprint sites and a reference site at which DNase I reactivity was invariant across the titration generated values for the site intensities (I site ) and the reference intensity (I ref ). The apparent fractional occupancy (θ app ) of the sites were calculated using the equation: where I site ° and I ref ° are the site and reference intensities, respectively, from a DNase I control lane to which no polyamide was added. The ([L] tot , θ app ) data were fit to a Langmuir binding isotherm (eq. 2, n=l) by minimizing the difference between θ app and θ fit , using the modified Hill equation: θfit — θmin + (θmax - θmin ) (2) 1 + a n [L]" tot where [L tot ] is the total polyamide concentration, K a is the equilibrium association constant, and θ min and θ max are the experimentally determined site saturation values when the site is unoccupied or saturated, respectively. The data were fit using a nonlinear least-squares fitting procedure of KaleidaGraph software (v. 3.0.1, Abelbeck Software) with K a , θ max , and θ mm as the adjustable parameters. The goodness of fit of the binding curve to the data points is evaluated by the correlation coefficient, with R > 0.97 as the criterion for an acceptable fit. Four sets of acceptable data were used in determining each association constant. All lanes from a gel were used unless a visual inspection revealed a data point to be obviously flawed relative to neighboring points. The data were normalized using the following equation: A: d = 0.20 μM K d = 0.56 μM K d = 0.33 μM K d = 0.59 μM The reported equilibrium dissociation constants are the mean values obtained from two DNase I footprint titration experiments. The assays were carried out at 22 °C, pH 7.0 in the presence of 10 mM TrisΗCl, 10 M KCl, 10 mM MgCl 2 , and 5 mM CaCl 2 . tRing pairing opposite T « A and A»T in the third position. ^Calculated as K d (5'-TGATT -3')/K d (5'-TGTTT-3'). EXAMPLE 5: PREPARATION OF A BIFUNCTIONAL Hp-Py-Im-POLYAMIDE. ImImOpPy-y-ImPyPyPy-β-Dp-NH2. ImlmOpPy-γ-ImPyPyPy-β-Pam-Resin was synthesized in a stepwise fashion by machine-assisted solid phase methods from Boc-β-Pam- Resin (0.66 mmol/g). Baird, E. E. & Dervan, P. B. describes the solid phase synthesis of polyamides containing imidazole and pyrrole amino acids. J. Am. Chem. Soc. 118, 6141-6146 (1996); also see PCT US 97/003332. 3-hydroxyρyrrole-Boc-amino acid (0.7 mmol) was incoφorated by placing the amino acid (0.5 mmol) and HBTU (0.5 mmol) in a machine synthesis cartridge. Upon automated delivery of DMF (2 mL) and DIEA (1 mL) activation occurs. A sample of ImlmOpPy-γ-ImPyPyPy-β-Parn-Resin (400 mg, 0.40 mmol/gram) was placed in a glass 20 mL peptide synthesis vessel and treated with neat 3,3'-diamino-N- methyldipropylamine (2 mL) and heated (55 °C) with periodic agitation for 16 h. The reaction mixture was then filtered to remove resin, 0.1% (wt/v) TFA added (6 mL) and the resulting solution purified by reversed phase HPLC. ImImOpPy-γ-ImPyPyPy-β-Dp-ΝH2 is recovered upon lyophilization of the appropriate fractions as a white powder (93 mg, 46% recovery). UV (H2O) λmax 246, 316 (66,000); ! H NMR (DMSO-ctø) δ 10.34 (s, 1 H), 10.30 (br s, 1 H), 10.25 (s, 1 H), 9.96 (s, 1 H), 9.95 (s, 1 H), 9.89 (s, 1 H), 9.24 (s, 1 H), 9.11 (s, 1 H), 8.08 (t, 1 H, J = 5.6 Hz), 8.0 (m, 5 H), 7.62 (s, 1 H), 7.53 (s, 1 H), 7.42 (s, 1 H), 7.23 (d, 1H, J = 1.2 Hz), 7.21 (m, 2 H), 7.15 (m, 2 H), 7.13 (d, 1 H), 7.11 (m, 2 H), 7.04 (d, 1 H), 6.84 (m, 3 H), 3.98 (s, 3 H), 3.97 (s, 3 H), 3.92 (s, 3 H), 3.82 (m, 6 H), 3.80 (s, 3 H), 3.77 (d, 6 H), 3.35 (q, 2 H, J= 5.8 Hz) 3.0-3.3 (m, 8 H), 2.86 (q, 2 H, J = 5.4 Hz), 2.66 (d, 3 H, J = 4.5 Hz), 2.31 (m, 4 H), 1.94 (quintet, 2 H, J = 6.2 Hz), 1.74 (m, 4 H); MALDI-TOF-MS (monoisotopic), 1296.0 (1296.6 calc. for C6OH78N23O11). ImlmOpPy-y-ImPyPyPy-β-Dp-EDTA. Excess EDTA-dianhydride (50 mg) was dissolved in DMSO/NMP (1 mL) and DIEA (1 mL) by heating at 55 °C for 5 min. The dianhydride solution was added to hnImOpPy-γ-ImPyPyPy-β-NH2 (13 mg, 10 μmol) dissolved in DMSO (750 μL). The mixture was heated (55 °C, 25 min.) and the remaining EDTA-anhydride hydrolyzed (0.1M NaOH, 3 mL, 55 °C, 10 min). Aqueous TFA (0.1% wt/v) was added to adjust the total volume to 8 mL and the solution purified directly by reversed phase HPLC to provide IrnlmOpPy-γ-ImPyPyPy-β-Dp-EDTA as a white powder upon lyophilization of the appropriate fractions (5.5 mg, 40% recovery). MALDI-TOF-MS (monoisotopic), 1570.9 (1570.7 calc. for C70H92N25OI8). ImlmHpPy-y-ImPyPyPy-β-Dp-EDTA. In order to remove the methoxy protecting group, a sample of ImlmOpPy-γ-ImPyPyPy-β-Dp-EDTA (5 mg, 3.1 μmol) was treated with sodium thiophenoxide at 100 °C for 2 h. DMF (1000 μL) and thiophenol (500 μL) were placed in a (13 x 100 mm) disposable Pyrex screw cap culture tube. A 60 % dispersion of sodium hydride in mineral oil (100 mg) was slowly added. Upon completion of the addition of the sodium hydride, ImlmOpPy-γ-ImPyPyPy-β-Dp-EDTA (5 mg) dissolved in DMF (500 μL) was added. The solution was agitated, and placed in a 100 °C heat block, and deprotected for 2 h. Upon completion of the reaction the culture tube was cooled to 0°C, and 7 ml of a 20 % (wt/v) solution of trifluoroacetic acid added. The aqueous layer is separated from the resulting biphasic solution and purified by reversed phase HPLC. ImlmHpPy-γ-ImPyPyPy-β-Dp-EDTA is recovered as a white powder upon lyophilization of the appropriate fractions (3.2 mg, 72 % recovery). UV (H2O) λ max 246, 312 (66,000); MALDI-TOF-MS (monoisotopic), 1556.6 (1556.7 calc. for C69H9θN25θl8)- ImImPyPy-y-ImOpPyPy-β-Dp-NH2. ImlmPyPy-γ-ImOpPyPy-β-Pam-Resin was synthesized in a stepwise fashion by machine-assisted solid phase methods from Boc-β-Pam- Resin (0.66 mmol/g). Baird, E. E. & Dervan, P. B. describes the solid phase synthesis of polyamides containing imidazole and pyrrole amino acids. J. Am. Chem. Soc. 118, 6141-6146 (1996); also see PCT US 97/003332. 3-hydroxypyrrole-Boc-amino acid (0.7 mmol) was incoφorated by placing the amino acid (0.5 mmol) and HBTU (0.5 mmol) in a machine synthesis cartridge. Upon automated delivery of DMF (2 mL) and DIEA (1 mL) activation occurs. A sample of ImlmPyPy-γ-ImOpPyPy-β-Pam-Resin (400 mg, 0.40 mmol/gram) was placed in a glass 20 mL peptide synthesis vessel and treated with neat 3,3'-diamino-N- methyldipropylamine (2 mL) and heated (55 °C) with periodic agitation for 16 h. The reaction mixture was then filtered to remove resin, 0.1% (wt/v) TFA added (6 mL) and the resulting solution purified by reversed phase HPLC. ImImPyPy-γ-ImOpPyPy-β-Dp-NH2 is recovered upon lyophilization of the appropriate fractions as a white powder (104 mg, 54% recovery). UV (H2O) λ ma χ 246, 316 (66,000); MALDI-TOF-MS (monoisotopic), 1296.6 (1296.6 calc. for C6OH78N23O11). ImlmPyPy-y-ImOpPyPy-β-Dp-EDTA. Excess EDTA-dianhydride (50 mg) was dissolved in DMSO/NMP (1 mL) and DIEA (1 mL) by heating at 55 °C for 5 min. The dianhydride solution was added to ImImPyPy-γ-ImOpPyPy-β-NH2 (13 mg, 10 μmol) dissolved in DMSO (750 μL). The mixture was heated (55 °C, 25 min.) and the remaining EDTA-anhydride hydrolyzed (0.1M NaOH, 3 mL, 55 °C, 10 min). Aqueous TFA (0.1% wt/v) was added to adjust the total volume to 8 mL and the solution purified directly by reversed phase HPLC to provide ImlmPyPy-γ-ImOpPyPy-β-Dp-EDTA as a white powder upon lyophilization of the appropriate fractions (5.9 mg, 42% recovery). MALDI-TOF-MS (monoisotopic), 1570.8 (1570.7 calc. for C70H92N25OI8). ImlmPyPy-y-ImHpPyPy- -Dp-EDTA. In order to remove the methoxy protecting group, a sample of ImlmPyPy-γ-ImOpPyPy-β-Dp-EDTA (5 mg, 3.1 μmol) was treated with sodium thiophenoxide at 100 °C for 2 h. DMF (1000 μL) and thiophenol (500 μL) were placed in a (13 x 100 mm) disposable Pyrex screw cap culture tube. A 60 % dispersion of sodium hydride in mineral oil (100 mg) was slowly added. Upon completion of the addition of the sodium hydride, ImlmPyPy-γ-ImOpPyPy-β-Dp-EDTA (5 mg) dissolved in DMF (500 μL) was added. The solution was agitated, and placed in a 100 °C heat block, and deprotected for 2 h. Upon completion of the reaction the culture tube was cooled to 0°C, and 7 ml of a 20 % (wt/v) solution of trifluoroacetic acid added. The aqueous layer is separated from the resulting biphasic solution and purified by reversed phase HPLC. ImlmPyPy-γ-ImHpPyPy-β-Dp-EDTA is recovered as a white powder upon lyophilization of the appropriate fractions (3.2 mg, 72 % recovery). UV (H2O) λ max 246, 312 (66,000); MALDI-TOF-MS (monoisotopic), 1555.9 (1556.7 calc. for C69H90N25O18). EXAMPLE 6: DETERMINATION OF POLYAMIDE BINDING ORIENTATION Affinity cleavage experiments using haiφin polyamides modified with EDTA*Fe(II) at either the C-terminus or on the γ-turn, were used to determine polyamide binding orientation and stoichiometry. The results of affinity cleavage experiments are consistent only with recognition of 6-bp by an 8-ring haiφin complex and rule out any extended 1 : 1 or overlapped complex formation. In addition, affinity cleavage experiments reveal haiφin formation supporting the claim that it is the Hp/Py and Py/Hp pairing which form at both match and mismatch sites to discriminate A»T from T»A. Affinity cleavage reactions were executed in a total volume of 40 μL. A stock solution of polyamide or H 2 O was added to a solution containing labeled restriction fragment (20,000 cpm), affording final solution conditions of 25 mM Tris- Acetate, 20 mM NaCl, 100 μM/bp calf thymus DNA, and pH 7.0. Solutions were incubated for a minimum of 4 hours at 22°C. Subsequently, 4 μL of freshly prepared 100 μM Fe(NH 4 ) 2 (SO 4 ) 2 was added and the solution allowed to equilibrate for 20 min. Cleavage reactions were initiated by the addition of 4 μL of 100 mM dithiothreitol, allowed to proceed for 30 min at 22 °C, then stopped by the addition of 10 μL of a solution containing 1.5 M NaOAc (pH 5.5), 0.28 mg/mL glycogen, and 14 μM base pairs calf thymus DNA, and ethanol precipitated. The reactions were resuspended in lx TBE/80% formamide loading buffer, denatured by heating at 85 °C for 15 min, and placed on ice. The reaction products were separated by electrophoresis on an 8% polyacrylamide gel (5% cross-link, 7 M urea) in lx TBE at 2000 V for 1.5 hours. Gels were dried and exposed to a storage phosphor screen. Relative cleavage intensities were determined by volume integration of individual cleavage bands using ImageQuant software. EXAMPLE 7: IMPROVEMENT TO POLYAMIDE SEQUENCE SPECIFICITY. The polyamides- of this invention provide improved specificity relative to existing polyamide technology. Turner, J. T., Baird, E. E., and Dervan, P.B. describe the recognition of seven base pair sequences in the minor groove of DNA by ten-ring pyrrole-imidazole polyamide haiφins J. Am. Chem. Soc. 1997 119, 7636. For example, quantitative DNasel footprint titrations reveal that the 10-ring haiφin ImPyPyPyPy-γ-ImPyPyPyPy-β-Dp binds a 5'- TGTAACA-3- sequence with an equlibrium dissociation constant of 0.083 nM, and 18-fold specificity versus a single base mismatch site. A number of other sites are also bound on the 252-bp DNA fragment used for the footprint titration experiments. (Figure 13). Introduction of a Hp/Py and Py/Hp pair in the 10-ring polyamide, ImHpPyPyPy-γ-ImHpPyPyPy-β-Dp, to recognize a T # A and A*T within the 7-bp target sequence, increases the sequence-specificty. For example, a single base mismatch site 5'-TGGAACA-3 is discriminated by > 5000-fold (Figure 13, Table 8). In fact all 245 7-bp mismatch sites present on the restriction fragment are discriminated > 5000- fold by the polyamide ImHpPyPyPy-γ-ImHpPyPyPy-β-Dp (Figure 13). For cases where three A,T base pairs are present in succession it is preferred to substitute Py/Py in place of at least one Hp/Py or Py/Hp to provide for recognition of A*T and T«A at a single position. TABLE 8 Equilibrium dissociation constants* Polyamidet 5'-TGGTCA-3' 5'-TGGACA-3' K^ 5 '-T G fa fl A JJ ϊl c A-3 ' 5 '-T G [C -1 T fl £1 C A-3" • 5 ϊ>O C SOC Hp Py +X X +X X XX Hr 1 >5000 3'-A C |j X y τX[5 fj G T-5" 3 ' -A C [ -J* l [J O T-5' ).2 n\ i AT d > l ( XX) n VI * The reported dissociation constants are the average values obtained from three DNase I footprint titration experiments. The standard deviation for each data set is less than 15% of the reported number. Assays were carried out in the presence of 10 mM Tris'HCL 10 mM KCl, 10 mM MgC12, and 5 mM CaC12 at pH 7.0 and 22 "C. tRing pairing opposite T»A and A « T in the fourth position. ^Calculated as K d (5'-TGGTACA-3')/K d (5'-TGTAACA-3'). EXAMPLE 8: USE OF PAIRING CODE There are 256 possible four base pair combinations of A, T, G, and C. Of these, there are a possible 240 four base pair sequences which contain at least 1 A«T or T«A base pair and therefore can advantageously use an Hp/Py, or Py Hp carboxamide binding. Polyamides binding to any of these sequences can be designed in accordance with the code of TABLE 2. Table 9 lists the sixteen eight-ring haiφin polyamides (1-16) which recognize the sixteen 5'- WGTNN -3' sequences (W = A or T, X = A, G, C, or T). Table 10 lists the sixteen eight-ring haiφin polyamides (17-32) which recognize the sixteen 5'-WGANNW-3' sequences (17-32). Table 11 lists the twelve eight-ring haiφin polyamides (33-44) which recognize twelve 5'- WGGNNW-3' sequences which contain at least one A,T base pair. Table 11 lists the four eight- ring haiφin polyamides (G1-G4) which target the four 5'-WGGNNW-3' sequences (G1-G4) which contain exclusively G » C base pairs. Table 12 lists the twelve eight-ring haiφin polyamides (45-56) which recognize twelve 5'-WGCNN -3' sequences which contain at least one A,T base pair. Table 12 lists the four eight-ring haiφin polyamides (G5-G8) which target the four 5'-WGCNNW-3' sequences (G5-G8) which contain exclusively G'C base pairs. Table 13 lists the sixteen eight-ring haiφin polyamides (57-72) which recognize the sixteen 5'- WTTNNW-3' sequences (57-72). Table 14 lists the sixteen eight-ring haiφin polyamides (73- 88) which recognize the sixteen S'-WTANNW-S' sequences (73-88). Table 15 lists the sixteen eight-ring haiφin polyamides (89-104) which recognize the sixteen 5'-WTGNNW-3' sequences (89-104). Table 16 lists the sixteen eight-ring haiφin polyamides (105-120) which recognize the sixteen 5'-WTCNNW-3' sequences (105-120). Table 17 lists the sixteen eight-ring haiφin polyamides (121-136) which recognize the sixteen 5'-WATNNW-3' sequences (121-136). Table 18 lists the sixteen eight-ring haiφin polyamides (137-152) which recognize the sixteen 5'-WAANNW-3' sequences (137-152). Table 19 lists the sixteen eight-ring haiφin polyamides (153-168) which recognize the sixteen 5'-WAGNNW-3' sequences (153-168). Table 20 lists the sixteen eight-ring haiφin polyamides (169-184) which recognize the sixteen 5'-WACNN - 3' sequences (169-184). Table 21 lists the sixteen eight-ring haiφin polyamides (185-200) which recognize the sixteen 5'-WCTNNW-3' sequences (185-200). Table 22 lists the sixteen eight-ring haiφin polyamides (201-216) which recognize the sixteen 5'-WCANNW-3' sequences (201-216). Table 23 lists the twelve eight-ring haiφin polyamides (217-228) which recognize the twelve 5'-WCGNNW-3' sequences which contain at least one A,T base pair. Table 23 lists the four eight-ring haiφin polyamides (G9-G12) which target the four 5'- WCGNNW-3' sequences (G9-G12) which contain exclusively C » G base pairs. Table 24 lists the twelve eight-ring haiφin polyamides (229-240) which recognize the twelve 5'-WCCNNW- 3' sequences which contain at least one A,T base pair. Table 24 lists the four eight-ring haiφin polyamides (G13-G16) which target the four 5'-WCCNNW-3' sequences (G13-G16) which contain exclusively C*G base pairs. TABLE 9: 8-ring Haiφin Polyamides for recognition of 6-bp 5'-WGTNNW-3' DNA sequence aromatic amino acid sequence 1) 5'- - G T T T W-3 1) ImHpHpHp-γ-PyPyPyPy 2) 5 « --W G T T A W-3 2) ImHpHpPy-γ-HpPyPyPy 3) 5' -W G T T G W-3 3) ImHpHpIm-γ-PyPyPyPy 4) 5' -W G T T C W-3 4) ImHpHpPy-γ-ImPyPyPy 5) 5' -W G T A T W-3 5) ImHpPyHp-γ-PyHpPyPy 6) 5' -W G T A A W-3 6) ImHpPyPy-γ-HpHpPyPy 7) 5' -W G T A G W-3 7) ImHpPylm-γ-PyHpPyPy 8) 5' -W G T A C W-3 8) ImHpPyPy-γ-ImHpPyPy 9) 5- -W G T G T W-3 9) ImHpImHp-γ-PyPyPyPy 10) 5' -W G T G A W-3 10) ImHpImPy-γ-HpPyPyPy 11) 5' -W G T G G W-3 11) ImHpImlm-γ-PyPyPyPy 12) 5' -W G T G C W-3 12) ImHpImPy-γ-ImPyPyPy 13) 5' -W G T C T W-3 13) ImHpPyHp-γ-PylmPyPy 14) 5' -W G T C A W-3 14) ImHpPyPy-γ-HpImPyPy 15) 5' -W G T C G W-3 15) ImHpPylm-γ-PylmPyPy 16) 5' -W G T C C W-3 16) ImHpPyPy-γ-ImlmPyPy TABLE 10: 8-ring Haiφin Polyamides for recognition of 6-bp 5'-WGANNW-3' DNA sequence aromatic amino acid sequence 17) 5'- -W G A T T W-3 17) ImPyHpHp-γ-PyPyHpPy 18) 5'--W G A T A W-3 18) ImPyHpPy-γ-HpPyHpPy 19) 5' -W G A T G W-3 19) ImPyHpIm-γ-PyPyHpPy 20) 5' -W G A T C W-3 20) ImPyHpPy-γ-ImPyHpPy 21) 5' -W G A A T W-3 21) ImPyPyHp-γ-PyHpHpPy 22) 5' -W G A A A W-3 22) ImPyPyPy-γ-HpHpHpPy 23) 5' -W G A A G W-3 23) ImPyPylm-γ-PyHpHpPy 24) 5' -W G A A C W-3 24) ImPyPyPy-γ-ImHpHpPy 25) 5' -W G A G T W-3 25) ImPylmHp-γ-PyPyHpPy 26) 5" -W G A G A W-3 26) ImPylmPy-γ-HpPyHpPy 27) 5' -W G A G G W-3 27) ImPylmlm-γ-PyPyHpPy 28) 5' -W G A G C W-3 28) ImPylmPy-γ-ImPyHpPy 29) 5' -W G A C T W-3 29) ImPyPyHp-γ-PylmHpPy 30) 5' -W G A C A W-3 30) ImPyPyPy-γ-HpImHpPy 31) 5' -W G A C G W-3 31) ImPyPylm-γ-PylmHpPy 32) 5' -W G A C C W-3 32) I PyPyPy-γ-ImlmHpPy TABLE 11 : 8-ring Haiφin Polyamides for recognition of 6-bp S'-WGGNNW-S' DNA sequence aromatic amino acid sequence 33) 5" -W G G T T W-3' 33) ImlmHpHp-γ-PyPyPyPy 34) 5' -W G G T A W-3' 34) ImlmHpPy-γ-HpPyPyPy 35) 5" -W G G T G W-3' 35) ImlmHpIm-γ-PyPyPyPy 36) 5' -W G G T C W-3 1 36) ImlmHpPy-γ-ImPyPyPy 37) 5' -W G G A T W-3' 37) ImlmPyHp-γ-PyHpPyPy 38) 5' -W G G A A W-3' 38) ImlmPyPy-γ-HpHpPyPy 39) 5' -W G G A G W-3' 39) ImlmPylm-γ-PyHpPyPy 40) 5' -W G G A C W-3' 40) ImlmPyPy-γ-ImHpPyPy 41) 5' -W G G G T W-3' 41) imlmlmHp-γ-PyPyPyPy 42) 5' -W G G G A W-3» 42) ImlmlmPy-γ-HpPyPyPy 43) 5' -W G G C T W-3 1 43) ImlmPyHp-γ-PylmPyPy 44) 5 -W G G C A W-3' 44) ImlmPyPy-γ-HpImPyPy Gl) 5 -W G G G G W-3 1 Gl) Imlmlmlm-γ-PyPyPyPy G2) 5 -W G G G C W-3' G2) ImlmlmPy-γ-ImPyPyPy G3) 5 -W G G C G W-3' G3) imlmPylm-γ-PylmPyPy G4) 5 -W G G C C W-3" G4) ImlmPyPy-γ-ImlmPyPy TABLE 12: 8-ring Haiφin Polyamides for recognition of 6-bp 5^WGCNNW-3' DNA sequence aromatic amino acid sequence 45) 5' -W G C T T W-3' 45) ImPyHpHp-γ-PyPylmPy 46) 5' -W G C T A W-3' 46) ImPyHpPy-γ-HpPylmPy 47) 5' -W G C T G W-3' 47) ImPyHpIm-γ-PyPylmPy 48) 5' -W G C T C W-3' 48) ImPyHpPy-γ-ImPylmPy 49) 5' -W G C A T W-3' 49) ImPyPyHp-γ-PyHpImPy 50) 5' -W G C A A W-3' 50) ImPyPyPy-γ-HpHpImPy 51) 5' -W G C A G W-3' 51) ImPyPylm-γ-PyHpImPy 52) 5' -W G C A C W-3' 52) ImPyPyPy-γ-ImHpImPy 53) 5' -W G C G T W-3' 53) ImPylmHp-γ-PyPyl Py 54) 5' -W G C G A W-3' 54) ImPylmPy-γ-HpPylmPy 55) 5" -W G C C T W-3' 55) ImPyPyHp-γ-PylmlmPy 56) 5" -W G C C A W-3' 56) ImPyPyPy-γ-HpImlmPy G5) 5 -W G C G G W-3' G5) ImPylmlm-γ-PyPylmPy G6) 5 -W G C G C W-3' G6) ImPylmPy-γ-ImPylmPy G7) 5 '-W G C C G W-3' G7) ImPyPylm-γ-PylmlmPy G8) 5 " -W G C C C W-3' G8) ImPyPyPy-γ-ImlmlmPy TABLE 13: 8-ring Haiφin Polyamides for recognition of 6-bρ S'-WTTNNW-S DNA sequence aromatic amino acid sequence 57) 5' -W T T T T W-3 57) HpHpHpHp-γ-PyPyPyPy 58) 5'-W T T " T A W-3 58) HpHpHpPy-γ-HpPyPyPy 59) 5'-W T T T G W-3 59) HpHpHpIm-γ-PyPyPyPy 60) 5'-W T T T C W-3 60) HpHpHpPy-γ-ImPyPyPy 61) 5'-W T T A T W-3 61) HpHpPyHp-γ-PyHpPyPy 62) 5'-W T T A A W-3 62) HpHpPyPy-γ-HpHpPyPy 63) 5'-W T T A G W-3 63 ) HpHpPylm-γ-PyHpPyPy 64) 5'-W T T A C W-3 64 ) HpHpPyPy-γ- ImHpPyPy 65) 5'-W T T G T W-3 65)HpHpImHp-γ-PyPyPyPy 66) 5'-W T T G A W-3 1 66 ) HpHpImPy-γ-HpPyPyPy 67) 5'-W T T G G W-3 1 67)HpHpImIm-γ-PyPyPyPy 68) 5'-W T T G C W-3 1 68) HpHpImPy-γ-ImPyPyPy 69) 5'-W T T C T W-3 1 69) HpHpPyHp-γ-PyImPyPy 70) 5'-W T T C A W-3' 70) HpHpPyPy-γ-HpImPyPy 71) 5'-W T T C G W-3' 7l) HpHpPyIm-γ-PylmPyPy 72) 5' -W T T C C W-3 • 72 ) HpHpPyPy-γ- ImlmPyPy TABLE 14: 8-ring Haiφin Polyamides for recognition of 6-bp S'-WTANNW-S' DNA sequence aromatic amino acid sequence 73) 5'- •W T A T T W-3 73 ) HpPyHpHp -γ - PyPyHpPy 74) 5' -W T " A T A W-3 74 ) HpPyHpPy-γ-HpPyHpPy 75) 5' -W T A T G W-3 75 ) HpPyHpIm-γ-PyPyHpPy 76) 5' -W T A T C W-3 76 ) HpPyHpPy-γ- ImPyHpPy 77) 5' -W T A A T W-3 77) HpPyPyHp-γ-PyHpHpPy 78) 5' -W T A A A W-3 78 ) HpPyPyPy-γ-HpHpHpPy 79) 5' -W T A A G W-3 79 ) HpPyPylm-γ- PyHpHpPy 80) 5" -W T A A C W-3 80 ) HpPyPyPy-γ- ImHpHpPy 81) 5' -W T A G T W-3 81)HpPyImHp-γ-PyPyHpPy 82) 5' -W T A G A W-3 82)HpPyImPy-γ-HpPyHpPy 83) 5' -W T A G G W-3 83)HpPyImIm-γ-PyPyHpPy 84) 5' -W T A G C W-3 84 ) HpPylmPy-γ- ImPyHpPy 85) 5' -W T A C T W-3 85 ) HpPyPyHp -γ - Py ImHpPy 86) 5' -W T A C A W-3 86) HpPyPyPy-γ-HpImHpPy 87) 5 1 -W T A C G W-3 87 ) HpPyPylm-γ-PylmHpPy 88) 5' -W T A C C W-3 88 ) HpPyPyPy-γ- Iml HpPy TABLE 15: 8-ring Haiφin Polyamides for recognition of 6-bp 5'-WTGNNW-3' DNA sequence aromatic amino acid sequence 89) 5" -w T σ T T w-3' 89) HpImHpHp-γ-PyPyPyPy 90) 5' -W T G T A W-3' 90) HpImHpPy-γ-HpPyPyPy 91) 5' -W T G T G W-3' 91) HpImHpIm-γ-PyPyPyPy 92) 5' -W T G T C W-3' 92) HpImHpPy-γ- ImPyPyPy 93) 5' -W T G A T W-3' 93) HpImPyHp-γ-PyHpPyPy 94) 5' -W T G A A W-3' 94) HpImPyPy-γ-HpHpPyPy 95) 5' -W T G A G W-3' 95) HpImPylm-γ-PyHpPyPy 96) 5' -W T G A C W-3' 96) HpImPyPy-γ-ImHpPyPy 97) 5' -W T G G T W-3 1 97) HpImlmHp-γ-PyPyPyPy 98) 5 -W T G G A W-3' 98) HpImlmPy-γ-HpPyPyPy 99) 5 -W T G C T W-3' 99) HpImPyHp-γ-PylmPyPy 100) 5 -W T G C A W-3' 100) HpImPyPy-γ-HpImPyPy 101) 5 -W T G G G W-3' 101) HpImlmlm-γ-PyPyPyPy 102) 5 -W T G G C W-3' 102) HpImlmPy-γ-ImPyPyPy 103) 5 1 -W T G C G W-3' 103) HpImPylm-γ-PylmPyPy 104) 5 '-W T G C C W-3' 104) HpImPyPy-γ-ImlmPyPy TABLE 16: 8-ring Haiφin Polyamides for recognition of 6-bp 5'-WTCNNW-3' DNA sequence aromatic amino acid sequence 105) 5' -W T C T T W-3 105) HpPyHpHp-γ-PyPylmPy 106) 5'-W T C T A W-3 106) HpPyHpPy-γ-HpPylmPy 107) 5'-W T C T G W-3 107)HpPyHpIm-γ-PyPyImPy 108) 5'-W T C T C W-3 108)HpPyHpPy-γ-ImPyImPy 109) 5--W T C A T W-3 109) HpPyPyHp-γ-PyHpImPy 110) 5'-W T C A A W-3 110) HpPyPyPy-γ-HpHpImPy 111) 5'-W T C A G W-3 111) HpPyPylm-γ-PyHpImPy 112) 5' -W T C A C W-3 112) HpPyPyPy-γ-ImHpImPy 113) 5'-W T C G T W-3 113)HpPyImHp-γ-PyPyImPy 114) 5'-W T C G A W-3 114)HpPyImPy-γ-HpPyImPy 115) 5'-W T C C T W-3 115)HpPyPyHp-γ-PyImImPy 116) 5--W T C C A W-3 116)HpPyPyPy-γ-HpImImPy 117) 5'-W T C G G W-3 117) HpPylmlm-γ-PyPylmPy 118) 5'-W T C G C W-3 118)HpPyImPy-γ-ImPyImPy 119) 5'-W T C C G W-3 119) HpPyPylm-γ-PylmlmPy 120) 5'-W T C C C W-3 120) HpPyPyPy-γ-ImlmlmPy TABLE 17: 8-ring Haiφin Polyamides for recognition of 6-bp 5 , -WATNN -3' DNA sequence aromatic amino acid sequence 121) 5' -W A T T T W-3' 121) PyHpHpHp-γ-PyPyPyHp 122) 5' -W A T T A W-3' 122) PyHpHpPy-γ-HpPyPyHp 123) 5" -W A T T G W-3' 123) PyHpHpIm-γ-PyPyPyHp 124) 5' -W A T T C W-3' 124 ) PyHpHpPy-γ-ImPyPyHp 125) 5' -W A T A T W-3' 125) PyHpPyHp-γ-PyHpPyHp 126) 5' -W A T A A W-3' 126) PyHpPyPy-γ-HpHpPyHp 127) 5' -W A T A G W-3' 127) PyHpPylm-γ-PyHpPyHp 128) 5 -W A T A C W-3' 128) PyHpPyPy-γ-ImHpPyHp 129) 5 -W A T G T W-3' 129) PyHpImHp-γ-PyPyPyHp 130) 5 -W A T G A W-3' 130) PyHpImPy-γ-HpPyPyHp 131) 5 -W A T G G W-3' 131) PyHpImlm-γ-PyPyPyHp 132) 5 -W A T G C W-3' 132) PyHpImPy-γ-ImPyPyHp 133) 5 '-W A T C T W-3' 133) PyHpPyHp-γ-PylmPyHp 134) 5 ' -W A T C A W-3' 134) PyHpPyPy-γ-HpImPyHp 135) 5 '-W A T C G W-3' 135) PyHpPylm-γ-PylmPyHp 136) 5 ■ -W A T C C W-3' 136) PyHpPyPy-γ-ImlmPyHp TABLE 18: 8-ring Haiφin Polyamides for recognition of 6-bp 5'-WAANNW-3' DNA sequence aromatic amino acid sequence 137 ) 5 -W A A T T W-3 137) PyPyHpHp-γ-PyPyHpHp 138) 5 -W A A T A W-3 138) PyPyHpPy-γ-HpPyHpHp 139) 5 -W A A T G W-3 139) PyPyHpIm-γ-PyPyHpHp 140) 5 -W A A T C W- 3 140) PyPyHpPy-γ- ImPyHpHp 141) 5 -W A A A T W-3 141) PyPyPyHp-γ-PyHpHpHp 142 ) 5 -W A A A A W- 3 142 ) PyPyPyPy-γ-HpHpHpHp 143 ) 5 -W A A A G W- 3 143 ) PyPyPylm-γ-PyHpHpHp 144) 5 -W A A A C W-3 144) PyPyPyPy-γ-ImHpHpHp 145 ) 5 -W A A G T W- 3 145) PyPylmHp-γ-PyPyHpHp 146 ) 5 -W A A G A W- 3 146) PyPylmPy-γ-HpPyHpHp 147) 5 -W A A G G W-3 147) PyPylmlm-γ-PyPyHpHp 148) 5 -W A A G C W-3 148) PyPylmPy-γ- ImPyHpHp 149) 5 -W A A C T W- 3 149) PyPyPyHp-γ-PylmHpHp 150) 5 -W A A C A W-3 150) PyPyPyPy-γ-HpImHpHp 151) 5 -W A A C G W- 3 151) PyPyPylm-γ-PylmHpHp 152 ) 5 -W A A C C W- 3 152) PyPyPyPy-γ-ImlmHpHp TABLE 19: 8-ring Haiφin Polyamides for recognition of 6-bp 5'-WAGNNW-3' DNA sequence aromatic amino acid sequence 153) 5'-W A G T T W-3 153 ) PylmHpHp-γ-PyPyPyHp 154) 5'-W A G T A W-3 154) PylmHpPy-γ-HpPyPyHp 155) 5'-W A G T G W-3 155) PylmHpIm-γ-PyPyPyHp 156) 5'-W A G T C W-3 156) PylmHpPy-γ-ImPyPyHp 157) 5'-W A G A T W-3 157) PylmPyHp-γ-PyHpPyHp 158) 5'-W A G A A W-3 158) PylmPyPy-γ-HpHpPyHp 159) 5'-W A G A G W-3 159) PylmPylm-γ-PyHpPyHp 160) 5'-W A G A C W-3 160) PylmPyPy-γ-ImHpPyHp 161) 5'-W A G G T W-3 161) PylmlmHp-γ-PyPyPyHp 162) 5'-W A G G A W-3 162 ) PylmlmPy-γ-HpPyPyHp 163) 5'-W A G C T W-3 163) PylmPyHp-γ-PylmPyHp 164) 5'-W A G C A W-3 1 164) PylmPyPy-γ-HpImPyHp 165) 5'-W A G G G W-3' 165) Pylmlmlm-γ-PyPyPyHp 166) 5'-W A G G C W-3' 166) PylmlmPy-γ-ImPyPyHp 167) 5'-W A G C G W-3' 167) PylmPylm-γ-PylmPyHp 168) 5'-W A G C C W-3' 168) PylmPyPy-γ-ImlmPyHp TABLE 20: 8-ring Haiφin Polyamides for recognition of 6-bρ 5'-WACNNW-3' DNA sequence aromatic amino acid sequence 169) 5' -W A C T T W-3' 169) PyPyHpHp-γ-PyPylmHp 170) 5' -W A C T A W-3' 170) PyPyHpPy-γ-HpPylmHp 171) 5' -W A C T G W-3' 171) PyPyHpIm-γ-PyPylmHp 172) 5' -W A C T C W-3' 172) PyPyHpPy-γ-ImPylmHp 173) 5' -W A C A T W-3' 173 ) PyPyPyHp-γ-PyHpImHp 174) 5 -W A C A A W-3' 174) PyPyPyPy-γ-HpHpImHp 175) 5 -W A C A G W-3' 175) PyPyPylm-γ-PyHpImHp 176) 5 -W A C A C W-3' 176) PyPyPyPy-γ-ImHpImHp 177) 5 -W A C G T W-3' 177) PyPylmHp-γ-PyPylmHp 178) 5 -W A C G A W-3' 178) PyPylmPy-γ-HpPylmHp 179) 5 '-W A C C T W-3' 179) PyPyPyHp-γ-PylmlmHp 180) 5 ' -W A C C A W-3' 180) PyPyPyPy-γ-HpImlmHp 181) 5 ' -W A C G G W-3' 181) PyPylmlm-γ-PyPylmHp 182) 5 • -W A C G C W-3' 182) PyPylmPy-γ-ImPylmHp 183) 5 ' -W A C C G W-3' 183) yPyPylm-γ-PylmlmHp 184) 5 ' -W A C C C W-3' 184) PyPyPyPy-γ-ImlmlmHp TABLE 21: 8-ring Haiφin Polyamides for recognition of 6-bp 5'-WCTNN -3' DNA sequence aromatic amino acid sequence 185) 5'-W C T T T W-3 185) PyHpHpHp-γ-PyPyPylm 186) 5'-W C T T A W-3 186) PyHpHpPy-γ-HpPyPylm 187) 5' -W C T T G W-3 187) PyHpHpIm-γ-PyPyPylm 188) 5'-W C T T C W-3 188) PyHpHpPy-γ-ImPyPylm 189) 5'-W C T A T W-3 189) PyHpPyHp-γ-PyHpPylm 190) 5'-W C T A A W-3 190) PyHpPyPy-γ-HpHpPylm 191) 5' -W C T A G W-3 191) PyHpPylm-γ-PyHpPylm 192) 5' -W C T A C W-3 192) PyHpPyPy-γ-ImHpPylm 193) 5' -W C T G T W-3 193) PyHpImHp-γ-PyPyPylm 194) 5'-W C T G A W-3 194) PyHpImPy-γ-HpPyPylm 195) 5' -W C T G G W-3 195) PyHpImlm-γ-PyPyPylm 196) 5' -W C T G C W-3 196) PyHpImPy-γ-ImPyPylm 197) 5' -W C T C T W-3 197) PyHpPyHp-γ-PylmPylm 198) 5'-W C T C A W-3 198) PyHpPyPy-γ-HpImPylm 199) 5'-W C T C G W-3 199) PyHpPylm-γ-PylmPylm 200) 5' -W C T C C W-3 200) PyHpPyPy-γ-ImlmPylm TABLE 22: 8-ring Haiφin Polyamides for recognition of 6-bp 5^WCANNW-3' DNA sequence aromatic amino acid sequence 201) 5' -W C A T T W-3' 201) PyPyHpHp-γ-PyPyHpIm 202) 5' -W C " A T A W-3' 202) PyPyHpPy-γ-HpPyHpIm 203) 5' -W C A T G W-3' 203) PyPyHpIm-γ-PyPyHpIm 204) 5' -W C A T C W-3' 204 ) PyPyHpPy-γ- ImPyHpIm 205) 5' -W C A A T W-3' 205) PyPyPyHp-γ-PyHpHpIm 206) 5' -W C A A A W-3' 206) PyPyPyPy-γ-HpHpHpIm 207) 5' -W C A A G W-3' 207) PyPyPylm-γ-PyHpHpIm 208) 5' -W C A A C W-3' 208) PyPyPyPy-γ-ImHpHpIm 209) 5' -W C A G T W-3' 209) PyPylmHp-γ-PyPyHpIm 210) 5' -W C A G A W-3' 210) PyPylmPy-γ-HpPyHpIm 211) 5 -W C A G G W-3 1 211) PyPylmlm-γ-PyPyHpIm 212) 5 -W C A G C W-3' 212) PyPylmPy-γ-ImPyHpIm 213) 5 -W C A C T W-3 1 213) PyPyPyHp-γ-PylmHpIm 214) 5 -W C A C A W-3 1 214) yPyPyPy-γ-HpImHpIm 215) 5 1 -W C A C G W-3 1 215) PyPyPylm-γ-PylmHpIm 216) 5 1 -W C A C C W-3 1 216) PyPyPyPy-γ-ImlmHpIm TABLE 23: 8-ring Haiφin Polyamides for recognition of 6-bp 5'-WCGNNW-3' DNA sequence aromatic amino acid sequence 217) 5'-W C G T T W-3 217) PylmHpHp-γ-PyPyPylm 218) 5 ( -W C G T A W-3 218) PylmHpPy-γ-HpPyPylm 219) 5'-W C G T G W-3 219) PylmHpIm-γ-PyPyPylm 220) 5'-W C G T C W-3 220) PylmHpPy-γ-ImPyPylm 221) 5'-W C G A T W-3 221) PylmPyHp-γ-PyHpPylm 222) 5'-W C G A A W-3 222) PylmPyPy-γ-HpHpPylm 223) 5"-W C G A G W-3 223) PylmPylm-γ-PyHpPylm 224) 5 ! -W C G A C W-3 224) PylmPyPy-γ-ImHpPylm 225) 5'-W C G G T W-3 225) PylmlmHp-γ-PyPyPylm 226) 5'-W C G G A W-3 226) PylmlmPy-γ-HpPyPylm 227) 5'-W C G C T W-3 227) PylmPyHp-γ-PylmPylm 228) 5'-W C G C A W-3' 228) PylmPyPy-γ-HpImPylm G9) 5'-W C G G G W-3' G9) Pylmlmlm-γ-PyPyPylm G10) 5'-W C G G C W-3' G10) PylmlmPy-γ-ImPyPylm Gil) 5 1 -W C G C G W-3 1 Gil) PylmPylm-γ-PylmPylm G12) 5'-W C G C C W-3 1 G12 ) PylmPyPy-γ- ImlmPylm TABLE 24: 8-ring Haiφin Polyamides for recognition of 6-bp S'-WCCN -S' DNA sequence aromatic amino acid sequence 229) 5'-W C C T T W-3 229) PyPyHpHp-γ-PyPylmlm 230) 5'-W C-C T A W-3 230) PyPyHpPy-γ-HpPylmlm 231) 5'-W C C T G W-3 231) PyPyHpIm-γ-PyPylmlm 232) 5'-W C C T C W-3 232 ) PyPyHpPy-γ- ImPylmlm 233) 5'-W C C A T W-3 233) yPyPyHp-γ-PyHpImlm 234) 5'-W C C A A W-3 234) PyPyPyPy-γ-HpHpImlm 235) 5'-W C C A G W-3 235) PyPyPylm-γ-PyHpImlm 236) 5'-W C C A C W-3 236) PyPyPyPy-γ-ImHpImlm 237) 5'-W C C G T W-3 237) PyPylmHp-γ-PyPylmlm 238) 5'-W C C G A W-3 238) PyPylmPy-γ-HpPylmlm 239) 5'-W C C C T W-3 239) PyPyPyHp-γ-Pylmlmlm 240) 5'-W C C C A W-3 240) PyPyPyPy-γ-HpImlmlm G13) 5'-W C C G G W-3 G13) PyPylmlm-γ-PyPylmlm G14) 5'-W C C G C W-3 G14) PyPylmPy-γ-ImPylmlm G15) 5' -W C C C G W-3 G15) PyPyPylm-γ-Pylmlmlm G16) 5'-W C C C C W-3 G16) PyPyPyPy-γ-Imlmlmlm EXAMPLE 9: Aliphatic/Aromatic amino acid pairing for recognition of the DNA minor groove. Selective placement of an aliphatic β-alanine (β) residue paired side-by-side with either a pyrrole (Py) or imidazole (Im) aromatic amino acid is found to compensate for sequence composition effects for recognition of the minor groove of DNA by haiφin pyrrole-imidazole polyamides. A series of polyamides were prepared which contain pyrrole and imidazole aromatic amino acids, as well as γ-aminobutyric acid (γ) "turn" and β-alanine "spring" aliphatic amino acid residues. The binding affinities and specificities of these polyamides are regulated by the placement of paired β/β Py/β and Im/β residues. Quantitative footprint titrations demonstrate that replacing two Py/Py pairings in a 12-ring haiφin (6-γ-6) with two Py/β pairings affords 10- fold enhanced affinity and similar sequence specificity for an 8-bp target sequence. Table 25 Equilibrium association constants (M" ' ) for polyamιdes. α c Polyamide 5'-TGTTAACA-3' 5'-TGTGAACA-3' Sp∞ficit ' ^o S^S^ i - 7 " 10 ' 0 2 - 7 x 10 ' 6 ^$8S2*> <- s * 10 ' 0 77 * >° 9 6 ^ •gZZ 2 - 7 x l0 '° " x .O* 5 ^g$$g ) * > x K>» s l x .O* 1 " Values reported are the mean values obtained from three DNase 1 footprint titration experiments * The assays were earned out at 22 °C at pH 70 in the presence of 10 mM Tπs-HCl, 10 mM KCl. 10 mM MgCl 2 , and 5 mM CaCli c Match site association constants and specificities higher than the parent hairpin are shown in boldtype ''Specificity is calculated as K a (match) / K a (mιsmatch) The 6-γ-6 haiφin ImPylmPyPyPy-γ-ImPyPyPyPyPy-β-Dp, which contains six consecutive amino acid pairings, is unable to discriminate a single-base-pair mismatch site 5'- TGTTAACA-3' from a 5'-TGTGAACA-3' match site. The haiφin polyamide Im-β- ImPyPyPy-γ-ImPyPyPy-β-Py-β-Dp binds to the 8-bp match sequence 5'-TGTGAACA-3' with an equilibrium association constant of K a = 2.4 x 10 M " and > 48-fold specificity versus the 5'-TGTTAACA-3' single-base-pair mismatch site. Table 26 Equilibrium association constants (M~ M for polyamides °" c Polyamide 5'-TG AACA-3' 5'-TGTGAACA- 3 ' Specificity'' ^ gSS8$ 5 x , ° 9 5 * 109 ' a Values reported for 1, 5, and 10 are the mean values obtained from three DNase 1 footprint titration experiments & The assays were earned out at 22 °C at pH 7.0 in the presence of 10 mM Tπs-HCl, 10 mM KCl, 10 mM MgClj, and 5 mM CaC^. c Match sice association constants and specificities higher than parent haiφins are shown in boldtype. ''Specificity is calculated as K-^matcrQ / K a (mιsmatch) Modeling indicates that the β-alanine residue relaxes ligand curvature, providing for optimal hydrogen bond formation between the floor of the minor groove and both Im-residues within the Im-β-Im polyamide subunit. This observation provided the basis for design of a haiφin polyamide, Im-β-ImPy-γ-Im-β-ImPy-β-Dp, which incoφorates Im/β pairings to recognize a "problematic" 5'-GCGC-3' sequence at subnanomolar concentrations. Table 27 Equilibrium association constarnts (M " ' ) for polyamides.""* Polyamide 5'-TGCGCA-3' 5i'-TGGCCA-3' 5'-TGGGGA-3' + θ 5 - : # 3.7 x lO 7 < 10 7 < 10 7 D 0 Q ~»~ " ^ ) 3.7 x lO 9 1.4 x lO 8 1.1 x 10 8 a Values reported are the mean values obuained from a minimum of three DNase I footpnnt titration experiments. '''The assays were carried out at 22 °C at pH 7.0 in the presence of 10 mM! Tris-HCl, 10 mM KCl, 10 mM MgCl 2 , and 5 mM CaCl 2 . These results identify Im β and β/Im pairings that respectively discriminate G*C and C»G from A«T T»A as well as Py/β and β/Py pairings that discriminate A # T/T » A from G # C/C»G. These aliphatic/aromatic amino acid pairings will facilitate the design of haiφin polyamides which recognize both a larger binding site size as well as a more diverse sequence repertoire. EXAMPLE 10: POLYAMIDE BIOTIN CONJUGATES Bifunctional conjugates prepared between sequence specific DNA binding polyamides and biotin are useful for a variety of applications. First, such compounds can be readily attached to a variety of matrices through the strong interaction of biotin with the protein streptavidin. Readily available strepdavidin-derivatized matrices include magnetic beads for separations as well as resins for chromatography. A number of such polyamide-biotin conjugates have been synthesized by solid phase synthetic methods outlined in detail above. Following resin cleavage with a variety of diamines, the polyamides were reacted with various biotin carboxylic acid derivatives to yield bifunctional conjugates. The bifunctional conjugates were purified by HPLC and characterized by MALDI-TOF mass spectroscopy and 'H NMR. The scheme for the synthesis of an exemplary biotin-polyamide conjugate is shown below. The foregoing is intended to be illustrative of the present invention, but not limiting. Numerous variations and modifications of the present invention may be effected without departing from the true spirit and scope of the invention.

Claims: What is claimed is: 1. In a polyamide having at least three consecutive carboxamide pairs for binding to at least three DNA base pairs in the minor groove of a duplex DNA sequence having 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 of the duplex DNA sequence or selecting a Py/Hp carboxamide pair to bind to an A»T DNA base pair in the minor groove of the duplex DNA sequence. 2. The polyamide of claim 1 wherein at least four consecutive carboxamide pairs bind to at least four DNA base pairs. 3. The polyamide of claim 1 wherein at least five consecutive carboxamide pairs bind to at least five DNA base pairs. 4. The polyamide of claim 1 wherein at least six consecutive carboxamide pairs bind to at least six DNA base pairs. 5. The polyamide of claim 1 wherein the A»T or T»A base pair has a G»C or C«G base pair on either side. 6. The polyamide of claim 1 wherein the duplex DNA sequence is a regulatory sequence. 7. The polyamide of claim 1 wherein the duplex DNA sequence is a promoter sequence. 8. The polyamide of claim 1 wherein the duplex DNA sequence is a coding sequence. 9. The polyamide of claim 1 wherein the duplex DNA sequence is a non- coding sequence. 10. The polyamide of claim 1 wherein the binding of the carboxamide pairs to the DNA base pairs modulates the expression of a gene. 11. A composition comprising an effective amount of the polyamide of claim 1 and a pharmologically suitable excipient. 12. A diagnostic kit comprising the polyamide of claim 1. 13. A polyamide according to claim 1 having the formula: XlX 2 X3X4-Y-X5X6X7X8 wherein γ is -NH-CH2-CH2-CH2-CONH- haiφin linkage derived from γ-aminobutyric acid or a chiral haiφin linkage derived from R-2,4- diaminobutyric acid; X4/X5, X3/X6, X2/X7, and X\/X$ represent carboxamide binding pairs which bind the DNA base pairs wherein at least one binding pair is Hp/Py or Py/Hp and the other binding pairs are selected from Py/Im Im/Py to correspond to the DNA base pair in the minor groove to be bound. 14. The polyamide of claim 13 wherein there is at least one β-alanine in a non- Hp containing binding pair. 15. The polyamide of claim 13 wherein dimethylaminopropylamide is covalently bound to Xi or X%. 16. A polyamide selected from those listed in Tables 9-24 as compounds 1 through 240. 17. A polyamide selected from shown in Fig. 4.

Inventors: BAIRD ELDON E, DERVAN PETER B, BAIRD, ELDON, E., DERVAN, PETER, B.
Applicants: BAIRD ELDON E, CALIFORNIA INST OF TECHN, DERVAN PETER B, BAIRD, ELDON, E., CALIFORNIA INSTITUTE OF TECHNOLOGY, DERVAN, PETER, B.
Application year: 1998
Main classification: C07D207
IPC codes: A61K38, A61K47, C07D207, C07D233, C07D403, C07K5, C07K7, C08G69, C12Q1