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Title:
IMPROVED POLYAMIDES FOR BINDING IN THE MINOR GROOVE OF DOUBLE STRANDED DNA
Document Type and Number:
WIPO Patent Application WO/1998/037066
Kind Code:
A1
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.

Inventors:
BAIRD ELDON E (US)
DERVAN PETER B (US)
Application Number:
PCT/US1998/001006
Publication Date:
August 27, 1998
Filing Date:
January 21, 1998
Export Citation:
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Assignee:
CALIFORNIA INST OF TECHN (US)
BAIRD ELDON E (US)
DERVAN PETER B (US)
International Classes:
A61K31/40; A61K31/415; A61K31/4178; A61K31/785; A61K38/04; A61K38/00; A61K47/48; A61K49/00; A61P43/00; C07D207/34; C07D233/90; C07D403/12; C07D403/14; C07K5/078; C07K7/02; C07K7/04; C08G69/00; C08G69/08; C12N15/09; C12Q1/68; (IPC1-7): C07D207/34; C07D233/90; A61K31/415; C07D403/14; C12Q1/68
Domestic Patent References:
WO1996005196A11996-02-22
Foreign References:
Other References:
J. W. TRAUGER ET AL: "Recognition of DNA by designed ligands at subnanomolar concentrations", NATURE, vol. 382, no. 6591, 8 August 1996 (1996-08-08), pages 559 - 561, XP002066256
S.E.SWALLEY ET AL: "Recognition of a 5'-(A,T)GGG(A,T)2-3' sequence in the minor groove of DNA by an eight-ring hairpin polyamide", JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, vol. 118, no. 35, 4 September 1996 (1996-09-04), pages 8198 - 8206, XP002066377
E. B. BAIRD ET AL: "Solid phase synthesis of polyamides containing imidazole and pyrrole amino acids", JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, vol. 118, no. 26, July 1996 (1996-07-01), pages 6141 - 6146, XP000674666
M. E. PARKS ET AL: "Optimization of the hairpin polyamide design for recognition of the minor groove of DNA", JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, vol. 118, no. 26, July 1996 (1996-07-01), pages 6147 - 6152, XP000674668
M. E. PARKS ET AL: "Recognition of 5'-(A,T)GG(AT)2-3' sequences in the minor groove of DNA by hairpin polyamides", JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, vol. 118, no. 26, July 1996 (1996-07-01), DC US, pages 6153 - 6159, XP000674667
S. E. SWALLEY ET AL: "Discrimination of 5'-GGGG-3', and 5'-GGCC-3' sequences in the minor groove of DNA by eight-ring hairpin polyamides", JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, vol. 119, no. 30, 30 July 1997 (1997-07-30), DC US, pages 6953 - 6961, XP002066260
W. L. WALKER ET AL: "Estimation of the DNA sequence discriminatory ability of hairpin-linked lexitropsins", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES,U.S.A., vol. 94, no. 11, May 1997 (1997-05-01), pages 5634 - 5639, XP002066261
Attorney, Agent or Firm:
Berkman, Charles S. (Suite 4700 633 West Fifth Stree, Los Angeles CA, US)
Moon, Donald Keith (Quality House Quality Cour, Chancery Lane London WC2A 1HT, GB)
Download PDF:
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 AT 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 AsT 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 AT or TA base pair has a GC or CG 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 : X1X2X3X4YX5X6X7X8 wherein y isNHCH2CH2CH2CONHhairpin linkage derived from yaminobutyric acid or a chiral hairpin linkage derived from R2, 4 diaminobutyric acid ; X4/X5, X3/X6, X2/X7, and X1/Xg 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 (3alanine in a nonHp containing binding pair.
15. The polyamide of claim 13 wherein dimethylaminopropylamide is covalently bound to X1 or Xg.
16. A polyamide selected from those listed in Tables 924 as compounds 1 through 240.
17. A polyamide selected from shown in Fig. 4.
Description:
IMPROVED POLYAMIDES 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 GC from C*G and both of these from AeT or T*A base pairs. Wade, W. S., Mrksich, M. & Dervan, P. B. describes the design of peptides that bind in the minor groove of DNA at 5'- (A, T) G (A, T) C (A, T)-3' sequences by a dimeric side-by-side motif. J. Am. Chem. Soc. 114, 8783-8794 (1992) ; Mrksich, M. et al. describes antiparallel side-by-side motif for sequence specific-recognition in the minor groove of DNA by the designed peptide 1-methylimidazole-2-carboxamidenetropsin. Proc. Natl. Acad. Sci. USA 89, 7586-7590 (1992) ; Trager, 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 GC but does not distinguish AT from TA. 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-TEA 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 AT from T*A. The replacement of a single hydrogen atom on the pyrrole with a hydroxy group in a Hp/Py pair regulates affinity and specificity of a polyamide by an order of magnitude. Utilizing Hp together with Py and Im in polyamides to form four aromatic amino acid pairs (Im/Py, Py/Im, Hp/Py, and Py/Hp) provides a code to distinguish all four Watson-Crick base pairs in the minor groove of DNA.

SUMMARY OF THE INVENTION The invention encompasses improved polyamides for binding to the minor groove of double stranded ("duplex") DNA. The polyamides are in the form of a hairpin comprising two groups of at least three consecutive carboxamide residues, the two groups covalently linked by an aliphatic amino acid residue, preferably y-aminobutyric acid or 2, 4 diaminobutyric acid, the consecutive carboxamide residues of the first group pairing in an antiparallel manner with the consecutive carboxamide residues of the second group in the minor groove of double stranded DNA. The improvement relates to the inclusion of a binding pair of Hp/Py carboxamides in the polyamide to bind to a TA base pair in the minor groove of double stranded DNA or Py/Hp carboxamide binding pair in the polyamide to bind to an AT 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 AT 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 AT 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 AcT, TA, 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 y-aminobutyric acid or a substituted y- aminobutyric acid to form a hairpin with a member of each carboxamide pairing on each side of it. Preferably the substituted y-aminobutyric acid is a chiral substituted y-aminobutyric acid such as (R)-2, 4-diaminobutyric acid. In addition, the polyamides may contain an aliphatic amino acid residue, preferably a (3-alanine residue, in place of a non-Hp carboxamide. The ß- alanine residue is represented in formulas as P. The (3-alanine residue becomes a member of a carboxamide binding pair. The invention further includes the substitution as a POP binding pair for non-Hp containing binding pair. Thus, binding pairs in addition to the Hp/Py and Py/Hp are Im/p, P/Im, Py/ß, ß/Py, and ß/ß.

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 L 2 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 DNaseI 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 Laboratow 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"d 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, a 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 Vl, 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, l9th 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-a-lipoic acid, acridine, captothesin, pyrene, mitomycin, texas red, anthracene, anthrinilic acid, avidin, DAPI, isosulfan blue, malachite green, psoralen, ethyl red, 4- (psoraen-8-yloxy)-butyrate, tartaric acid, (+)-a-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. ImImPyPy-y-ImPyPyPy-p-Dp (1), ImImPyPy-y-ImHpPyPy-p-Dp (2), and ImlmHpPy-y-ImPyPyPy-p-Dp (3). (Hp = 3- hydroxy-N-methylpyrrole, Im = N-methylimidazole, Py = N-methylpyrrole, P = (3-alanine, y = y-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'H 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' (AT 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 y-aminobutyric acid, the diamond represents p-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 3'32P labeled 250-bp pJK6 EcoRl/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 L. A polyamide stock solution or H20 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 MgCl2, 5 mM CaC12, 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 AT/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 y-aminobutyric acid (y), the diamond represents P-alanine (p), 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*HCI, 10 mM KCI, 10 mM MgCl2, and 5 mM CaC12 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 ImImPyPy-y-ImHpPyPy-p-Dp starting from commercially available Boc-p-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-y-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 ImImPyPy-y-ImOpPyPy--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 ImImPyPy-y-ImOpPyPy-p-Dp and ImImPyPy-y-ImHpPyPy-p-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 ImImPyPy-y-ImOpPyPy-p-Dp and ImImPyPy-y- ImHpPyPy-p-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-y-OpPyPy-p-Dp and ImPyPy-y-HpPyPy-p-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-y-PyHpPy-p-Dp and ImHpPy-y-PyPyPy-p-Dp on the 3'-32P labeled 370-bp pDEH1 EcoRI/PvuII 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 uM, 5 uM, 2 uM, 1 uM, 500 nM, 200 nM, 100 nM, 50 nM, 20 nM, 10 nM ImPyPy-y-PyPyPy-p-Dp, respectively or 1 uM, 500 nM, 200 nM, 100 nM, 50 nM, 20 nM, 10 nM, 5 nM, 2 nM, 1 nM ImHpPy-y- PyPyPy-p-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 pL. A polyamide stock solution or H2O was added to an assay buffer containing radiolabeled restriction fragment, with the final solution conditions of 10 mM Tris-HC1. 10 mM KC1, 10 mM MgCl2, 5 mM Cad2, 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. describe the pairing rules for recognition in the minor groove of DNA by pyrrole-imidazole polyamides. Chemistrv & Biology 4, 569-578 (1997).

Figure 11 shows the synthesis of a bifunctional polyamide which incorporates the Hp/Py pair. Treatment of a sample of ImImPyPy-y-ImHpPyPy-p-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) EDTA-dianhydride, DMSO/NMP, DIEA, 55 °C ; (ii) 0. 1M NaOH, followed by reverse phase HPLC purification provides the Op- Py-Im-polyamide-EDTA 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 ImImPyPy-y-ImHpPyPy-p-Dp-EDTAFe (II) 2-EFe (II) and ImImHpPy-y-ImPyPyPy-p-Dp- EDTA-Fe (II) 3-EFe (II) by affinity cleaving footprint titration. Top and bottom left : Affinity cleavage experiments on a 3 32p labeled 250-bp pJK6 EcoRl/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 uM, 1. 0 RM, 100 nM, 10 nM, 1 nM polyamide 2-E*Fe (II) ; lane 7, intact restriction fragment, no polyamide added. Bottom left : lane 1, A reaction ; lanes 2-6, 8. 5 uM, 1. 0 uM, 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 uL. A stock solution of polyamide or H20 was added to a solution containing 20 kcpm labeled restriction fragment, affording final solution conditions of 25 mM Tris-Acetate, 20 mM NaCI, 100 pM/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 AT/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-EFe (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 p-alanine moiety.

A curved line represents the y-aminobutyric acid, and the + represents the positively charged dimethylaminopropylamide tail group. The boxed Fe denotes the EDTA (II) cleavage moiety.

Figure 13 shows quantitative DNase I footprint titration experiments with the polyamides ImPyPyPyPy-y-ImPyPyPyPy-p-Dp and ImHpPyPyPy-y-ImHpPyPyPy-ß-Dp on the 31 32 P labeled 252-bp pJK7 EcoRS/Pvu II restriction fragment. For ImPyPyPyPy-y- ImPyPyPyPy-p-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 uM, 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-y-ImHpPyPyPy-p-Dp gel (right) : lane 1, intact DNA ; lane 2, DNase I digestion products in the absence of polyamide ; lanes 3-19, 1. 0 uM, 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

L. A polyamide stock solution or Hr0 was added to an assay buffer containing radiolabeled restriction fragment, with the final solution conditions of 10 mM Tris-HCI, 10 mM KCI, 10 mM MgCl2, 5 mM CaCl2, 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 AT/TA 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 hairpin template is shown at the top. A polyamide having the formula XlX2X3X4-y-X5X6X7X8 wherein y is the-NH-CH2-CH2-CH2-CONH-hairpin linkage derived from y-aminobutyric acid or a chiral hairpin linkage derived from R-2, 4- diaminobutyric acid ; X4/X5, X3/X6, X2/X7, and XI/Xg 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 hairpin 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 hairpin 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 y-aminobutyric acid.

Fig. 15 shows the 8-ring Hp-Py-Im-polyamide hairpins described by the pairing code of the present invention. The eight ring hairpin template is shown at the top. A polyamide having the formula XlX2X3X4-y-X5X6X7Xs wherein y is the-NH-CH2-CH2-CH2-CONH-hairpin linkage derived from y-aminobutyric acid or a chiral hairpin linkage derived from R-2, 4- diaminobutyric acid ; X4/X5, X3/X6, X2/X7, and X,/X8 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 hairpin 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 hairpin 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 y-aminobutyric acid.

Fig. 16 shows the 8-ring Hp-Py-Im-polyamide hairpins described by the pairing code of the present invention. The eight ring hairpin template is shown at the top. A polyamide having the formula XlX2X3X4-y-X5X6X7X8 wherein y is the-NH-CH2-CH2-CH2-CONH-hairpin linkage derived from y-aminobutyric acid or a chiral hairpin linkage derived from R-2, 4- diaminobutyric acid ; X4/X5, X3/X6, X2/X7, and Xl/X8 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 hairpin 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 hairpin 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 y-aminobutyric acid.

Fig. 17 shows the 8-ring Hp-Py-Im-polyamide hairpins described by the pairing code of the present invention. The eight ring hairpin template is shown at the top. A polyamide having the formula XlX2X3X4-y-X5X6X7X8 wherein y is the-NH-CH2-CH2-CH2-CONH-hairpin linkage derived from y-aminobutyric acid or a chiral hairpin linkage derived from R-2, 4- diaminobutyric acid ; X4/X5, X3/X6, X2/X7, and X,/X8 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 hairpin 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 hairpin 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 y-aminobutyric acid.

Four-ring polyamide subunits, covalently coupled to form eight-ring hairpin 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 hairpin polyamides. J. Am. Chem. Soc.

119, 6953-6961 (1997). The DNA-binding affinities of three eight-ring hairpin 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 TA and AT has been determined. Equilibrium dissociation constants (Kd) for ImImPyPy-y-ImPyPyPy-ß-Dp 1, ImIrnPyPy-y-ImHpPyPy-ß-Dp 2, ImImHpPy-y-ImPyPyPy-p-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 Kd values were determined by quantitative DNase I footprint titration experiments : on a 3'32P-labeled 250-bp DNA fragment containing the target sites, 5'- TGGACA-3'and 5'-TGGTCA-3'which differ by a single AT base pair in the fourth position.

The DNase footprint gels are shown in Figure 3.

TABLE 1 Equilibrium dissociation constants* Polyamide 5'-TGGTCA-3'5'-TGGACA-3'K, 5'-T G G T C A-3'5'-T G G A C A-3' 1 pypy + ? v + 2. 0 3'-A C C G T-5'3'-A C G T-5' Kd=0. 077nM Kd=0. 15nM 5'-T G G T C A-3'S'-T G ti A C A-3' H 0. 06 3'-A C C A G T-5'3'-A C C t G T-5' Kd = 15 nM Kd = 0. 83 nM 5'-T G G T C A-3'5'-T G G A C A-3' H H 3 Hp/Py + 77 3 Hp/Py +) <O C J +< C J 77 3'-A C C A G T-5'3'-A C C T G T-5' Kd = 0. 48 nM Kd =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*HCI, 10 mM KCI, 10 mM MgC12, and 5 mM CaC12 at pH 7. 0 and 22 °C. tRing pairing opposite T-A and AT in the fourth position.

$Calculated as Kd (5'-TGGACA-3')/Kd (5'-TGGTCA-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 TeA 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 (Kd = 0. 077 or 0. 15 nM respectively). In contrast, polyamide 2 (Py/Hp) binds to 5'-TGGTCA-3'and 5'-TGGACA-3'with dissociation constants which differ by a factor of 18 (Kd =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 (KD= 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 zu 100 nM, indicating that the Hp/Py and Py/Hp ring pairings do not bind opposite G*C or CoG. The A*T vs. TeA discrimination is achieved preferably when the two neighboring base pairs are G*C and CeG (GTC vs. GAC).

The specificity of polyamides 2 and 3 for sites which differ by a single AT/TA 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'-TGGTCA-3' by 191-fold, a free energy difference of 3. 1 kcal mol l. Interaction of 2 with 5'-TGGACA-3'is destabilized only 6-fold relative to 1, a free energy difference of 1. 1 kcal mol. 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''. Interaction of 3 with 5'TGGTCA-3'is destabilized only 6-fold relative to 1, a free energy difference of 1. 0 kcal mol''.

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 GC C*G TA AT Im/Py +--- <BR> <BR> <BR> <BR> <BR> <BR> Py/Im'+ Hp/Py--+- <BR> <BR> <BR> <BR> <BR> <BR> Py/Hp---f- * favored (+), disfavored (-)

For certain GC rich sequences the affinity of polyamide-DNA complexes may be enhanced by substitution of an Im/ß pair for Im/Py at G*C and p/Im for Py/Im at C*G. At AT and TA base pairs, either a Py/ß, ß/Py, and 0/0 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/ß Py/Im P/Im Hp/Py Py/p, P/Py, Hp/p, P/P Py/Hp Py/ß, P/Py, R/Hp, ß/ß 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 (3-alanine in place of a pyrrole amino acid in the synthetic methods provides aromatic/aliphatic pairing (Im/ß, (3/Im, Py/ß, and ß/Py) and aliphatic/aliphatic pairing (P/P) substitution. The use of y-aminobutyric acid, or a substituted y-aminobutyric acid such as (R)-2, 4 diaminobutyric acid, provides for preferred hairpin 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. 1H NMR and 13C NMR spectra were recorded on a General Electric-QE 300 NMR spectrometer in CD30D or DMSO-d6, with chemical shifts reported in parts per million relative to residual CHD20D or DMSO-ds, 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 0 OH Ethyl 4-carboxy-3-hydroxy-1-HO methylpyrrole-2-carboxylate. o H Ethyl 4-[(Benzyloxycarbonyl) amino]-3-CO N UOH I I hydroxy-1-methylpyrrole-2-carboxylate 0 ° >° 0 0 N OMe 0 H OMe Ethyl 4- [ (Benzvloxycarbonyl) aminol-3- methoxy-l-methylpyrrole-2-carboxylate0 - \ I O Ethyl 4-[(tert-Butyloxycarbonyl) amino]-3-XO H OMe methoxy-1-methylpyrrole-2-carboxylate \ Y tf O OU o O \ _o_ _N OMe 4- [ (tert-Butyloxycarbonyl) amino]-3-methoxy -1-methylpyrrole-2-carboxylic acid i o Ethyl 4- (benzyloxycarbonyl) amino]-3-hydroxy-I-methylpyrrole-2-carboxylate Ethyl-4- carboxy-3-hydroxy-1-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%) 1H NMR (DMSO-d6) 8 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 Ci6HisN205).

Ethyl 4- (tert-butoxycarbonyl) amino]-3-methoxy-1-methylpyrrole-2- carboxylate. Ethyl 4-[(benzyloxycarbonyl)amino]-3-hydroxy-1-methylpyrrole-2-car boxylate (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 1N H2SO4. The aqueous layer was extracted with diethyl ether. Organic layers were combined, washed with 10% H2SO4, dried over MgS04, 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 atm) 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 MgS04. Solvent was removed in vacuo to give a white solid (8. 94 g, 71%) H NMR (DMSO-d6) 8 8. 43 (s, IH), 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 Ci4H22N205).

Ethyl 4- (benzyloxycarbonyl) aminoJ-3-hydroxy-I-methylpyrrole-2-carboxylate Ethyl 4- [(tert-butoxyvarbonyl) amino]-3-methoxy-1-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 MgS04, and solvent removed in vacuo to give a white solid (6. 0 g, 20. 5 mmol, 87% based on recovered SM) IH NMR (DMSO-d6) 8 12. 14 (s, 1H), 8. 37 (s, IH), 6. 98 (s, 1H), 3. 69 (s, 3H), 3. 66 (s, 3H), 1. 42 (s, 9H) ; MS mle 293. 112 (M+H 293. 104 calcd. for C 12H 18N2°5)- EXAMPLE 2 : SOLID PHASE SYNTHESIS OF 3-HYDROXYPYRROLE POLYAMIDES.

Cycling protocols were optimized to afford high stepwise coupling yields (>99%).

Deprotection by aminolysis 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- 1-yl)-1, 1, 3, 3-tetramethyluronium hexa-fluorophosphate (HBTU) and 0. 2 mmol/gram Boc-p- alanine- (-4-carboxamidomethyl)-benzyl-ester-copoly (styrene-divinylbenzene) resin (Boc-p- 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 (R)-2-amino-4-Boc-diaminobutyric acid were purchased from Bachem. N, N-diisopropylethylamine (DIEA), N, N-dimethylformamide (DMF), N- methylpyrrolidone (NMP), DMSO/NMP, Acetic anhydride (Ac2O), 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)-a-methoxy-a- (trifuoromethyl) phenylacetic acid ( (R) MPTA) and (S)-a-methoxy-a- (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.'H NMR spectra were recorded on a General Electric-QE NMR 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 desorption/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 RAINEN C18, Microsorb MV, 5llm, 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 um C18 column equipped with a guard, 0. 1% (wt/v) TFA, 0. 25% acetonitrile/min. 18MQ water was obtained from a Millipore MilliQ water purification system, and all buffers were 0. 2 um 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, y-aminobutyric acid, Boc-glycine, and Boc-P- 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 lL, 4. 0 mmol), and acetic anhydride (380 AL, 4. 0 mmol) were combined immediately before use.

Manual Synthesis Protocol. Boc-B-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 the 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-ß-Dp. ImLrnOpPy-y-ImPyPyPy-ß-Pam-Resin was synthesized in a stepwise fashion by machine-assisted solid phase methods from Boc-p-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 incorporated 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 rnL) activation occurs. A sample of ImImOpPy-y-ImPyPyPy-ß-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.

ImImOpPy-y-ImPyPyPy-p-Dp is recovered upon lyophilization of the appropriate fractions as a white powder (97 mg, 49% recovery). UV (H20) Ex 246, 316 (66, 000) ;'H NMR (DMSO-d6)

8 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, 2H, J=5. 4Hz), 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 C58H72N22O11).

ImImHpPy-y-ImPyPyPy. In order to remove the methoxy protecting group, a sample of ImImOpPy-y-ImPyPyPy-p-Dp (5 mg, 3. 9 umol) was treated with sodium thiophenoxide at 100 °C for 2 h. DMF (1000 uL) and thiophenol (500 gel) 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, ImImOpPy-γ-ImPyPyPy- ß-Dp (5 mg) dissolved in DMF (500 1L) 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. ImImHpPy-y-ImPyPyPy-p-Dp is recovered as a white powder upon lyophilization of the appropriate fractions (3. 8 mg, 77 % recovery). UV (H20) Lx 246, 312 (66, 000) ; IH NMR (DMSO-d6) 8 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, 2H, J=5. 4Hz), 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 Cs7H7lN22oll) ImImPyPy-y-ImOpPyPy-p-Dp. ImImPyPy-y-ImOpPyPy-p-Pam-Resin was synthesized in a stepwise fashion by machine-assisted solid phase methods from Boc-p-Pam-Resin (0. 66 mmol/g) as described for ImImOpPy-y-ImPyPyPy-p-Dp. A sample of ImImPyPy-y-ImOpPyPy- ß-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. ImImPyPy-y- ImOpPyPy-p-Dp is recovered upon lyophilization of the appropriate fractions as a white powder (101 mg, 50% recovery). UV (H20) 4, 246, 316 (66, 000) ; MALDI-TOF-MS (monoisotopic), 1253. 6 (1253. 6 calc. for CsgH72N220))).

ImImPyPy-y-ImHpPyPy. A sample of ImImPyPy-y-ImOpPyPy-p-Dp (5 mg, 3. 9 µmol) was treated with sodium thiophenoxide and purified by reversed phase HPLC as described for ImImHpPy-y-ImPyPyPy-p-Dp. ImImPyPy-y-ImHpPyPy-ß-Dp is recovered upon lyophilization of the appropriate fractions as a white powder (3. 2 mg, 66 % recovery). W (H20) wx 246, 312 (66, 000) ; MALDI-TOF-MS (monoisotopic), 1239. 6 (1239. 6 calc. for Cs7H7, N22O").

ImPyPy-y-OpPyPy-p-Dp. ImPyPy-y-OpPyPy-p-Pam-Resin was synthesized in a stepwise fashion by machine-assisted solid phase methods from Boc-p-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 incorporated 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-y-OpPyPy-p-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-y-OpPyPy-p-Dp is recovered upon lyophilization of the appropriate fractions as a white powder (45 mg, 25% recovery). LJV (H20) 4lax 246, 310 (50, 000) ; 1H NMR (DMSO-d6) 5 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. 11 (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, 2H, J=5. 8Hz), 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 C48H6N, 609).

ImPyPy-y-HpPyPy. In order to remove the methoxy protecting group, a sample of ImPyPy-y-OpPyPy-p-Dp (5 mg, 4. 8 pmol) was treated with sodium thiophenoxide at 100 °C for 2 h. DMF (1000 uL) and thiophenol (500 uL) 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, ImImPyPy-y-ImOpPyPy- ß-Dp (5 mg) dissolved in DMF (500 1L) 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. ImImHpPy-y-ImHpPyPy-p-Dp is recovered as a white powder upon lyophilization of the appropriate fractions (2. 5 mg, 52 % recovery). UV (H20) EX 246, 310 (50, 000) ; 1H NMR (DMSO-d6) 5 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 C47H61HN16O9).

Table 5. Mass spectral characterization of Op and Hp polyamides, synthesized and purified as described for ImImOpPy-y-ImPyPyPy-p-Dp and ImImHpPy-γ-ImPyPyPy-ß-Dp.

POLYAMIDE FORMULA (M+H) CALCD FOUND ImOpPy-y-PyPyPy-ß-Dp C48H63NI609 1007. 5 1007. 5 ImHpPy-y-PyPyPy-p-Dp C47H61N16O9 993. 5 993. 2 ImPyOp-γ-PyPyPy-ß-Dp C48H63N16O9 1007. 5 1007. 5 ImPyHp-γ-PyPyPy-ß-Dp C47H61N16O9 993.5 993.4 ImPyPy-γ-OpPyPy-ß-Dp C48H63N16O9 1007. 5 1007. 6 ImPyPy-y-HpPyPy-R-Dp C47H6, N, 609 993. 5 993. 2 ImPyPy-γ-PyOpPy-ß-Dp C48H63N16O9 1007. 5 1007. 5 ImPyPy-y-PyHpPy-p-Dp C47H6iN, 609 993. 5 993. 4 ImOpOp-γ-PyPyPy-ß-Dp C49H65N16O10 1037. 5 1037. 5 ImHpHp-γ-PyPyPy-ß-Dp C47H61N16O10 1009. 5 1009. 4 ImImOpPy-y-ImPyPyPy-p-Dp Cs8H72N220u 1253. 6 1253. 5 ImImHpPy-y-ImPyPyPy-ß-Dp C57H7, N22011 1239. 6 1239. 6 ImImPyPy-y-ImOpPyPy-p-Dp C58H72N22011 1253. 6 1253. 6 ImImPyPy-y-ImHpPyPy-p-Dp C57H7) N220n 1239. 6 1239. 6 ImOpPyPy-y-ImOpPyPy-p-Dp C6oH76N21 O12 1282. 6 1282. 6 ImHpPyPy-γ-ImHpPyPy-ß-Dp C58H72N21O12 1254. 6 1254. 6 ImImOpPy-y-ImOpPyPy-p-Dp C59H75N22012 1283. 6 1283. 6 ImImHpPy-y-ImHpPyPy-p-Dp Cs7H7iN22012 1255. 6 1255. 5 ImOpPyPy-γ-PyPyPyPy-ß-Dp C60H75N20O11 1251. 6 1251. 5 ImPyPyPy-y-PyPyOpPy-p-Dp C60H75N20O11 1251. 6 1251. 5 ImImPyPy-y-ImPyOpPy-p-Dp C5gH72N22011 1253. 6 1253. 7 ImOpPyPyPy-y-ImOpPyPyPy-p-Dp C72H88N25014 1526. 7 1526. 6 ImHpPyPyPy-γ-ImHpPyPyPy-γ-Dp C70H84N25O14 1498. 7 1498. 0 ImImPyPyPy-y-ImOpOpPyPy-p-Dp C7lH87N26014 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 hairpin 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 AT or TA 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 pL (Brenowitz, M. et al., 1986). A polyamide stock solution or H20 (for reference lanes) was added to an assay buffer containing 3'32P radiolabeled restriction fragment (20, 000 cpm), affording final solution conditions of 10 mM TrisvHCl, 10 mM KCI, 10 mM MgCl2, 5 mM Cad2, 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 L 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 pL of a solution containing 2. 25 M NaCI, 150 mM EDTA, 23 uM 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 S0230 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 PhosphorImager 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 (Isite) and the reference intensity (Iref). The apparent fractional occupancy of the sites were calculated using the equation : G itollrd () am = 1-- (1) /. //r. f° where Isite° and Iref° are the site and reference intensities, respectively, from a DNase I control lane to which no polyamide was added.

The ( [L] tot, Oapp) data were fit to a Langmuir binding isotherm (eq. 2, n=1) by minimizing the difference between (3app and Of-, t, using the modified Hill equation : where [Lot] is the total polyamide concentration, Ka is the equilibrium association constant, and Omin and Omax 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 Ka, Omax, and Omin 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 : TABLE 6 Discrimination of 5'-TGTAA-3'and 5'-TGTTA-3'* Pairs 5'-TGTAA-3'S'-TGTTA-3'lrco 5'-T G T A A-3'5'-T G T T A-3' + + 2. 0 3'-A C A TIT-5'3'-A C A AT-5' 3'-A C A T T-5'3'-A C A A T-5' Kd = 0. 014 wM Kd = 0 007 WM 5'-T 4 A-3'5'-T X A-3' T G T T b24 3 A T T-5'3'-A C A a T-5' Kd = 0. 20 ßM Kd = 0. 56 M 5'-T X A-3'5'-T J A-3' H Hp/Py 14 3'-A C A T T-5'3'-A C A A T-5' Kd = 4. 0 gM Kd = 0. 2 1 he reported equilibrium dissociation constants are the mean values obtained from two DNase I footprint titration experiments on a 3 32p labeled 370-bp pDEHl EcoRI/PvulI DNA restriction fragment". The assays were carried out at 22 °C, pH 7. 0 in the presence of 10 mM TrisHCI, 10 mM KCI, 10 mM MgCl2, and 5 mM CaCl2. tRing pairing opposite T-A and A-T in the third position. tCalculated as K@(5'-TGTAA-3;)/K@(5'-TGTTA-3').

TABLE 7 Discrimination of 5'-TGTTT-3'and 5'-TGATT-3" Pairt 5'-TGATT-3'S'-TGTTT-3'Kro1 5'-T G T T-3'5'-T G T-3' + + 5. 2 Py/Py C', A 11-5'3'-A C A A A-5' Kd = 0. 026 ZM Rd = 0. 005 FM 5'-T G 0 T T-3'5'-T G N S T-3' T HP/PY 66 3'-A C A A-5'3'-A C A A-5' Kd S3pM=0. 008fiM 5'-T G ? T T-3'5'-T G T T T-3' CCCC Py/Hp H H 0. 56 3'-A A A-5'3'-A C A A-5' Kd=0. 33FM Kd=0. 59FM 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 Tris*HCI 10 mM KCI, 10 mM MgC12, and 5 mM CaCl tRing pairing opposite T*A and AT in the third position. tCalculated as Kd (5'-TGAlT-3')/Kd (5'-TGTTT-3').

EXAMPLE 5 : PREPARATION OF A BIFUNCTIONAL Hp-Py-Im-POLYAMIDE.

ImImOpPy-y-ImPyPyPy-ß-Dp-NH2. ImlmOpPy-y-ImPyPyPy-ß-Pam-Resin was synthesized in a stepwise fashion by machine-assisted solid phase methods from Boc-ß-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 incorporated 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 ImImOpPy-y-ImPyPyPy-ß-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. ImImOpPy-y-ImPyPyPy-ß-Dp-NH2 is recovered upon lyophilization of the appropriate fractions as a white powder (93 mg, 46% recovery). UV (H20) xmas 246, 316 (66, 000) ; 1H NMR (DMSO-d6) 8 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 C60H78N23Ol l) ImImOpPy-y-ImPyPyPy-p-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 ImImOpPy-y-ImPyPyPy-ß-NH2 (13 mg, 10 gmol) dissolved in DMSO (750 gel). The mixture was heated (55 °C, 25 min.) and the remaining EDTA-anhydride hydrolyzed (0. IM 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 ImImOpPy-y-ImPyPyPy-p-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. fbrC7oH92N250ig).

ImImHpPy-y-ImPyPyPy-p-Dp-EDTA. In order to remove the methoxy protecting group, a sample of ImImOpPy-y-ImPyPyPy-p-Dp-EDTA (5 mg, 3. 1 umol) was treated with sodium thiophenoxide at 100 °C for 2 h. DMF (1000 1L) and thiophenol (500 uL) 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, ImImOpPy-y-ImPyPyPy-p-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. ImImHpPy-y-ImPyPyPy-p-Dp-EDTA is recovered as a white powder upon lyophilization of the appropriate fractions (3. 2 mg, 72 % recovery). LTV (H20) kmax 246, 312 (66, 000) ; MALDI-TOF-MS (monoisotopic), 1556. 6 (1556. 7 calc. for C69H9ON2fol8) ImImPyPy-y-ImOpPyPy-p-Dp-NH2. ImImPyPy-y-ImOpPyPy-p-Pam-Resin was synthesized in a stepwise fashion by machine-assisted solid phase methods from Boc-p-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 incorporated 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 ImImPyPy-y-ImOpPyPy-p-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-y-ImOpPyPy-p-Dp-NH2 is recovered upon lyophilization of the appropriate fractions as a white powder (104 mg, 54% recovery). UV (H20) kmax 246, 316 (66, 000) ; MALDI-TOF-MS (monoisotopic), 1296. 6 (1296. 6 calc. for C60H78N2301 1) ImImPyPy-y-ImOpPyPy-p-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-y-ImOpPyPy-p-NH2 (13 mg, 10 Rmol) dissolved in DMSO (750 gel). The mixture was heated (55 °C, 25 min.) and the remaining EDTA-anhydride hydrolyzed (0. IM 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 ImImPyPy-y-ImOpPyPy-ß-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 C70H92N25o 18) ImImPyPy-y-ImHpPyPy-p-Dp-EDTA. In order to remove the methoxy protecting group, a sample of ImImPyPy-y-ImOpPyPy-p-Dp-EDTA (5 mg, 3. 1 gmol) was treated with sodium thiophenoxide at 100 °C for 2 h. DMF (1000 uL) and thiophenol (500 gel) 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, ImImPyPy-y-ImOpPyPy-p-Dp-EDTA (5 mg) dissolved in DMF (500 pL) 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. ImImPyPy-y-ImHpPyPy-p-Dp-EDTA is recovered as a white powder upon lyophilization of the appropriate fractions (3. 2 mg, 72 % recovery). UV (H20) kmax 246, 312 (66, 000) ; MALDI-TOF-MS (monoisotopic), 1555. 9 (1556. 7 calc. for C69H9oN25018).

EXAMPLE 6 : DETERMINATION OF POLYAMIDE BINDING ORIENTATION Affinity cleavage experiments using hairpin polyamides modified with EDTA-Fe (II) at either the C-terminus or on the y-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 hairpin complex and rule out any extended 1 : 1 or overlapped complex formation. In addition, affinity cleavage experiments reveal hairpin formation

supporting the claim that it is the Hp/Py and Py/Hp pairing which form at both match and mismatch sites to discriminate AT from TA.

Affinity cleavage reactions were executed in a total volume of 40 pL. A stock solution of polyamide or H20 was added to a solution containing labeled restriction fragment (20, 000 cpm), affording final solution conditions of 25 mM Tris-Acetate, 20 mM NaCI, 100 RM/bp calf thymus DNA, and pH 7. 0. Solutions were incubated for a minimum of 4 hours at 22°C.

Subsequently, 4 pL of freshly prepared 100 uM Fe (NH4) 2 (SO4) 2 was added and the solution allowed to equilibrate for 20 min. Cleavage reactions were initiated by the addition of 4 uL of 100 mM dithiothreitol, allowed to proceed for 30 min at 22 °C, then stopped by the addition of 10 I1L of a solution containing 1. 5 M NaOAc (pH 5. 5), 0. 28 mg/mL glycogen, and 14 pM 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 hairpins J. Am. Chem. Soc. 1997 119, 7636. For example, quantitative DNaseI footprint titrations reveal that the 10-ring hairpin ImPyPyPyPy-y-ImPyPyPyPy-ß-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, LmHpPyPyPy-y-ImHpPyPyPy-ß-Dp, to recognize a TA and AT 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-y-ImHpPyPyPy-ß-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 AT and TA at a single position.

TABLE 8 Equilibrium dissociation constants* Polyamide 5'-TGGTCA-3'5'-TGGACA-3'Kr 5'-T G-3'5'-T G G T t A-3' OOOOO DOCC Py/Py-9X000- ODOC 3'-A C Ai T 5 G T-5'3'-A C C A S G T-5' Kd = Kd = 1 5'-T G T A C A-3'5'-T G Q T A C A-3' H % Hp/Py +"+p- ( >5000 3'-A C A T T G T-5'3'-A C C A T G T-5' Kd=0. 2nM Kdo lOOOnM *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 TriswHCI, 10 mM KCI, 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. tCalculated as Kd (5'-TGGTACA-3')/Kd (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 AeT or TeA 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 hairpin polyamides (1-16) which recognize the sixteen 5'- WGTNNW-3'sequences (W = A or T, X = A, G, C, or T). Table 10 lists the sixteen eight-ring hairpin polyamides (17-32) which recognize the sixteen 5'-WGANNW-3'sequences (17-32).

Table 11 lists the twelve eight-ring hairpin 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 hairpin polyamides (G1-G4) which target the four 5'-WGGNNW-3'sequences (G1-G4) which contain exclusively GeC base pairs. Table 12 lists the twelve eight-ring hairpin polyamides (45-56) which recognize twelve 5'-WGCNNW-3'sequences which contain at least one A, T base pair. Table 12 lists the four eight-ring hairpin polyamides (G5-G8) which target the four 5'-WGCNNW-3'sequences (G5-G8) which contain exclusively GC base pairs. Table 13 lists the sixteen eight-ring hairpin polyamides (57-72) which recognize the sixteen 5'- WTTNNW-3'sequences (57-72). Table 14 lists the sixteen eight-ring hairpin polyamides (73- 88) which recognize the sixteen 5'-WTANNW-3'sequences (73-88). Table 15 lists the sixteen eight-ring hairpin polyamides (89-104) which recognize the sixteen 5'-WTGNNW-3'sequences (89-104). Table 16 lists the sixteen eight-ring hairpin polyamides (105-120) which recognize the sixteen 5'-WTCNNW-3'sequences (105-120). Table 17 lists the sixteen eight-ring hairpin polyamides (121-136) which recognize the sixteen 5'-WATNNW-3'sequences (121-136).

Table 18 lists the sixteen eight-ring hairpin polyamides (137-152) which recognize the sixteen 5'-WAANNW-3'sequences (137-152). Table 19 lists the sixteen eight-ring hairpin polyamides (153-168) which recognize the sixteen 5'-WAGNNW-3'sequences (153-168). Table 20 lists the sixteen eight-ring hairpin polyamides (169-184) which recognize the sixteen 5'-WACNNW- 3'sequences (169-184). Table 21 lists the sixteen eight-ring hairpin polyamides (185-200) which recognize the sixteen 5'-WCTNNW-3'sequences (185-200). Table 22 lists the sixteen eight-ring hairpin polyamides (201-216) which recognize the sixteen 5'-WCANNW-3' sequences (201-216). Table 23 lists the twelve eight-ring hairpin 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 hairpin polyamides (G9-G12) which target the four 5'- WCGNNW-3'sequences (G9-G12) which contain exclusively CG base pairs. Table 24 lists the twelve eight-ring hairpin 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 hairpin polyamides (G13-G16) which target the four 5'-WCCNNW-3'sequences (G13-G16) which contain exclusively CG base pairs.

TABLE 9 : 8-ring Hairpin Polyamides for recognition of 6-bp 5'-WGTNNW-3' DNA sequence aromatic amino acid sequence 1) 5'-W G T T T W-3'1) ImHpHpHp-y-PyPyPyPy 2) 5'-WGTTAW-3'2) ImHpHpPy-y-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-y-PyHpPyPy 6) 5'-W G T A A W-3' 6)ImHpPyPy-γ-HpHpPyPy 7) 5'-W G T A G W-3'7) ImHpPyIm-y-PyHpPyPy 8) 5'-W G T A C W-3'8) ImHpPyPy-y-ImHpPyPy 9) 5'-W G T G T W-3'9) ImHpImHp-y-PyPyPyPy 10) 5'-W G T G A W-3'10) ImHpImPy-y-HpPyPyPy 11) 5'-W G T G G W-3'11) ImHpImIm-y-PyPyPyPy 12) 5'-W G T G C W-3'12) ImHpImPy-y-ImPyPyPy 13) 5'-W G T C T W-3'13) ImHpPyHp-y-PyImPyPy 14) 5'-W G T C A W-3'14) ImHpPyPy-y-HpImPyPy 15) 5'-W G T C G W-3'15) ImHpPyIm-y-PyImPyPy 16) 5'-W G T C C W-3'16) ImHpPyPy-y-ImImPyPy

TABLE 10 : 8-ring Hairpin 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-Y-PyPyHpPy 18) 5'-W G A T A W-3'18) ImPyHpPy-y-HpPyHpPy 19) 5'-W G A T G W-3' 19) ImPyHpIm-y-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-y-PyHpHpPy 22) 5'-W G A A A W-3' 22) ImPyPyPy-y-HpHpHpPy 23) 5'-WGAAGW-3'23) ImPyPyIm-y-PyHpHpPy 24) 5'-W G A A C W-3' 24)ImPyPyPy-γ-InHpHpPy 25) 5'-W G A G T W-3' 25)ImPyImHp-γ-PyPyHpPy 26) 5'-W G A G A W-3' 26) ImPyImPy-γ-HpPyHpPy 27) 5'-W G A G G W-3' 27) ImPyImIm-γ-PyPyHpPy 28) 5'-W G A G C W-3'28) ImPyImPy-y-ImPyHpPy 29) 5'-W G A C T W-3' 29) ImPyPyHp-γ-PyImHpPy 30) 5'-W G A C A W-3'30) ImPyPyPy-y-HpImHpPy 31) 5'-W G A C G W-3' 31) ImPyPyIm-γ-PyImHpPy 32) 5'-W G A C C W-3' 32) ImPyPyPy-γ-ImImHpPy

TABLE 11 : 8-ring Hairpin Polyamides for recognition of6-bp 5'-WGGNNW-3' DNA sequence aromatic amino acid sequence 33) 5'-W G G T T W-3'33) ImImHpHp-y-PyPyPyPy 34) 5'-W G G T A W-3' 34) ImImHpPy-γ-HpPyPyPy 35) 5'-W G G T G W-3'35) ImImHpIm-y-PyPyPyPy 36) 5'-W G G T C W-3'36) ImImHpPy-y-ImPyPyPy 37) 5'-W G G A T W-3'37) ImImPyHp-y-PyHpPyPy 38) 5'-W G G A A W-3' 38) ImImPyPy-γ-HpHpPyPy 39) 5'-W G G A G W-3'39) ImImPyIm-y-PyHpPyPy 40) 5'-W G G A C W-3' 40) ImImPyPy-γ-ImHpPyPy 41) 5'-W G G G T W-3'41) ImImImHp-y-PyPyPyPy 42) 5'-W G G G A W-3' 42) ImImImPy-γ-HpPyPyPy 43) 5'-W G G C T W-3'43) ImImPyHp-y-PyImPyPy 44) 5'-W G G C A W-3' 44) ImImPyPy-γ-HpImPyPy Gl) 5'-W G G G G W-3'Gl) ImImImIm-y-PyPyPyPy G2) 5'-W G G G C W-3' G2) ImImImPy-γ-ImPyPyPy G3) 5'-W G G C G W-3' G3) ImImPyIm-γ-PyImPyPy G4) 5'-W G G C C W-3' G4) ImImPyPy-γ-ImImPyPy

TABLE 12 : 8-ring Hairpin 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-y-PyPyImPy 46) 5'-W G C T A W-3'46) ImPyHpPy-y-HpPyImPy 47) 5'-W G C T G W-3'47) ImPyHpIm-y-PyPyImPy 48) 5'-W G C T C W-3' 48) ImPyHpPy-γ-ImPyImPy 49) 5'-W G C A T W-3'49) ImPyPyHp-y-PyHpImPy 50) 5'-W G C A A W-3' 50) ImPyPyPy-γ-HpHpImPy 51) 5'-W G C A G W-3' 51) ImPyPyIm-γ-PyHpImPy 52) 5'-W G C A C W-3'52) ImPyPyPy-y-ImHpImPy 53) 5'-W G C G T W-3'53) ImPyImHp-y-PyPyImPy 54) 5'-W G C G A W-3' 54) ImPyImPy-y-HpPyImPy 55) 5'-W G C C T W-3'55) ImPyPyHp-y-PyImImPy 56) 5'-W G C C A W-3'56) ImPyPyPy-y-HpImImPy G5) 5'-W G C G G W-3'G5) ImPyImIm-y-PyPyImPy G6) 5'-W G C G C W-3' G6) ImPyImPy-γ-ImPyImPy G7) 5'-W G C C G W-3' G7)ImPyPyIm-γ-PyImImPy G8) 5'-W G C C C W-3'G8) ImPyPyPy-y-ImImImPy

TABLE 13 : 8-ring Hairpin Polyamides for recognition of 6-bp 5'-WTTNNW-3' DNA sequence aromatic amino acid sequence 57) 5'-W T T T T W-3'57) HpHpHpHp-y-PyPyPyPy 58) 5'-W T T T A W-3'58) HpHpHpPy-y-HpPyPyPy 59) 5'-W T T T G W-3'59) HpHpHpIm-y-PyPyPyPy 60) 5'-W T T T C W-3'60) HpHpHpPy-y-ImPyPyPy 61) 5'-W T T A T W-3'61) HpHpPyHp-y-PyHpPyPy 62) 5'-W T T A A W-3'62) HpHpPyPy-y-HpHpPyPy 63) 5'-W T T A G W-3'63) HpHpPyIm-y-PyHpPyPy . 64) 5'-W T T A C W-3'64) HpHpPyPy-y-ImHpPyPy 65) 5'-W T T G T W-3'65) HpHpImHp-y-PyPyPyPy 66) 5'-W T T G A W-3'66) HpHpImPy-y-HpPyPyPy 67) 5'-W T T G G W-3'67) HpHpImIm-y-PyPyPyPy 68) 5'-W T T G C W-3'68) HpHpImPy-y-ImPyPyPy 69) 5'-W T T C T W-3'69) HpHpPyHp-y-PyImPyPy 70) 5'-W T T C A W-3'70) HpHpPyPy-y-HpImPyPy 71) 5'-W T T C G W-3'71) HpHpPyIm-y-PyImPyPy 72) 5'-W T T C C W-3'72) HpHpPyPy-y-ImImPyPy

TABLE 14 : 8-ring Hairpin Polyamides for recognition of 6-bp 5'-WTANNW-3' DNA sequence aromatic amino acid sequence 73) 5'-W T A T T W-3'73) HpPyHpHp-y-PyPyHpPy 74) 5'-W T A T A W-3'74) HpPyHpPy-y-HpPyHpPy 75) 5'-W T A T G W-3'75) HpPyHpIm-y-PyPyHpPy 76) 5'-W T A T C W-3'76) HpPyHpPy-y-ImPyHpPy 77) 5'-W T A A T W-3'77) HpPyPyHp-y-PyHpHpPy 78) 5'-W T A A A W-3'78) HpPyPyPy-y-HpHpHpPy 79) 5'-W T A A G W-3'79) HpPyPyIm-y-PyHpHpPy 80) 5'-W T A A C W-3'80) HpPyPyPy-y-ImHpHpPy 81) 5'-W T A G T W-3' 81) HpHpImHp-γ-PyPyHpPy 82) 5'-W T A G A W-3'82) HpPyImPy-y-HpPyHpPy 83) 5'-W T A G G W-3'83) HpPyImIm-y-PyPyHpPy 84) 5'-W T A G C W-3'84) HpPyImPy-y-ImPyHpPy 85) 5'-W T A C T W-3'85) HpPyPyHp-y-PyImHpPy 86) 5'-W T A C A W-3'86) HpPyPyPy-y-HpImHpPy 87) 5'-W T A C G W-3'87) HpPyPyIm-y-PyImHpPy 88) 5'-W T A C C W-3'88) HpPyPyPy-y-ImImHpPy

TABLE 15 : 8-ring Hairpin Polyamides for recognition of 6-bp 5'-WTGNNW-3' DNA sequence aromatic amino acid sequence 89) 5'-W T G T T W-3'89) HpImHpHp-y-PyPyPyPy 90) 5'-W T G T A W-3'90) HpImHpPy-y-HpPyPyPy 91) 5'-W T G T G W-3'91) HpImHpIm-y-PyPyPyPy 92) 5'-W T G T C W-3'92) HpImHpPy-y-ImPyPyPy 93) 5'-W T G A T W-3'93) HpImPyHp-y-PyHpPyPy 94) 5'-W T G A A W-3'94) HpImPyPy-y-HpHpPyPy 95) 5'-W T G A G W-3'95) HpImPyIm-y-PyHpPyPy 96) 5'-W T G A C W-3'96) HpImPyPy-y-ImHpPyPy 97) 5'-W T G G T W-3'97) HpImImHp-y-PyPyPyPy 98) 5'-W T G G A W-3'98) HpImImPy-y-HpPyPyPy 99) 5'-W T G C T W-3'99) HpImPyHp-y-PyImPyPy 100) 5'-W T G C A W-3'100) HpImPyPy-y-HpImPyPy 101) 5'-W T G G G W-3'101) HpImImIm-y-PyPyPyPy 102) 5'-W T G G C W-3'102) HpImImPy-y-ImPyPyPy 103) 5'-W T G C G W-3'103) HpImPyIm-y-PyImPyPy 104) 5'-W T G C C W-3'104) HpImPyPy-y-ImImPyPy

TABLE 16 : 8-ring Hairpin 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-y-PyPyImPy 106) 5'-W T C T A W-3' 106) HpPyHpPy-γ-HpPyImPy 107) 5'-W T C T G W-3'107) HpPyHpIm-y-PyPyImPy 108) 5'-W T C T C W-3'108) HpPyHpPy-y-ImPyImPy 109) 5'-W T C A T W-3'109) HpPyPyHp-y-PyHpImPy 110) 5'-W T C A A W-3'110) HpPyPyPy-y-HpHpImPy 111) 5'-W T C A G W-3' 111) HpPyPyIm-γ-PyHpImPy 112) 5'-W T C A C W-3'112) HpPyPyPy-y-ImHpImPy 113) 5'-W T C G T W-3'113) HpPyImHp-y-PyPyImPy 114) 5'-W T C G A W-3'114) HpPyImPy-y-HpPyImPy 115) 5'-W T C C T W-3'115) HpPyPyHp-y-PyImImPy 116) 5'-W T C C A W-3'116) HpPyPyPy-y-HpImImPy 117) 5'-W T C G G W-3'117) HpPyImIm-y-PyPyImPy 118) 5'-W T C G C W-3' 118) HpPyImPy-γ-ImPyImPy 119) 5'-W T C C G W-3' 119) HpPyPyIm-y-PyImImPy 120) 5'-W T C C C W-3'120) HpPyPyPy-y-ImImImPy

TABLE 17 : 8-ring Hairpin Polyamides for recognition of 6-bp 5'-WATNNW-3' DNA sequence aromatic amino acid sequence 121) 5'-W A T T T W-3'1 = 122) 5'-W A T T A W-3'122) PyHpHpPy-y-HpPyPyHp 123) 5'-W A T T G W-3'123) PyHpHpIm-y-PyPyPyHp 124) 5'-W A T T C W-3'124) PyHpHpPy-y-ImPyPyHp 125) 5'-W A T A T W-3'125) PyHpPyHp-y-PyHpPyHp 126) 5'-W A T A A W-3'126) PyHpPyPy-y-HpHpPyHp 127) 5'-W A T A G W-3'127) PyHpPyIm-y-PyHpPyHp 128) 5'-W A T A C W-3'128) PyHpPyPy-y-ImHpPyHp 129) 5'-W A T G T W-3'129) PyHpImHp-y-PyPyPyHp 130) 5'-W A T G A W-3'130) PyHpImPy-y-HpPyPyHp 131) 5'-W A T G G W-3'131) PyHpImIm-y-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-PyImPyHp 134) 5'-W A T C A W-3' 134) PyHpPyPy-γ-HpImPyHp 135) 5'-W A T C G W-3'135) PyHpPyIm-y-PyImPyHp 136) 5'-W A T C C W-3' 136) PyHpPyPy-γ-ImImPyHp

TABLE 18 : 8-ring Hairpin 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-y-PyPySpHp 138) 5'-W A A T A W-3'138) PyPyHpPy-y-HpPyHpHp 139) 5'-W A A T G W-3'139) PyPyHpIm-y-PyPyHpHp 140) 5'-W A A T C W-3'140) PyPyHpPy-y-ImPyHpHp 141) 5'-W A A A T W-3'141) PyPyPyHp-y-PyHpHpHp 142) 5'-W A A A A W-3'142) PyPyPyPy-y-HpHpHpHp 143) 5'-W A A A G W-3'143) PyPyPyIm-y-PyHpHpHp 144) 5'-W A A A C W-3'144) PyPyPyPy-y-ImHpHpHp 145) 5'-W A A G T W-3'145) PyPyImHp-y-PyPyHpHp 146) 5'-W A A G A W-3'146) PyPyImPy-y-HpPyHpHp 147) 5'-W A A G G W-3'147) PyPyImIm-y-PyPyHpHp 148) 5'-W A A G C W-3'148) PyPyImPy-y-ImPyHpHp 149) 5'-W A A C T W-3'149) PyPyPyHp-y-PyImHpHp 150) 5'-W A A C A W-3'150) PyPyPyPy-y-HpImHpHp 151) 5'-W A A C G W-3'151) PyPyPyIm-y-PyImHpHp 152) 5'-W A A C C W-3'152) PyPyPyPy-y-mImHpHp

TABLE 19 : 8-ring Hairpin 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) PyImHpHp-γ-PyPyPyHp 154) 5'-W A G T A W-3' 154) PyImHpPy-γ-HpPyPyHp 155) 5'-W A G T G W-3' 155) PyImHpIm-γ-PyPyPyHp 156) 5'-W A G T C W-3' 156) PyImHpPy-γ-ImPyPyHp 157) 5'-W A G A T W-3' 157) PyImPyHp-γ-PyHpPyHp 158) 5'-W A G A A W-3'158) PyImPyPy-y-HpHpPyHp 159) 5'-W A G A G W-3' 159) PyImPyIm-γ-PyHpPyHp 160) 5'-W A G A C W-3' 160) PyImPyPy-γ-ImHpPyHp 161) 5'-W A G G T W-3'161) PyImImHp-y-PyPyPyHp 162) 5'-W A G G A W-3' 162) PyImImPy-γ-HpPyPyHp 163) 5'-W A G C T W-3'163) PyImPyHp-y-PyImPyHp 164) 5'-W A G C A W-3' 164) PyImPyPy-γ-HpImPyHp 165) 5'-W A G G G W-3' 165) PyImImIm-γ-PyPyPyHp 166) 5'-W A G G C W-3'166) PyImImPy-y-ImPyPyHp 167) 5'-W A G C G W-3'167) PyImPyIm-y-PyImPyHp 168) 5'-W A G C C W-3' 168) PyImPyPy-γ-ImImPyHp

TABLE 20 : 8-ring Hairpin Polyamides for recognition of 6-bp 5'-WACNNW-3' DNA sequence aromatic amino acid sequence 169) 5'-W A C T T W-3' 169) PyPyHpHp-γ-PyPyImHp 170) 5'-W A C T A W-3' 170) PyPyHpPy-γ-HpPyImHp 171) 5'-W A C T G W-3' 171) PyPyHpIm-γ-PyPyImHp 172) 5'-W A C T C W-3'172) PyPyHpPy-y-ImPyImHp 173) 5'-W A C A T W-3'173) PyPyPyHp-y-PyHpImHp 174) 5'-W A C A A W-3'174) PyPyPyPy-y-HpHpImHp 175) 5'-W A C A G W-3'175) PyPyPyIm-y-PyHpImHp 176) 5'-W A C A C W-3'176) PyPyPyPy-y-ImHpImHp 177) 5'-W A C G T W-3'177) PyPyImHp-y-PyPyImHp 178) 5'-W A C G A W-3'178) PyPyImPy-y-HpPyImHp 179) 5'-W A C C T W-3'179) PyPyPyHp-y-PyImImHp 180) 5'-W A C C A W-3'180) PyPyPyPy-y-HpImImHp 181) 5'-W A C G G W-3' 181) PyPyImIm-y-PyPyImHp 182) 5'-W A C G C W-3'182) PyPyImPy-y-ImPyImHp 183) 5'-W A C C G W-3' 183) PyPyPyIm-γ-PyImImHp 184) 5'-W A C C C W-3' 184) PyPyPyPy-y-ImImImHp

TABLE 21 : 8-ring Hairpin Polyamides for recognition of 6-bp 5'-WCTNNW-3' DNA sequence aromatic amino acid sequence 185) 5'-W C T T T W-3'185) PyHpHpHp-y-PyPyPyIm 186) 5'-W C T T A W-3'186) PyHpHpPy-y-HpPyPyIm 187) 5'-W C T T G W-3'187) PyHpHpIm-y-PyPyPyIm 188) 5'-W C T T C W-3'188) PyHpHpPy-y-ImPyPyIm 189) 5'-W C T A T W-3' 189) PyHpPyHp-γ-PyHpPyIm 190) 5'-W C T A A W-3'190) PyHpPyPy-y-HpHpPyIm 191) 5'-W C T A G W-3'l91) PyHpPyIm-y-PyHpPyIm 192) 5'-W C T A C W-3'192) PyHpPyPy-y-ImHpPyIm 193) 5'-W C T G T W-3'193) PyHpImHp-y-PyPyPyIm 194) 5'-W C T G A W-3' 194) PyHpImPy-γ-HpPyPyIm 195) 5'-W C T G G W-3' 195) PyHpImIm-γ-PyPyPyIm 196) 5'-W C T G C W-3' 196) PyHpImPy-γ-ImPyPyIm 197) 5'-W C T C T W-3'197) PyHpPyHp-y-PyImPyIm 198) 5'-W C T C A W-3'198) PyHpPyPy-y-HpImPyIm 199) 5'-W C T C G W-3'199) PyHpPyIm-y-PyImPyIm 200) 5'-W C T C C W-3' 200) PyHpPyPy-γ-ImImPyIm

TABLE 22 : 8-ring Hairpin 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-y-PyPyHpIm 202) 5'-W C A T A W-3 202) PyPyHpPy-y-HpPyHpIm 203) 5'-W C A T G W-3' 203) PyPyHpIm-y-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) PyPyPyIm-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) PyPyImHp-γ-PyPyHpIm 210) 5'-W C A G A W-3' 210) PyPyImPy-γ-HpPyHpIm 211) 5'-W C A G G W-3' 211) PyPyImIm-γ-PyPyHpIm 212) 5'-W C A G C W-3' 212) PyPyImPy-γ-ImPyHpIm 213) 5'-W C A C T W-3' 213) PyPyPyHp-γ-PyImHpIm 214) 5'-W C A C A W-3' 214) PyPyPyPy-γ-HpImHpIm 215) 5'-W C A C G W-3' 215) PyPyPyIm-γ-PyImHpIm 216) 5'-W C A C C W-3' 216) PyPyPyPy-γ-ImImHpIm

TABLE 23 : 8-ring Hairpin 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) PyImHpHp-y-PyPyPyIm 218) 5'-W C G T A W-3'218) PyImHpPy-y-HpPyPyIm 219) 5'-W C G T G W-3'219) PyImHpIm-y-PyPyPyIm 220) 5'-W C G T C W-3'220) PyImHpPy-y-ImPyPyIm 221) 5'-W C G A T W-3'221) PyImPyHp-y-PyHpPyIm 222) 5'-W C G A A W-3'222) PyImPyPy-y-HpHpPyIm 223) 5'-W C G A G W-3'223) PyImPyIm-y-PyHpPyIm 224) 5'-W C G A C W-3'224) PyImPyPy-y-ImHpPyIm 225) 5'-W C G G T W-3'225) PyImImHp-y-PyPyPyIm 226) 5'-W C G G A W-3' 226) PyImImPy-γ-HpPyPyIm 227) 5'-W C G C T W-3'227) PyImPyHp-y-PyImPyIm 228) 5'-W C G C A W-3' 228) PyImPyPy-γ-HpImPyIm G9) 5'-W C G G G W-3' G9) PyImImIm-γ-PyPyPyIm G10) 5'-W C G G C W-3'G10) PyImImPy-y-ImPyPyIm Gll) 5'-W C G C G W-3'Gll) PyImPyIm-y-PyImPyIm G12) 5'-W C G C C W-3' G12) PyImPyPy-γ-ImImPyIm

TABLE 24 : 8-ring Hairpin Polyamides for recognition of 6-bp 5'-WCCNNW-3' DNA sequence aromatic amino acid sequence 229) 5'-W C C T T W-3'229) PyPyHpHp-y-PyPyImIm 230) 5'-W C C T A W-3'230) PyPyHpPy-y-HpPyImIm 231) 5'-W C C T G W-3'231) PyPyHpIm-y-PyPyImIm 232) 5'-W C C T C W-3'232) PyPyHpPy-y-ImPyImIm 233) 5'-W C C A T W-3'233) PyPyPyHp-y-PyHpImIm 234) 5'-W C C A A W-3'234) PyPyPyPy-y-HpHpImIm 235) 5'-W C C A G W-3'235) PyPyPyIm-y-PyHpImIm 236) 5'-W C C A C W-3'236) PyPyPyPy-y-ImHpImIm 237) 5'-W C C G T W-3'237) PyPyImHp-y-PyPyImIm 238) 5'-W C C G A W-3'238) PyPyImPy-y-HpPyImIm 239) 5'-W C C C T W-3'239) PyPyPyHp-y-PyImImIm 240) 5'-W C C C A W-3'240) PyPyPyPy-y-HpImImIm G13) 5'-W C C G G W-3'G13) PyPyImIm-y-PyPyImIm G14) 5'-W C C G C W-3'G14) PyPyImPy-y-ImPyImIm G15) 5'-W C C C G W-3'G15) PyPyPyIm-y-PyImImIm G16) 5'-W C C C C W-3'G16) PyPyPyPy-y-ImImImIm EXAMPLE 9 : Aliphatic/Aromatic amino acid pairing for recognition of the DNA minor groove.

Selective placement of an aliphatic p-alanine (p) 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 hairpin pyrrole-imidazole polyamides. A series of polyamides were prepared which contain pyrrole and imidazole aromatic amino acids, as well as y-aminobutyric acid (y)"turn"and ß-alanine'; spring"aliphatic amino acid residues. The binding affinities and specificities of these polyamides are regulated by the placement of paired ß/ß Py/ß and Im/ß residues. Quantitative footprint titrations demonstrate that replacing two Py/Py pairings in a 12-ring hairpin (6-y-6) with two Py/ß

pairings affords 10-fold enhanced affinity and similar sequence specificity for an 8-bp target sequence.

Table25 Equilibrium association constants (M-1) for polyamides. a-c Polyamide 5'-TGTTAACA-3'5'-TGTGAACA-3'Specificityd 2. 5 x 109 3. 9 x 108 6 1. 3 x 109 2. 0 x 108 7 = 1. 7x101° 2. 7xl09 6 1x10"2. 2xt055 6. 6 x 109 2. 5 x 109 26 = 4. 5x101° 7. 7xlO9 6 **4xin'°77x) n6 004. SX1U/./X1U0 g2. 7xt0"'5. 7xS s I x 108--I x 108 1 -00-00-'s t x) Us t x tU "Values reported are the mean values obtained from three DNase I footprint titration experiments. b The assays were carried out at 22 °C at pH 7. 0 in the presence of 10 mM Tris-HCI, IO mM KCI. 10 mM MgCI2, and 5 mM CaCI-.'Match site association constants and specificities higher than the parent hairpin are shown in boldtype dSpecificity is calculated as Ka (match)/Ka (mismatch).

The 6-y-6 hairpin ImPyImPyPyPy-y-ImPyPyPyPyPy-ß-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 hairpin polyamide Im-ß- ImPyPyPy-γ-ImPyPyPy-ß-Py-ß-Dp binds to the 8-bp match sequence 5'-TGTGAACA-3'with an equilibrium association constant of Ka = 2. 4 x 10 and > 48-fold specificity versus the 5'-TGTTAACA-3'single-base-pair mismatch site.

Table 26 Equilibrium association constants (M-1) for polyamides.a-c Polyamide 5'-TGTTAACA-3'S'-TGTGAACA-3'Specificityd 2. S x 109 3. 9 x (O8 6 6. 6 x 109 2. 5 x 108 26 HW x **-O-O-O-D-C-W a Values reported for 1, 5, and 10 are the mean values obtained from three DNase I footprint titration experiments. bThe assays were carried out at 22 °C at pH 7. 0 in the presence of 10 mM Tris-HCI, 10 mM KCI, 10 mM MgCI2, and 5 mM CaCI2* CMatch site association constants and specificities higher than parent hairpins are shown in boldtype. dSpecificity is calculated as Ka (match)/Ka (mismatch).

Modeling indicates that the ß-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-ß-Im polyamide subunit. This observation provided the basis for design of a hairpin polyamide, Im-ß-ImPy-γ-Im-ß-ImPy-ß-Dp, which incorporates Im/ß pairings to recognize a"problematic"5'-GCGC-3'sequence at subnanomolar concentrations.

Table 27 Equilibrium association constamts (M*') for polyamides. a-b Polyamide 5'-TGCGCA-3'5i'-TGGCCA-3'5'-TGGGGA-3' 3. 7 x 10 < 10 < 10 3. 7 x 109 1. 4 x 108 1. 1 x lOR a Values reported are the mean values obtained from a minimum of three DNase I footprint titration experiments. tbThe assays were carried out at 22 °C at pH 7. 0 in the presence of 10 mM Tris-HCI, tO mM KC1, 10 mM MgCI2, and 5 mM Cadiz.

These results identify Im/p and p/Im pairings that respectively discriminate GC and C-G from AT/T-A as well as Py/p and ß/Py pairings that discriminate AT/TvA from G#C/C#G. These aliphatic/aromatic amino acid pairings will facilitate the design of hairpin 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. --P-Py-Py-Py-tm-y-Py-Py-Py-tm Resin HzNNNHy 0 o HN NH 0 H H H O ,,, il NO- 0 0 o DMF, DIEA 12h RT HN NH \ 0 Q \ Htt, H 0 0 o N N---0N 0 \ 9,'NLHiHH Fi H H \\ O O N H N N H N II H H P H H 1 O N v '0 . . HN-rb N') t-J N tSNSH 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.