Eight-ring Hairpin Research Articles

Correlation of Local Effects of DNA Sequence and Position of β-Alanine Inserts with Polyamide–DNA Complex Binding Affinities and Kinetics

To improve our understanding of the effects of β-alanine (β) substitution and the number of heterocycles on DNA binding affinity and selectivity, we investigated the interactions of an eight-ring hairpin polyamide (PA) and two β derivatives as well as a six-heterocycle analogue with their cognate DNA sequence, 5'-TGGCTT-3'. Binding selectivity and the effects of β have been investigated with the cognate and five mutant DNAs. A set of powerful and complementary methods have been employed for both energetic and structural evaluations: UV melting, biosensor surface plasmon resonance, isothermal titration calorimetry, circular dichroism, and a DNA ligation ladder global structure assay. The reduced number of heterocycles in the six-ring PA weakens the binding affinity; however, the smaller PA aggregates significantly less than the larger PAs and allows us to obtain the binding thermodynamics. The PA-DNA binding enthalpy is large and negative with a large negative ΔC(p) and is the primary driving component of the Gibbs free energy. The complete SPR binding results clearly show that β substitutions can substantially weaken the binding affinity of hairpin PAs in a position-dependent manner. More importantly, the changes in the binding of PA to the mutant DNAs further confirm the position-dependent effects on the PA-DNA interaction affinity. Comparison of mutant DNA sequences also shows a different effect in recognition of T·A versus A·T base pairs. The effects of DNA mutations on binding of a single PA as well as the effects of the position of β substitution on binding tell a clear and very important story about sequence-dependent binding of PAs to DNA.

Quantitative Microarray Profiling of DNA-Binding Molecules

A high-throughput Cognate Site Identity (CSI) microarray platform interrogating all 524 800 10-base pair variable sites is correlated to quantitative DNase I footprinting data of DNA binding pyrrole-imidazole polyamides. An eight-ring hairpin polyamide programmed to target the 5 bp sequence 5'-TACGT-3' within the hypoxia response element (HRE) yielded a CSI microarray-derived sequence motif of 5'-WWACGT-3' (W = A,T). A linear beta-linked polyamide programmed to target a (GAA)3 repeat yielded a CSI microarray-derived sequence motif of 5'-AARAARWWG-3' (R = G,A). Quantitative DNase I footprinting of selected sequences from each microarray experiment enabled quantitative prediction of Ka values across the microarray intensity spectrum.

Toward artificial developmental regulators.

A polyamide-peptide conjugate is designed which recruits sequence specifically the developmental regulator Exd to a cognate DNA site. In particular, an eight-ring hairpin polyamide (Im-Im-Py(C3H6NHR)-Py-gamma-Im-Py-Py-Py-beta-Dp) with a heptapeptide (R = Ac-Phe-Tyr-Pro-Trp-Met-Lys-Gly-) attached on a central ring was shown to induce cooperative binding of the Drosophila Hox protein cofactor Exd with a Kd of 4.4 nM in vitro, an order of magnitude more efficient than the natural Hox protein partner Ubx. The conjugate joins two sequence specific domains, one for DNA and one for the protein. This small molecule thus serves as a cooperative protein-DNA dimerizer, which mimics the natural Hox family of developmental regulators.

DNA Binding Hairpin Polyamides with Antifungal Activity

Eight-ring hairpin polyamides containing N-methylimidazole (Im) and N-methylpyrrole (Py) amino acids have been shown to bind with subnanomolar affinity to discrete DNA sites and to modulate a variety of DNA-dependent biological processes. We show here that addition of a second positive charge at the C terminus of an 8-ring hairpin polyamide confers activity against a number of clinically relevant fungal strains in vitro, and activity against Candida albicans in a mouse model. Control experiments indicate that the observed antifungal activity results from a DNA binding mechanism-of-action that does not involve DNA damage or disruption of chromosomal integrity. Hairpin activity is shown to be proportional to yeast DNA content (ploidy). Transcriptional interference is proposed as the likely explanation for fungal cytotoxicity. Experiments with sensitized yeast strains indicate the potential for discrete sites of action rather than global effects.

Imidazopyridine/Pyrrole and hydroxybenzimidazole/pyrrole pairs for DNA minor groove recognition.

The DNA binding properties of fused heterocycles imidazo[4,5-b]pyridine (Ip) and hydroxybenzimidazole (Hz) paired with pyrrole (Py) in eight-ring hairpin polyamides are reported. The recognition profile of Ip/Py and Hz/Py pairs were compared to the five-membered ring pairs Im/Py and Hp/Py on a DNA restriction fragment at four 6-base pair recognition sites which vary at a single position 5'-TGTNTA-3', where N = G, C, T, A. The Ip/Py pair distinguishes G.C from C.G, T.A, and A.T, and the Hz/Py pair distinguishes T.A from A.T, G.C, and C.G, affording a new set of heterocycle pairs to target the four Watson-Crick base pairs in the minor groove of DNA.

Cell-free and Cellular Activities of a DNA Sequence Selective Hairpin Polyamide-CBI Conjugate

Alkylating agents are generally highly reactive with DNA but demonstrate limited DNA sequence selectivity. In contrast, synthetic pyrrole-imidazole polyamides recognize specific DNA sequences with high affinity but are unable to permanently damage DNA. An eight-ring hairpin polyamide conjugated to the alkylating moiety cyclopropylpyrroloindole, related to the natural product CC-1065, affords a conjugate 1-CBI (polyamide 1-CBI (1-(chloromethyl)-5-hydroxyl-1,2-dihydro-3H-benz[e]indole) conjugate), which binds to specific sequences in the minor groove of DNA and alkylates a single adenine flanking the polyamide binding site. In this study, we show that 1-CBI alkylates DNA in both plasmid and intracellular minichromosomal form and inhibits DNA replication under both cell-free and cellular conditions. In addition, it inhibits cell growth and arrests cells in the G2/M phase of the cell cycle.

Expanding the recognition of the minor groove of DNA by incorporation of β-alanine in hairpin polyamides

In order to expand the recognition code by hairpin polyamides to include DNA sequences of the type 5′-CWWC-3′ two polyamides, PyPyPyPy-(R) H 2N γ-ImPyPyIm-β-Dp ( 1) and PyPyPyPy-(R) H 2N γ-ImPy-β-Im-β-Dp ( 2) were synthesized which have in common an Py/Im pair in the terminal position for targeting C·G but differ with respect to internal placement of a β-alanine residue. The equilibrium association constants ( K a) were determined at four DNA sites which differ at a single common position, 5′-TNTACA-3′ (N=T, A, G, C). Quantitative DNase I footprint titration experiments reveal that the eight-ring hairpin PyPyPyPy-(R) H 2N γ-ImPyPyIm-β-Dp ( 1) binds the four binding sites with similar affinities, K a=1.3–1.9×10 10 M −1 indicating that there is no preference for the position N. In contrast, a redesigned polyamide PyPyPyPy-(R) H 2N γ-ImPy-β-Im-β-Dp ( 2) that places an internal flexible aliphatic β-alanine to the 5′-side of a key imidazole group bound the match site 5′-TCTACA-3′ with high affinity and good sequence discrimination ( K a(match)=4.9×10 10 M −1 and the single base pair mismatch sites with 5- to 25-fold lower affinity). These results expand the repertoire of sequences targetable by hairpins and emphasize the importance of β-alanine as a key element for minor groove recognition.

Alternative heterocycles for dna recognition: a 3-pyrazole/pyrrole pair specifies for g·c base pairs

Synthetic ligands comprising three aromatic amino acids, pyrrole (Py), imidazole (Im), and hydroxypyrrole (Hp), specifically recognize predetermined sequences as side-by-side pairs in the minor groove of DNA. To expand the repertoire of aromatic rings that may be utilized for minor groove recognition, three five-membered heterocyclic rings, 3-pyrazolecarboxylic acid (3-Pz), 4-pyrazolecarboxylic acid (4-Pz), and furan-2-carboxylic acid (Fr), were examined at the N-terminus of eight-ring hairpin polyamide ligands. The DNA binding properties of 3-Pz, 4-Pz, and Fr each paired with Py were studied by quantitative DNase I footprinting titrations on a 283 bp DNA restriction fragment containing four 6-bp binding sites 5′-A T NCCTA A-3′ (N=G, C, A, or T; 6-bp polyamide binding site is underlined). The pair 3-Pz/Py has increased binding affinity and sequence specificity for G·C bp compared with Im/Py.

Hydroxybenzamide/Pyrrole Pair Distinguishes T·A from A·T Base Pairs in the Minor Groove of DNA

A new aromatic pair, 2-hydroxy-6-methoxybenzamide/1-methylpyrrole at the terminal position of hairpin polyamides has been designed for distinguishing T·A from A·T base pairs and both from G·C/C·G in the minor groove of DNA. Four eight-ring hairpin polyamides with benzamide (Bz), 2-hydroxybenzamide (Hb-1), 2-hydroxy-6-methylbenzamide (Hb-2), and 2-hydroxy-6-methoxybenzamide (Hb-3) at the N-terminal position were synthesized. The equilibrium association constants (K_a) were determined at four DNA sites which differ at a single common position, 5‘-TNTACA-3‘ (N = T, A, G, C). Quantitative DNase I footprint titration experiments reveal that (Hb-3)PyPyPy-(R)^H2^Nγ-ImPyPyPy-β-Dp (4) bound the sequences 5‘-TTTACA-3‘ and 5‘-TATACA-3‘ with high affinity; K_a = 2.6 × 10^(10) M^(-1) and K_a = 8.4 × 10^9 M^(-1), respectively, a 3-fold specificity for T vs A was found. Importantly, the sequences 5‘-TGTACA-3‘ and 5‘-TCTACA-3‘ are bound with 50 and 200 times lower affinity, revealing an overall specificity of Hb-3/Py of T > A ≫ G > C. These results expand the repertoire of sequences targetable by hairpin polyamides.

Recognition of the DNA minor groove by pyrrole-imidazole polyamides: comparison of desmethyl- and N-methylpyrrole

Polyamides consisting of N-methylpyrrole (Py), N-methylimidazole (Im), and N-methyl-3-hydroxypyrrole (Hp) are synthetic ligands that recognize predetermined DNA sequences with affinities and specificities comparable to many DNA-binding proteins. As derivatives of the natural products distamycin and netropsin, Py/Im/Hp polyamides have retained the N-methyl substituent, although structural studies of polyamide:DNA complexes have not revealed an obvious function for the N-methyl. In order to assess the role of the N-methyl moiety in polyamide:DNA recognition, a new monomer, desmethylpyrrole (Ds), where the N-methyl moiety has been replaced with hydrogen, was incorporated into an eight-ring hairpin polyamide by solid-phase synthesis. MPE footprinting, affinity cleavage, and quantitative DNase I footprinting revealed that replacement of each Py residue with Ds resulted in identical binding site size and orientation and similar binding affinity for the six-base-pair (bp) target DNA sequence. Remarkably, the Ds-containing polyamide exhibited an 8-fold loss in specificity for the match site versus a mismatched DNA site, relative to the all-Py parent. Polyamides with Ds exhibit increased water solubility, which may alter the cell membrane permeability properties of the polyamide. The addition of Ds to the repertoire of available monomers may prove useful as polyamides are applied to gene regulation in vivo. However, the benefits of Ds incorporation must be balanced with a potential loss in specificity.

Strand Selective Cleavage of DNA by Diastereomers of Hairpin Polyamide-seco-CBI Conjugates

Pyrrole-imidazole polyamides are synthetic ligands that bind predetermined DNA sequences with subnanomolar affinity. We report the synthesis and characterization of an eight-ring hairpin polyamide conjugated at the turn to both enantiomers of 1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benz[e]indole (seco-CBI), an alkylating moiety related to CC-1065. Alkylation yields and specificity were determined on a restriction fragment containing six base pair match and mismatch sites. Alkylation was observed at a single adenine flanking the polyamide binding site, and strand selective cleavage could be achieved based on the enantiomer of seco-CBI chosen. At 1 nM concentrations of polyamide-seco-CBI conjugate, near quantitative cleavage was observed after 12 h. These bifunctional molecules could be useful for targeting coding regions of genes and inhibition of transcription.

Selective inhibition of DNA gyrase in vitro by a GC specific eight-ring hairpin polyamide at nanomolar concentration

The influence of an eight-ring hairpin DNA minor groove binder on the gyrase mediated DNA supercoiling and cleavage reaction step of the enzyme was investigated. The results demonstrate that supercoiling is affected by the hairpin polyamide in the millimolar concentration range while the enzyme catalyzed cleavage of a 162 bp fragment of pBR322 containing a single strong gyrase site is effectively inhibited at nanomolar concentration. As demonstrated by footprint analysis the latter effect is caused by a specific binding of the hairpin forming polyamide to the enzyme recognition site (GGCC), which indicates that the gyrase activity to produce a double strand break is blocked at this site. The pyrrole–imidazole hairpin polyamide is the most potent inhibitor of the gyrase mediated cleavage reaction compared to other known anti-gyrase active DNA binding agents.

Sequence specific alkylation of DNA by hairpin pyrrole–imidazole polyamide conjugates

Background: Pyrrole–imidazole polyamides are synthetic ligands that recognize predetermined sequences in the minor groove of DNA with affinities and specificities comparable to those of DNA-binding proteins. As a result of their DNA-binding properties, polyamides could deliver reactive moieties for covalent reaction at specific DNA sequences and thereby inhibit DNA–protein interactions. Site-specific alkylation of DNA could be a useful tool for regulating gene expression. As a minimal first step, we set out to design and synthesize a class of hairpin polyamides equipped with DNA alkylating agents and characterize the specificity and yield of covalent modification.Results: Bis(dichloroethylamino)benzene derivatives of the well-characterized chlorambucil (CHL) were attached to the γ turn of an eight-ring hairpin polyamide targeted to the HIV-1 promoter. We found that a hairpin polyamide–CHL conjugate binds and selectively alkylates predetermined sites in the HIV promoter at subnanomolar concentrations. Cleavage sites were determined on both strands of a restriction fragment containing the HIV-1 promoter, revealing good specificity and a high yield of alkylation.Conclusions: The ability of polyamide–CHL conjugates to sequence specifically alkylate double-stranded DNA in high yield and at low concentrations sets the stage for testing their use as regulators of gene expression in cell culture and ultimately in complex organisms.

Inhibition of major-groove-binding proteins by pyrrole-imidazole polyamides with an Arg-Pro-Arg positive patch

Background: Gene-specific targeting of any protein-DNA complex by small molecules is a challenging goal at the interface of chemistry and biology. Polyamides containing N-methylimidazole and N-methylpyrrole amino acids are synthetic ligands that have an affinity and specificity for DNA comparable to many naturally occurring DNA-binding proteins. It has been shown that an eight-ring hairpin polyamide targeted to a specific minor-groove contact within a transcription factor binding site can inhibit protein-DNA binding and gene transcription. Polyamides and certain major-groove-binding proteins have been found to co-occupy the DNA helix, however. To expand the number of genes that can be targeted by pyrrole/imidazole polyamides, we set out to develop a class of polyamides that can selectively inhibit major-groove-binding proteins. Results: An eight-ring hairpin polyamide conjugated to a carboxy-terminal Arg-Pro-Arg tripeptide was designed to deliver a positive residue to the DNA backbone and interfere with protein-phosphate contacts. Gel mobility shift analysis demonstrated that a polyamide hairpin-Arg-Pro-Arg binding in the minor groove selectively inhibits binding of the transcription factor GCN4 (222–281) in the adjacent major groove. Substitution within the Arg-Pro-Arg revealed that each residue was required for optimal GCN4 inhibition. Conclusions: A pyrrole-imidazole polyamide that binds to a predetermined site in the DNA minor groove and delivers a positive patch to the DNA backbone can selectively inhibit a DNA-binding protein that recognizes the adjacent major groove. A subtle alteration of the DNA microenvironment targeted to a precise location within a specific DNA sequence could achieve both gene-specific and protein-specific targeting.

Recognition of Seven Base Pair Sequences in the Minor Groove of DNA by Ten-Ring Pyrrole−Imidazole Polyamide Hairpins

A new upper limit of binding site size is defined for the hairpin polyamide-DNA motif. Ten-ring hairpin polyamides containing pyrrole (Py) and imidazole (Im) amino acids were designed for recognition of seven base pair (bp) sequences in the minor groove of DNA. The DNA binding properties of two polyamides, ImPyPyPyPy-γ-ImPyPyPyPy-β-Dp, and ImImPyPyPy-γ-ImPyPyPyPy-β-Dp were analyzed by footprinting and affinity cleavage on a DNA fragment containing the respective match sites 5‘-TGTAACA-3‘ and 5‘-TGGAACA-3‘. Quantitative footprint titrations demonstrate that ImPyPyPyPy-γ-ImPyPyPyPy-β-Dp binds the 7-bp match sequence 5‘-TGTAACA-3‘ with an equilibrium association constant (K_a) of K_a = 1.2 × 10^(10) M^(-1) and 18-fold specificity versus the single base pair mismatch sequence 5‘-TGGAACA-3‘. ImImPyPyPy-γ-ImPyPyPyPy-β-Dp differs from ImPyPyPyPy-γ-ImPyPyPyPy-β-Dp by a single amino acid substitution and binds its match 5‘-TGGAACA-3‘ site with K_a = 3.6 × 10^9 M^(-1) and 300-fold specificity versus its corresponding single base pair mismatch sequence 5‘-TGTAACA-3‘. Ten-ring hairpin polyamides have binding affinities similar to those of eight-ring hairpin polyamides. These results indicate that the affinity of hairpin binding ceases to increase as the length of the polyamide subunits increases beyond four rings, analogous to the behavior of unlinked subunits. Therefore, recognition of seven base pairs by a ten-ring hairpin polyamide most likely represents an upper limit to the effective targetable site size of the hairpin polyamide-DNA motif.

Discrimination of 5‘-GGGG-3‘, 5‘-GCGC-3‘, and 5‘-GGCC-3‘ Sequences in the Minor Groove of DNA by Eight-Ring Hairpin Polyamides

Eight-ring hairpin polyamides which differ only by the linear arrangement of pyrrole (Py) and imidazole (Im) amino acids were designed for recognition of six base pair DNA sequences containing four contiguous G,C base pairs. The respective DNA binding properties of three polyamides, ImImPyPy-γ-ImImPyPy-β-Dp, ImPyImPy-γ-ImPyImPy-β-Dp, and ImImImIm-γ-PyPyPyPy-β-Dp, were analyzed by footprinting and affinity cleavage on a DNA fragment containing the respective match sites 5‘-TGGCCA-3‘, 5‘-TGCGCA-3‘, and 5‘-TGGGGA-3‘. Quantitative footprint titrations demonstrate that ImImPyPy-γ-ImImPyPy-β-Dp binds the designed match site 5‘-TGGCCA-3‘ with an equilibrium association constant of Ka = 1 × 1010 M-1 and >250-fold specificity versus the mismatch sequences, 5‘-TGCGCA-3‘ and 5‘-TGGGGA-3‘. The polyamides ImPyImPy-γ-ImPyImPy-β-Dp and ImImImIm-γ-PyPyPyPy-β-Dp recognize their respective 5‘-TGCGCA-3‘ and 5‘-TGGGGA-3‘ match sites with reduced affinity relative to ImImPyPy-γ-ImImPyPy-β-Dp, but again with high specificity with regard to mismatch sites. These results expand the DNA sequence repertoire targeted by pyrrole-imidazole polyamides and identify sequence composition effects which will guide further second-generation polyamide design for DNA recognition.

Recognition of a 5‘-(A,T)GGG(A,T)2-3‘ Sequence in the Minor Groove of DNA by an Eight-Ring Hairpin Polyamide

The use of pyrrole−imidazole polyamides for the recognition of core 5‘-GGG-3‘ sequences in the minor groove of double stranded DNA is described. Two hairpin pyrrole−imidazole polyamides, ImImIm-γ-PyPyPy-β-Dp and ImImImPy-γ-PyPyPyPy-β-Dp (Im = N-methylimidazole-2-carboxamide, Py = N-methylpyrrole-2-carboxamide, β = β-alanine, γ = γ-aminobutyric acid, and Dp = ((dimethylamino)propyl)amide), as well as the corresponding EDTA affinity cleaving derivatives, were synthesized and their DNA binding properties analyzed. Quantitative DNase I footprint titrations demonstrate that ImImIm-γ-PyPyPy-β-Dp binds the formal match sequence 5‘-AGGGA-3‘ with an equilibrium association constant of K_a = 5 × 10^6 M^(-1) (10 mM Tris·HCl, 10 mM KCl, 10 mM MgCl_2, and 5 mM CaCl_2, pH 7.0 and 22 °C). ImImImPy-γ-PyPyPyPy-β-Dp binds the same site, 5‘-AGGGAA-3‘, approximately two orders of magnitude more tightly than the six ring polyamide, with an equilibrium association constant of K_a = 4 × 10^8 M^(-1). The eight-ring hairpin polyamide demonstrates greater specifity for single base pair mismatches than does the six ring hairpin. Polyamides with an EDTA·Fe(II) moiety at the carboxy terminus confirm that each hairpin binds in a single orientation. The high affinity recognition of a 5‘-GGG-3‘ core sequence by an eight ring polyamide containing three contiguous imidazole amino acids demonstrates the versatility of pyrrole−imidazole polyamides and broadens the sequence repertoire for DNA recognition.