Solid-phase synthesis of DNA binding polyamides on oxime resin

Control of the energetics and specificity of DNA binding polyamides is necessary for inhibition of protein-DNA complex formation and gene regulation studies. Typically, solid-phase methods using Boc monomers for synthesis have depended on Boc-beta-Ala-PAM resin which affords a beta-alanine-Dp tail at the C-terminus, after cleavage with N,N-dimethylaminopropylamine (Dp). To address the energetic consequences of this tail for DNA minor groove binding, we describe an alternative solid phase method employing the Kaiser oxime resin which allows the synthesis of polyamides with incrementally shortened C-terminal tails. Polyamides without Dp and having methyl amide tails rather than beta-alanine show similar affinity relative to the standard beta-Dp tail. The truncated tail diminishes the A,T base pair energetic preference of the beta-Dp tail which will allow a greater variety of DNA sequences to be targeted by hairpin polyamides.     

Combinatorial determination of sequence specificity for nanomolar DNA-binding hairpin polyamides

Development of sequence-specific DNA-binding drugs is an important pharmacological goal, given the fact that numerous existing DNA-directed chemotherapeutic drugs rely on the strength and selectivity of their DNA interactions for therapeutic activity. Among the DNA-binding antibiotics, hairpin polyamides represent the only class of small molecules that can practically bind any predetermined DNA sequence. DNA recognition by these ligands depends on their side-by-side amino acid pairings in the DNA minor groove. Extensive studies have revealed that these molecules show extremely high affinity for sequence-directed, minor groove interaction. However, the specificity of such interactions in the presence of a large selection of sequences such as the human genome is not known. We used the combinatorial selection method restriction endonuclease protection, selection, and amplification (REPSA) to determine the DNA binding specificity of two hairpin polyamides, ImPyPyPy-gamma-PyPyPyPy-beta-Dp and ImPyPyPy-gamma-ImPyPyPy-beta-Dp, in the presence of more than 134 million different sequences. These were verified by restriction endonuclease protection assays and DNase I footprinting analysis. Our data showed that both hairpin polyamides preferentially selected DNA sequences having consensus recognition sites as defined by the Dervan pairing rules. These consensus sequences were rather degenerate, as expected, given that the stacked pyrrole-pyrrole amino acid pairs present in both polyamides are unable to discriminate between A.T and T.A base pairs. However, no individual sequence within these degenerate consensus sequences was preferentially selected by REPSA, indicating that these hairpin polyamides are truly consensus-specific DNA-binding ligands. We also discovered a preference for overlapping consensus binding sites among the sequences selected by the hairpin polyamide ImPyPyPy-gamma-PyPyPyPy-beta-Dp, and confirmed by DNase I footprinting that these complex sites provide higher binding affinity. These data suggest that multiple hairpin polyamides can cooperatively bind to their highest-affinity sites.     

Novel diamino imidazole and pyrrole-containing polyamides: Synthesis and DNA binding studies of mono- and diamino-phenyl-ImPy*Im polyamides designed to target 5'-ACGCGT-3'

Pyrrole- and imidazole-containing polyamides are widely investigated as DNA sequence selective binding agents that have potential use as gene control agents. The key challenges that must be overcome to realize this goal is the development of polyamides with low molar mass so the molecules can readily diffuse into cells and concentrate in the nucleus. In addition, the molecules must have appreciable water solubility, bind DNA sequence specifically, and with high affinity. It is on this basis that the orthogonally positioned diamino/dicationic polyamide Ph-ImPy*Im 5 was designed to target the sequence 5'-ACGCGT-3'. Py* denotes the pyrrole unit that contains a N-substituted aminopropyl pendant group. The DNA binding properties of diamino polyamide 5 were determined using a number of techniques including CD, ΔT(M), DNase I footprinting, SPR and ITC studies. The effects of the second amino moiety in Py* on DNA binding affinity over its monoamino counterpart Ph-ImPyIm 3 were assessed by conducting DNA binding studies of 3 in parallel with 5. The results confirmed the minor groove binding and selectivity of both polyamides for the cognate sequence 5'-ACGCGT-3'. The diamino/dicationic polyamide 5 showed enhanced binding affinity and higher solubility in aqueous media over its monoamino/monocationic counterpart Ph-ImPyIm 3. The binding constant of 5, determined from SPR studies, was found to be 1.5 × 10(7)M(-1), which is ～3 times higher than that for its monoamino analog 3 (4.8 × 10(6)M(-1)). The affinity of 5 is now approaching that of the parent compound f-ImPyIm 1 and its diamino equivalent 4. The advantages of the design of diamino polyamide 5 over 1 and 4 are its sequence specificity and the ease of synthesis compared to the N-terminus pyrrole analog 2.     

Polyamide recognition-mass spectrometry for distinguishing hairpin DNA from coil DNA

The discrimination between hairpin DNA and coil DNA has been well achieved through polyamides as probes by electrospray ionization (ESI) mass spectrometry. ESI mass spectra showed that polyamides bind to hairpin DNA with high selectivity, and almost no binding with coil DNA. In addition, the noncovalent interaction between polyamides and hairpin DNA was also studied; the results show that hairpin DNA with longer stem and polyamides with more heterocycles have higher binding affinity and stability in gas phase.     

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.     

DNA sequence selectivity of hairpin polyamide turn units

A class of hairpin polyamides linked by 3,4-diaminobutyric acid, resulting in a β-amine residue at the turn unit, showed improved binding affinities relative to their α-amino-γ-turn analogs for particular sequences. We incorporated β-amino-γ-turns in six-ring polyamides and determined whether there are any sequence preferences under the turn unit by quantitative footprinting titrations. Although there was an energetic penalty for G·C and C·G base pairs, we found little preference for T·A over A·T at the β-amino-γ-turn position. Fluorine and hydroxyl substituted α-amino-γ-turns were synthesized for comparison. Their binding affinities and specificities in the context of six-ring polyamides demonstrated overall diminished affinity and no additional specificity at the turn position. We anticipate that this study will be a baseline for further investigation of the turn subunit as a recognition element for the DNA minor groove.     

Kinetic consequences of covalent linkage of DNA binding polyamides

Polyamides composed of N-methylpyrrole (Py) and N-methylimidazole (Im) subunits can bind in the minor groove of DNA at predetermined sequences with subnanomolar affinity and high specificity. Covalent linkage of polymer subunits using a gamma-aminobutyric acid linker has been shown to increase both the affinity and specificity of polyamides. Using a fluorescence detected stopped-flow assay, we have studied the differences in association and dissociation kinetics of a series of polyamides representing unlinked, hairpin and cyclic analogues of the four ring polyamide ImPyPyPy-beta-Dp. Whereas the large differences seen in the equilibrium association constants between the unlinked and covalently linked polyamides are primarily due to higher association rate constants, discrimination between matched and mismatched sites by each polyamide can be ascribed in large part to differences in their dissociation rate constants. The consequences of this kinetic behavior for future design are discussed.     

Alpha-diaminobutyric acid-linked hairpin polyamides

A hairpin polyamide-chlorambucil conjugate linked by alpha-diaminobutyric acid (alpha-DABA) has been shown to have interesting biological properties in cellular and small animal models. Remarkably, this new class of hairpin polyamides has not been previously characterized with regard to energetics and sequence specificity. Herein we present a series of pyrrole-imidazole hairpin polyamides linked by alpha-DABA and compare them to polyamides containing the standard gamma-DABA turn unit. The alpha-DABA hairpins have overall decreased binding affinities. However, alpha-DABA polyamide-chlorambucil conjugates are sequence-specific DNA alkylators with increased specificities. Affinity cleavage studies of alpha-DABA polyamide-EDTA conjugates confirmed their preference for binding DNA in a forward hairpin conformation. In contrast, an unsubstituted glycine-linked polyamide prefers to bind in an extended binding mode. Thus, substitution on the turn unit locks the alpha-DABA polyamide into the forward hairpin binding motif.     

Sequence specific and high affinity recognition of 5'-ACGCGT-3' by rationally designed pyrrole-imidazole H-pin polyamides: thermodynamic and structural studies

Imidazole (Im) and Pyrrole (Py)-containing polyamides that can form stacked dimers can be programmed to target specific sequences in the minor groove of DNA and control gene expression. Even though various designs of polyamides have been thoroughly investigated for DNA sequence recognition, the use of H-pin polyamides (covalently cross-linked polyamides) has not received as much attention. Therefore, experiments were designed to systematically investigate the DNA recognition properties of two symmetrical H-pin polyamides composed of PyImPyIm (5) or f-ImPyIm (3e, f=formamido) tethered with an ethylene glycol linker. These compounds were created to recognize the cognate 5'-ACGCGT-3' through an overlapped and staggered binding motif, respectively. Results from DNaseI footprinting, thermal denaturation, circular dichroism, surface plasmon resonance and isothermal titration microcalorimetry studies demonstrated that both H-pin polyamides bound with higher affinity than their respective monomers. The binding affinity of formamido-containing H-pin 3e was more than a hundred times greater than that for the tetraamide H-pin 5, demonstrating the importance of having a formamido group and the staggered motif in enhancing affinity. However, compared to H-pin 3e, tetraamide H-pin 5 demonstrated superior binding preference for the cognate sequence over its non-cognates, ACCGGT and AAATTT. Data from SPR experiments yielded binding constants of 1.6x10(8)M(-1) and 2.0x10(10)M(-1) for PyImPyIm H-pin 5 and f-ImPyIm H-pin 3e, respectively. Both H-pins bound with significantly higher affinity (ca. 100-fold) than their corresponding unlinked PyImPyIm 4 and f-ImPyIm 2 counterparts. ITC analyses revealed modest enthalpies of reactions at 298 K (DeltaH of -3.3 and -1.0 kcal mol(-1) for 5 and 3e, respectively), indicating these were entropic-driven interactions. The heat capacities (DeltaC(p)) were determined to be -116 and -499 cal mol(-1)K(-1), respectively. These results are in general agreement with DeltaC(p) values determined from changes in the solvent accessible surface areas using complexes of the H-pins bound to (5'-CCACGCGTGG)(2). According to the models, the H-pins fit snugly in the minor groove and the linker comfortably holds both polyamide portions in place, with the oxygen atoms pointing into the solvent. In summary, the H-pin polyamide provides an important molecular design motif for the discovery of future generations of programmable small molecules capable of binding to target DNA sequences with high affinity and selectivity.     

DNA sequence recognition of thiazole-containing cross-linked polyamides can be favored

The binding ability of cross-linked thiazolated polyamides (containing the base sequence-reading elements thiazole(Th)-pyrrole(Py)-pyr-role(Py) and thiazole(Th)-imidazole(Im)-pyrrol(Py) to various DNA dodecamers has been investigated. CD titration experiments at high salt concentration demonstrate that the dimers with a heptanediyl linker (C7 dimer) show a significantly higher sequence specificity than their corresponding monomers. The dimer of Th-Py-Py primarily prefers binding to pure AT sequences and that of Th-Im-Py to the dodecamer sequences containing a GC pair within the central sequence (e.g. AACGTT). Surprisingly, the sequence binding ability is strongly influenced by the presence of a T-A step: e.g. Th-Py-Py has a similar affinity to the sequences TTTAAA and ATCGTA; likewise Th-Im-Py shows a preference for these sequences. The CD results correlate with footprinting data. Related biochemical studies on the effect of polyamides on DNA gyrase activity in vitro show that the C7 dimers most effectively inhibit the enzyme activity compared with the monomers and the natural reference minor groove binder distamycin. The highest inhibitory potency is observed for the Th-Py-Py-dimer. The role of the T-A step in binding of the cross-linked dimer to the minor groove is discussed in light of the sequence recognition of the TATA box binding protein.     

Specific targeting of insect and vertebrate telomeres with pyrrole and imidazole polyamides.

DNA minor groove-binding compounds (polyamides) that target insect and vertebrate telomeric repeats with high specificity were synthesized. Base pair recognition of these polyamides is based on the presence of the heterocyclic amino acids pyrrole and imidazole. One compound (TH52B) interacts uniquely and with excellent specificity (K(d) = 0.12 nM) with two consecutive insect-type telomeric repeats (TTAGG). A related compound, TH59, displays high specificity (K(d) = 0.5 nM) for tandem vertebrate (TTAGGG) and insect telomeric repeats. The high affinity and specificity of these compounds were achieved by bidentate binding of two flexibly linked DNA-binding moieties. Epifluorescence microscopy studies show that fluorescent derivatives of TH52B and TH59 stain insect or vertebrate telomeres of chromosomes and nuclei sharply. Importantly, the telomere-specific polyamide signals of HeLa chromosomes co-localize with the immunofluorescence signals of the telomere-binding protein TRF1. Our results demonstrate that telomere-specific compounds allow rapid estimation of relative telomere length. The insect-specific compound TH52 was shown to be incorporated rapidly into growing Sf9 cells, underlining the potential of these compounds for telomere biology and possibly human medicine.     

Alternative heterocycles for DNA recognition: an N-methylpyrazole/N-methylpyrrole pair specifies for A.T/T.A base pairs

Side-by-side pairs of three five-membered rings, N-methylpyrrole (Py), N-methylimidazole (Im), and N-methylhydroxy-pyrrole (Hp), have been demonstrated to distinguish each of the four Watson Crick base pairs in the minor groove of DNA. However, not all DNA sequences targeted by these pairing rules achieve affinities and specificities comparable to DNA binding proteins. We have initiated a search for new heterocycles which can expand the sequence repetoire currently available. Two heterocyclic aromatic amino acids. N-methylpyrazole (Pz) and 4-methylthiazole (Th), were incorporated into a single position of an eight-ring polyamide of sequence ImImXPy-gamma-lmPyPyPy-beta-Dp to examine the modulation of affinity and specificity for DNA binding by a Pz/Py pair and or a Th/Py pair. The X/Py pairings Pz/Py and Th/Py were evaluated by quantitative DNase I footprint titrations on a DNA fragment with the four sites 5'-TGGNCA-3' (N=T, A, G, C). The Pz/Py pair binds T.A and A.T with similar affinity to a Py/Py pair but with improved specificity. disfavoring both G.C and C.G by about 100-fold. The Th/Py pair binds poorly to all four Watson Crick base pairs. These results demonstrate that in some instances new heterocyclic aromatic amino acid pairs can be incorporated into imidazole-pyrrole polyamides to mimic the DNA specificity of Py/Py pairs which may be relevant as biological criteria in animal studies become important.     

Binding of hairpin pyrrole and imidazole polyamides to DNA: relationship between torsion angle and association rate constants

N-methylpyrrole (Py)-N-methylimidazole (Im) polyamides are small organic molecules that bind to DNA with sequence specificity and can be used as synthetic DNA-binding ligands. In this study, five hairpin eight-ring Py–Im polyamides 1–5 with different number of Im rings were synthesized, and their binding behaviour was investigated with surface plasmon resonance assay. It was found that association rate (k_a) of the Py–Im polyamides with their target DNA decreased with the number of Im in the Py–Im polyamides. The structures of four-ring Py–Im polyamides derived from density functional theory revealed that the dihedral angle of the Py amide carbonyl is 14∼18°, whereas that of the Im is significantly smaller. As the minor groove of DNA has a helical structure, planar Py–Im polyamides need to change their conformation to fit it upon binding to the minor groove. The data explain that an increase in planarity of Py–Im polyamide induced by the incorporation of Im reduces the association rate of Py–Im polyamides. This fundamental knowledge of the binding of Py–Im polyamides to DNA will facilitate the design of hairpin Py–Im polyamides as synthetic DNA-binding modules.     

Comprehensive high-resolution analysis of hairpin polyamides utilizing a fluorescent intercalator displacement (FID) assay

Four hairpin polyamides bearing subtle N- and C-terminal substitutions were examined in a fluorescent intercalator displacement (FID) assay enlisting a library of 512 DNA hairpins that contain all possible five base pair sequences in a challenging probe of its capabilities for establishing DNA binding sequence selectivity. Not only did the assay define the global sequence selectivity expected based on known structural interactions and Dervan's pairing rules establishing the utility of the method for characterizing such polyamides, but previously unappreciated subtle substituent effects on global sequence selectivity were also revealed. Thus, we report the discovery of a novel five base pair high affinity binding site of the form 5'-WWCWW (vs 5'-WGWWW) for the polyamide ImPyPy-gamma-PyPyPy-beta-Dp and its structural basis.     

Orientation preferences of hairpin pyrrole–imidazole polyamides toward mCGG site

Hairpin pyrrole–imidazole (Py-Im) polyamides are promising medium-sized molecules that bind sequence-specifically to the minor groove of B-form DNA. Here, we synthesized a series of hairpin Py-Im polyamides and explored their binding affinities and orientation preferences to methylated DNA with the ^mCGG target sequence. Thermal denaturation assays revealed that the five hairpin Py-Im polyamides, which were anticipated to recognize ^mCGG in a forward orientation, bind to nontarget DNA, GG^mC, in a reverse orientation. Therefore, we designed five Py-Im polyamides that could recognize ^mCGG in a reverse orientation. We found that the two Py-Im polyamides containing Im/β pairs preferentially bound to ^mCGG in a reverse orientation. The reverse binding Py-Im polyamide successfully inhibited TET1 binding on the methylated DNA. Taken together, this study illustrated the importance of designing reverse binding Py-Im polyamides for the target sequence, ^mCGG, which paved the way for Py-Im polyamides that can be used with otherwise difficult to access DNA with CG sequences.     

The thermodynamics of polyamide-DNA recognition: hairpin polyamide binding in the minor groove of duplex DNA

Crescent-shaped synthetic ligands containing aromatic amino acids have been designed for specific recognition of predetermined DNA sequences in the minor groove of DNA. Simple rules have been developed that relate the side-by-side pairings of Imidazole (Im) and Pyrrole (Py) amino acids to their predicted target DNA sequences. We report here thermodynamic characterization of the DNA-binding properties of the six-ring hairpin polyamide, ImImPy-gamma-PyPyPy-beta-Dp (where gamma = gamma-aminobutyric acid, beta = beta-alanine, and Dp = dimethylaminopropylamide). Our data reveal that, at 20 degrees C, this ligand binds with a relatively modest 1.8-fold preference for the designated match site, 5'-TGGTA-3', over the single base pair mismatch site, 5'-TGTTA-3'. By contrast, we find that the ligand exhibits a 102-fold greater affinity for its designated match site relative to the double base pair mismatch site, 5'-TATTA-3'. These results demonstrate that the energetic cost of binding to a double mismatch site is not necessarily equal to twice the energetic cost of binding to a single mismatch site. Our calorimetrically measured binding enthalpies and calculated entropy data at 20 degrees C reveal the ligand sequence specificity to be enthalpic in origin. We have compared the DNA-binding properties of ImImPy-gamma-PyPyPy-beta-Dp with the hairpin polyamide, ImPyPy-gamma-PyPyPy-beta-Dp (an Im --> Py "mutant"). Our data reveal that both ligands exhibit high affinities for their designated match sites, consistent with the Dervan pairing rules. Our data also reveal that, relative to their corresponding single mismatch sites, ImImPy-gamma-PyPyPy-beta-Dp is less selective than ImPyPy-gamma-PyPyPy-beta-Dp for its designated match site. This result suggests, at least in this case, that enhanced binding affinity can be accompanied by some loss in sequence specificity. Such systematic comparative studies allow us to begin to establish the thermodynamic database required for the rational design of synthetic polyamides with predictable DNA-binding affinities and specificities.