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.     

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.     

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.     

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.     

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.     

Monovalent cation size and DNA conformational stability

The effect of monovalent cations on the thermal stability of a small model DNA hairpin has been measured by capillary electrophoresis, using an oligomer with 16 thymine residues as an unstructured control. The melting temperature of the model hairpin increases approximately linearly with the logarithm of increasing cation concentration in solutions containing Na(+), K(+), Li(+), NH(4)(+), Tris(+), tetramethylammonium (TMA(+)), or tetraethylammonium (TEA(+)) ions, is approximately independent of cation concentration in solutions containing tetrapropylammonium (TPA(+)) ions, and decreases with the logarithm of increasing cation concentration in solutions containing tetrabutylammonium (TBA(+)) ions. At constant cation concentration, the melting temperature of the DNA model hairpin decreases in the order Li(+) ～ Na(+) ～ K(+) > NH(4)(+) > TMA(+) > Tris(+) > TEA(+) > TPA(+) > TBA(+). Isothermal studies indicate that the decrease in the hairpin melting temperature with increasing cation hydrophobicity is not due to saturable, site-specific binding of the cation to the random coil conformation, but to the concomitant increase in cation size with increasing hydrophobicity. Larger cations are less effective at shielding the charged phosphate residues in B-form DNA because they cannot approach the DNA backbone as closely as smaller cations. By contrast, larger cations are relatively more effective at shielding the phosphate charges in the random coil conformation, where the phosphate-phosphate distance more closely matches cation size. Hydrophobic interactions between alkylammonium ions interacting electrostatically with the phosphate residues in the coil may amplify the effect of cation size on DNA thermal stability.     

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.     

DNA minor-groove recognition by 3-methylthiophene/pyrrole pair

Hairpin polyamides are synthetic oligomers, which fold and bind to specific DNA sequences in a programmable manner. Internal side-by-side pairings of the aromatic amino acid residues 1-methyl-1H-pyrrole (Py), 1-methyl-1H-imidazole (Im), and 3-hydroxy-1-methyl-1H-pyrrole (Hp) confer the ability to distinguish between all four Watson-Crick base pairs in the minor groove of B-form DNA. In a broad search to expand the heterocycle repertoire, we found that when 3-methylthiophene (Tn), which presents a S-atom to the minor groove, is paired with Py, it exhibits a modest threefold specificity for TA>AT presumably by shape-selective recognition. In this study, we explore the scope and limitations of this lead by incorporating multiple Tn residues within a single hairpin polyamide. It was found that hairpin polyamides containing more that one Tn/Py pair exhibit lowered affinities and specificities for their match sites. It appears that little deviation is permissible from the parent five-membered ring 1-methyl-1H-pyrrole-2-carboxamide scaffold for DNA recognition.     

Investigation and improvement of DNA cleavage models of polyamide + Cu(II) nuclease + OOH<Superscript>-</Superscript> ligands bound to DNA

Background  

Copper nucleases as a famous class of artificial metallonucleases have attracted considerable interest in relation to their diverse potentials not only as therapeutic agents but also in genomic researches. Copper nucleases present high efficient oxidative cleavage of DNA, in which DNA strand scission occurs generally after hydrogen atom abstracted from a sugar moiety. In order to achieve the selective cleavage of DNA sequences by copper nucleases, the DNA specific recognition agents of the Dervan-type hairpin and cyclic polyamides can be considered as proper carriers of copper nucleases. Investigation of the DNA cleavage selectivity of copper nucleases assisted by the hairpin and cyclic polyamides at the molecular level has not yet been elucidated.     

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.     

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.     

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.     

Recognition of a 10 base pair sequence of DNA and stereochemical control of the binding affinity of chiral hairpin polyamide-Hoechst 33258 conjugates

Chiral hairpin polyamides linked to a Hoechst 33258 analogue at the -position of the hairpin turn amino acid (1, 2) were synthesized on solid phase by adopting Fmoc and ivDde techniques. The DNA-binding properties of enantiomeric conjugates 1 and 2, and N-terminal linked conjugate 3 for 8–14 bp sequences were determined by spectrofluorometric and thermal melting studies. Conjugates 1 and 2 recognize a 10 bp sequence, while conjugate 3 recognizes a 9 bp sequence. Interestingly, R-enantiomer 1 exhibited 10- to 30-fold higher binding affinities than S-enantiomer 2 for the DNA sequences studied. These binding differences were accounted for by molecular modeling studies, which revealed that the amide proton nearest to the chiral center in R-conjugate 1 is better positioned to form hydrogen bonds to the DNA bases, while S-conjugate 2 does not.     

Sequence-specific covalent labelling of DNA

Sequence-specific DNA modification is of significance for applications in bio- and nano-technology, medical diagnostics and fundamental life sciences research. Preferentially, labelling should be performed covalently, which avoids doubts about label dissociation from the DNA under various conditions. Several methods to label native DNA have been developed in the last two decades. Triple-helix-forming oligodeoxynucleotides and hairpin polyamides that bind DNA sequences specifically in the major and minor groove respectively were used as targeting devices for subsequent covalent labelling. In addition, enzyme-directed labelling approaches utilizing nicking endonucleases in combination with DNA polymerases or DNA methyltransferases have been employed. This review summarizes various techniques useful for functionalization of long native DNA.     

The effects of loop size on Sac7d-hairpin DNA interactions

Hairpin structure is a common feature of DNA molecules. They are located near functional loci, such as regulation and promotion sites, as well as in cruciform structures, and they provide potential binding sites for endogenous proteins. The effects of different hairpin loops that are composed of one to five thymidines, designated as L1-L5, and have a common self-complementary stem, CTATATAG, on the interactions with Sac7d were studied. In thermostability studies, Sac7d stabilized a tetra-loop hairpin DNA and hairpin DNA with GTTC tetra-loop regions better than it stabilized tri- and penta-loops. Circular dichroism (CD) spectra showed that hairpins retained primarily a B-type conformation upon Sac7d binding. Intermolecular interactions between hairpins were likely decreased, due to the Sac7d-induced kinks, as shown by an increase at 220nm in the CD spectra. Surface plasmon resonance (SPR) observations suggested that the rates of Sac7d binding to hairpin DNA depend on the loop size of the hairpin duplexes. At a fixed stem length, Sac7d binds to tetra-loop hairpin DNA duplexes with a higher association rate and lower dissociation rate, compared with their tri- and penta-loop counterparts. In addition, the tri-loop and GTC tri-loop hairpin DNA had lower affinity for Sac7d because of the smaller and tighter loop size. Our study indicates that Sac7d binding affinity to hairpin DNA is primarily determined by loop size and stem integrity, and the results presented here provide a model for studies concerning other minor groove DNA-binding proteins that kink hairpin DNA.     

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.