Search

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.

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.

Pyrrole-imidazole polyamides are ligands that bind in the minor groove of DNA with high affinity and sequence selectivity. Molecules of this class have been shown to disrupt specific transcription factor-DNA interactions and to inhibit basal and activated transcription from various RNA polymerase II and III promoters. A set of eight-ring hairpin-motif pyrrole-imidazole polyamides has been designed to bind within the binding site for the human cytomegalovirus (CMV) UL122 immediate early protein 2 (IE86). IE86 represses transcription of the CMV major immediate early promoter (MIEP) through its cognate cis recognition sequence (crs) located between the TATA box and the transcription initiation site. The designed polyamides bind to their target DNA sequence with nanomolar affinities and with a high degree of sequence selectivity. The polyamides effectively block binding of IE86 protein to the crs in DNase I footprinting experiments. A mismatch polyamide, containing a single imidazole to pyrrole substitution, and also a polyamide binding to a site located 14 base pairs upstream of the repressor binding site, do not prevent IE86 binding to the crs. IE86-mediated transcriptional repression in vitro is relieved by a match polyamide but not by a mismatch polyamide. Transcription from a DNA template harboring a mutation in the crs is not affected either by IE86 protein or by the match polyamides. These results demonstrate that this new class of small molecules, the pyrrole-imidazole polyamides, are not only effective inhibitors of basal and activated transcription, but also can be used to activate transcription by blocking the DNA-binding activity of a repressor protein.

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.

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.

The design of synthetic ligands that read the information stored in the DNA double helix has been a long-standing goal at the interface of chemistry and biology. Cell-permeable small molecules that target predetermined DNA sequences offer a potential approach 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 analogues have been shown to interfere with gene expression, the triple-helix approach is limited to recognition of purines and suffers from poor cellular uptake. The subsequent development of pairing rules for minor-groove binding polyamides containing pyrrole (Py) and imidazole (Im) amino acids offers a second code to control sequence specificity. An Im/Py pair distinguishes G x C from C x G and both of these from A x T/T x A base pairs. A Py/Py pair specifies A,T from G,C but does not distinguish AT from T x A. To break this degeneracy, we have added a new aromatic amino acid, 3-hydroxypyrrole (Hp), to the repertoire to test for pairings that discriminate A x T from T x A. We find that replacement of a single hydrogen atom with a hydroxy group in a Hp/Py pairing regulates affinity and specificity by an order of magnitude. By incorporation of this third amino acid, hydroxypyrrole-imidazole-pyrrole polyamides form four ring-pairings (Im/Py, Py/Im, Hp/Py and Py/Hp) which distinguish all four Watson-Crick base pairs in the minor groove of DNA.

Background: Cell-permeable small molecules that target predetermined DNA sequences with high affinity and specificity have the potential to control gene expression. A binary code has been developed to correlate DNA sequence with side-by-side pairings between N-methylpyrrole (Py) and N-methylimidazole (Im) carboxamides in the DNA minor groove. We set out to determine the relative energetics of pairings of Im/Py, Py/Im, Im/Im, and Py/Py for targeting G.C and A.T base pairs. A key specificity issue, which has not been previously addressed, is whether an Im/Im pair is energetically equivalent to an Im/Py pair for targeting G.C base pairs., Results: Equilibrium association constants were determined at two five-base-pair sites for a series of four six-ring hairpin polyamides, in order to test the relative energetics of the four aromatic amino-acid pairings opposite G.C and A.T base pairs in the central position. We observed that a G.C base pair was effectively targeted with Im/Py but not Py/Im, Py/Py, or Im/Im. The A.T base pair was effectively targeted with Py/Py but not Im/Py, Py/Im, or Im/Im., Conclusions: An Im/Im pairing is energetically disfavored for the recognition of both A.T and G.C. This specificity will create important limitations on undesirable slipped motifs that are available for unlinked dimers in the minor groove. Baseline energetic parameters will thus be created which, using the predictability of the current pairing rules for specific molecular recognition of double-helical DNA, will guide further second-generation polyamide design for DNA recognition.

Small molecules that target specific DNA sequences have the potential to control gene expression. Ligands designed for therapeutic application must bind any predetermined DNA sequence with high affinity and permeate living cells. Synthetic polyamides containing N-methylimidazole and N-methylpyrrole amino acids have an affinity and specificity for DNA comparable to naturally occurring DNA-binding proteins. We report here that an eight-ring polyamide targeted to a specific region of the transcription factor TFIIIA binding site interferes with 5S RNA gene expression in Xenopus kidney cells. Our results indicate that pyrrole-imidazole polyamides are cell-permeable and can inhibit the transcription of specific genes.

Pairing rules have been developed to predict the sequence specificity of minor groove binding polyamides containing pyrrole (Py) and imidazole (Im) amino acids. An Im/Py pair distinguishes G.C from C.G and both of these from A.T/T.A base pairs. A Py/Py pair appears not to distinguish A.T from T.A base pairs. To test the extent of this degeneracy, the affinity and binding orientation of the hairpin polyamide ImPyPy-gamma-PyPyPy-beta-Dp were measured for eight possible five base pair 5'-TG(A,T)(3)-3' match sites. Affinity cleavage experiments using a polyamide with an EDTA.Fe(II) moiety at the carboxy terminus, ImPyPy-gamma-PyPyPy-beta-Dp-EDTA.Fe(II), are consistent with formation of an oriented 1:1 hairpin polyamide complex at all eight 5'-TG(A,T)(3)-3' binding sites [20 mM HEPES, 200 mM NaCl, 50 mg/ml glycogen, pH 7.0, 22 degrees C, 5 mM DTT, 1 mM Fe(II)]. Quantitative DNase I footprint titration experiments reveal that ImPyPy-gamma-PyPyPy-beta-Dp binds all eight 5'-TG(A,T)(3)-3' target sites with only a 12-fold difference in the equilibrium association constants between the strongest site, 5'-TGTTT-3' (Ka = 2.1 x 10(8) M-1), and the weakest site, 5'-TGAAT-3' (Ka = 1.8 x 10(7) M-1) (10 mM Tris.HCl, 10 mM KCl, 10 mM MgCl2, 5 mM CaCl2, pH 7.0, 22 degrees C). This relatively small range indicates that the Py/Py pair is approximately degenerate for recognition of A,T base pairs, affording generality with regard to targeting sequences of mixed A.T/T.A composition.

Background: Three-ring polyamides containing N-methylimidazole and N-methylpyrrole amino acids bind sequence-specifically to double-helical DNA by forming side-by-side complexes in the minor groove. Simple pairing rules relate the amino-acid sequence of a pyrrole-imidazole polyamide to its expected DNA target site, and polyamides that target a wide variety of DNA sequences have been synthesized. We have shown previously that two three-ring subunits could be linked together by an aliphatic amino acid, increasing the binding affinity of the polyamide and, in some cases, increasing the length of the target sequence. We set out to determine whether different types of linkers could be used in a single molecule to generate a nine-ring polyamide molecule that would bind to specific DNA sequences., Results: A nine-ring pyrrole-imidazole polyamide, containing two different amino acid linkers, beta-alanine and gamma-aminobutyric acid, has been synthesized and shown to specifically bind a designated nine-base-pair target site at subnanomolar concentration in a novel extended hairpin conformation., Conclusions: The amino acids gamma-aminobutyric acid and beta-alanine optimally link three-ring pyrrole-imidazole subunits in 'hairpin' and 'extended' conformations, respectively. Both aliphatic amino acids can be combined to generate a nine-ring polyamide that specifically recognizes a nine-base-pair target site with very high affinity.