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Synthetic polyamides composed of three types of aromatic amino acids, N-methylimidazole (Im), N-methylpyrrole (Py) and N-methyl-3-hydroxypyrrole (Hp) bind specific DNA sequences as antiparallel dimers in the minor groove. The side-by-side pairings of aromatic rings in the dimer afford a general recognition code that allows all four base-pairs to be distinguished. To examine the structural consequences of changing the DNA sequence context on T.A recognition by Hp/Py pairs in the minor groove, crystal structures of polyamide dimers (ImPyHpPy)(2) and the pyrrole counterpart (ImPyPyPy)(2) bound to the six base-pair target site 5'-AGATCT-3' in a ten base-pair oligonucleotide have been determined to a resolution of 2.27 and 2.15 A, respectively. The structures demonstrate that the principles of Hp/Py recognition of T.A are consistent between different sequence contexts. However, a general structural explanation for the non-additive reduction in binding affinity due to introduction of the hydroxyl group is less clear. Comparison with other polyamide-DNA cocrystal structures reveals structural themes and differences that may relate to sequence preference., (Copyright 2000 Academic Press.)

The human T-cell leukemia virus type-1 (HTLV-I)-encoded Tax protein enhances viral gene transcription through interaction with three repeated DNA elements located in the viral promoter. These elements, called viral CREs, are composed of an off-consensus eight base-pair cyclic AMP response element (CRE), immediately flanked by sequences that are rich in guanine and cytosine residues. Recent biochemical experiments have demonstrated that in the presence of the cellular protein CREB, Tax directly binds the viral CRE G+C-rich sequences via interaction with the minor groove. To determine the functional significance of the Tax-DNA interaction, we synthesized minor groove-binding pyrrole-imidazole polyamides which bind specifically to the G+C-rich sequences in the viral CREs. At concentrations where the polyamides specifically protect the G+C-rich sequences from MPE:Fe cleavage, the polyamides block the Tax-DNA interaction. At precisely these same concentrations, the polyamides specifically inhibit Tax transactivation in vitro, without altering CREB-activated transcription or basal transcription from the same promoter. Together, these data provide strong evidence that Tax-viral CRE interaction is essential for Tax function in vitro, and suggest that targeted disruption of the Tax-DNA minor groove interaction with polyamides may provide a novel approach for inhibiting viral replication in vivo., (Copyright 1999 Academic Press.)

Transcription factor IIIB (TFIIIB) is composed of the TATA box binding protein (TBP) and class III gene-specific TBP-associated factors (TAFs). TFIIIB is brought to a site centered approximately 35 bp upstream from the transcription start site of tRNA genes via protein-protein interactions with the intragenic promoter-recognition factor TFIIIC. Since TBP interacts with TATA elements through the minor groove of DNA, we asked whether TFIIIB interacts with DNA in the minor groove. Polyamides containing pyrrole (Py) and imidazole (Im) amino acids are synthetic DNA ligands that bind to predetermined sequences in the minor groove of double helical DNA. These small molecules have been shown to interfere with protein-DNA interactions in the minor groove. A series of DNA constructs was generated in which the binding site for a Py-Im polyamide was placed at various distances upstream from a tRNA gene transcription start site. We find that a match polyamide will effectively inhibit tRNA gene transcription when its binding site is located within 33 bp of the transcription start site of the Xenopus TyrD tRNA gene. Moreover, in the presence of polyamide, RNA polymerase III is redirected to a new transcription initiation site located approximately one DNA helical turn downstream from the native start site. Our results suggest that a subunit of TFIIIB, possibly TBP, makes an essential minor groove DNA contact centered approximately 30 bp upstream from the tRNA gene., (Copyright 1999 Academic Press.)

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 gene-specific transcription factor IIIA (TFIIIA) binds to the internal promoter element of the 5 S rRNA gene through nine zinc fingers which make specific DNA contacts. Seven of the nine TFIIIA zinc fingers participate in major groove DNA contacts while two fingers, 4 and 6, have been proposed to bind in or across the minor groove. Pyrrole-imidazole polyamides are minor groove binding ligands that recognize predetermined DNA sequences with affinity and specificity comparable to natural DNA-binding proteins. We have examined the DNA binding activity of nine finger TFIIIA and shorter recombinant analogs in the presence of polyamides that bind six base-pair sequences (Kd = 0.03 to 1.7 nM) in the minor groove of the binding site for zinc finger 4. DNase I footprint titrations demonstrate that the polyamides and a recombinant protein containing the three amino-terminal zinc fingers of TFIIIA (zf1-3) co-occupy the TFIIIA binding site, in agreement with the known location of zf1-3 in the major groove. In contrast, the polyamides block the specific interaction of TFIIIA or zf1-4 with the 5 S RNA gene, supporting a model for minor groove occupancy by zinc finger 4. Minor groove binding polyamides targeted to specific DNA sequences may provide a novel chemical approach to probing multidomain protein-DNA interactions.

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.

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.