Aliphatic/Aromatic Amino Acid Pairings for Polyamide Recognition in the Minor Groove of DNA

Abstract

Selective placement of an aliphatic β-alanine (β) residue paired side-by-side with either a pyrrole (Py) or imidazole (Im) aromatic amino acid is found to compensate for sequence composition effects for recognition of the minor groove of DNA by hairpin pyrrole−imidazole polyamides. A series of polyamides were prepared which contain pyrrole and imidazole aromatic amino acids, as well as γ-aminobutyric acid (γ) “turn” and β-alanine “spring” aliphatic amino acid residues. The binding affinities and specificities of these polyamides are regulated by the placement of paired β/β, Py/β, and Im/β residues. Quantitative footprint titrations demonstrate that replacing two Py/Py pairings in a 12-ring hairpin (6-γ-6) with two Py/β pairings affords 10-fold enhanced affinity and similar sequence specificity for an 8-bp target sequence. The 6-γ-6 hairpin ImPyImPyPyPy-γ-ImPyPyPyPyPy-β-Dp, which contains six consecutive amino acid pairings, is unable to discriminate a single-base-pair mismatch site 5‘-TGT T AACA-3‘ from a 5‘-TGTGAACA-3‘ match site. The hairpin polyamide Im-β-ImPyPyPy-γ-ImPyPyPy-β-Py-β-Dp binds to the 8-bp match sequence 5‘-TGTGAACA-3‘ with an equilibrium association constant of K a = 2.4 × 1010 M-1 and ≥48-fold specificity versus the 5‘-TGT T AACA-3‘ single-base-pair mismatch site. Modeling indicates that the β-alanine residue relaxes ligand curvature, providing for optimal hydrogen bond formation between the floor of the minor groove and both Im residues within the Im-β-Im polyamide subunit. This observation provided the basis for design of a hairpin polyamide, Im-β-ImPy-γ-Im-β-ImPy-β-Dp, which incorporates Im/β pairings to recognize a “problematic” 5‘-GCGC-3‘ sequence at subnanomolar concentrations. These results identify Im/β and β/Im pairings that respectively discriminate G·C and C·G from A·T/T·A as well as Py/β and β/Py pairings that discriminate A·T/T·A from G·C/C·G. These aliphatic/aromatic amino acid pairings will facilitate the design of hairpin polyamides which recognize both a larger binding site size as well as a more diverse sequence repertoire.

Structure of a β-Alanine-linked Polyamide Bound to a Full Helical Turn of Purine Tract DNA in the 1:1 Motif

“…needed to reset the register for the following (C-terminal) residue. 21 It appears that the b residue is necessary for the two flanking Im residues in Im -b-Im to orient properly in order to form hydrogen bonds. The Im -b-Py subunit forms only one Im·G hydrogen bond, and therefore the flexibility is not as critical.…”

“…The aliphatic amino acid b-alanine (b), previously used to relax the over-curvature of long contiguous aromatic subunits, 18 -20 was incorporated between imidazole groups as Im -b-Im to target 5 0 -GNG-3 0 successfully. 21 In a breakthrough report, the 1:1 motif was revived by Laemmli and co-workers and used to effect phenotypic changes in Drosophila melanogaster by binding to 5 0 -GAGAA-3 0 repeats in satellite regions of the fly genome. 22 In the Laemmli design, b-alanine residues were used to add flexibility between ring subunits, allowing the Im residues to register optimally with the DNA helix.…”

Inhibition of Ets-1 DNA Binding and Ternary Complex Formation between Ets-1, NF-κB, and DNA by a Designed DNA-binding Ligand

“…Sequence-specific DNA recognition depends on side-byside pairing of pyrrole (Py) 1 and imidazole (Im) amino acids; a Py opposite an Im targets a C⅐G base pair, and an Im opposite a Py targets a G⅐C base pair (1,2). Py/Py, Py/␤-alanine (␤), and ␤/␤ pairs binds both A⅐T and T⅐A base pairs (2,3). Recent studies have shown that A/T degeneracy can be overcome by replacing one pyrrole ring of the Py/Py combination with 3-hydroxypyrrole (Hp), with the result that the Hp/Py prefers T⅐A over A⅐T base pairs (4).…”

Sequence-specific DNA binding by long hairpin pyrrole–imidazole polyamides containing an 8-amino-3,6-dioxaoctanoic acid unit

“…Various types of sequencespecific Py-Im polyamides have been developed to control gene expression by competitive binding against DNA-binding proteins such as transcription factor. [7][8][9][10][11][12][13][14][15][16][17][18] The introduction of a -alanine moiety within the hairpin motif allows the crescent-shaped Py-Im polyamides to fit into the minor groove of dsDNA, and antiparallel pairings of /, Py/-pair recognize both T-A and A-T. 19 In general, the -linker was set in the hairpin motif to extend the DNA sequence recognition. Although long hairpin Py-Im polyamides have interesting biological properties, they tend to  Corresponding author.…”

“…Polyamide 1 is a standard design with one -alanine inserted within the structure to relax the distortion between three rings. 19 Polyamides 2 and 3 each has one AO unit in either the N-or C-side strand, respectively. Notably, the introduction of the AO unit can decrease the number of coupling steps because the moiety covers two base pairs.…”