Aliphatic/aromatic amino acid pairings for polyamide recognition in the minor groove of DNA

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‘-TGTTAACA-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 Ka = 2.4 × 1010 M-1 and ≥48-fold specificity versus the 5‘-TGTTAACA-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

Recognition of the Minor Groove of DNA by Hairpin Polyamides Containing α-Substituted-β-Amino Acids

Incorporation of the flexible amino acid β-alanine (β) into hairpin polyamides composed of N-methylpyrrole (Py) and N-methylimidazole (Im) amino acids is required for binding to DNA sequences longer than seven base pairs with high affinity and sequence selectivity. Pairing the α-substituted-β-amino acids (S)-isoserine (^SIs), (R)-isoserine (^RIs), β-aminoalanine (Aa), and α-fluoro-β-alanine (Fb) side-by-side with β in hairpin polyamides alters DNA binding affinity and selectivity relative to the parent polyamide containing a β/β pairing. Quantitative DNase I footprinting titration studies on a restriction fragment containing the sequences 5‘-TGCNGTA-3‘ (N = A, T, G, and C) show that the polyamide ImPy^SIsImPy-γ-PyPyβImPy-β-Dp (^SIs/β pairing) binds to N = T (K_a = 4.5 × 10^9 M^(-1)) in preference to N = A (K_a = 6.2 × 10^8 M^(-1)). This result stands in contrast to the essentially degenerate binding of the parent ImPyβImPy-γ-PyPyβImPy-β-Dp (β/β pairing) to N = T and N = A, and to the slight preference of ImPyβImPy-γ-PyPy^SIsImPy-β-Dp (β/^SIs pairing) to N = A over N = T. Additionally, this study reveals that incorporation of ^RIs, Aa, and Fb into polyamides significantly reduces binding affinity. Therefore, DNA binding in the minor groove is sensitive to the stereochemistry, steric bulk, and electronics of the substituent at the α-position of β-amino acids in hairpin polyamides containing β/β pairs

Recognition of a 5‘-(A,T)GGG(A,T)_2-3‘ Sequence in the Minor Groove of DNA by an Eight-Ring Hairpin Polyamide

The use of pyrrole−imidazole polyamides for the recognition of core 5‘-GGG-3‘ sequences in the minor groove of double stranded DNA is described. Two hairpin pyrrole−imidazole polyamides, ImImIm-γ-PyPyPy-β-Dp and ImImImPy-γ-PyPyPyPy-β-Dp (Im = N-methylimidazole-2-carboxamide, Py = N-methylpyrrole-2-carboxamide, β = β-alanine, γ = γ-aminobutyric acid, and Dp = ((dimethylamino)propyl)amide), as well as the corresponding EDTA affinity cleaving derivatives, were synthesized and their DNA binding properties analyzed. Quantitative DNase I footprint titrations demonstrate that ImImIm-γ-PyPyPy-β-Dp binds the formal match sequence 5‘-AGGGA-3‘ with an equilibrium association constant of K_a = 5 × 10^6 M^(-1) (10 mM Tris·HCl, 10 mM KCl, 10 mM MgCl_2, and 5 mM CaCl_2, pH 7.0 and 22 °C). ImImImPy-γ-PyPyPyPy-β-Dp binds the same site, 5‘-AGGGAA-3‘, approximately two orders of magnitude more tightly than the six ring polyamide, with an equilibrium association constant of K_a = 4 × 10^8 M^(-1). The eight-ring hairpin polyamide demonstrates greater specifity for single base pair mismatches than does the six ring hairpin. Polyamides with an EDTA·Fe(II) moiety at the carboxy terminus confirm that each hairpin binds in a single orientation. The high affinity recognition of a 5‘-GGG-3‘ core sequence by an eight ring polyamide containing three contiguous imidazole amino acids demonstrates the versatility of pyrrole−imidazole polyamides and broadens the sequence repertoire for DNA recognition

A pyrrole-imidazole polyamide motif for recognition of eleven base pair sequences in the minor groove of DNA

A new upper limit of binding site size is defined for the 2:1 overlapped polyamide:DNA motif. Eight-ring polyamides composed of four-ring subunits containing pyrrole (Py) and imidazole (Im) amino acids linked by a central beta-alanine (beta) spacer (''4-beta-4 ligands'') were designed for recognition of eleven base pair sequences as antiparallel dimer (4-beta-4)(2) DNA complexes in the minor groove. The DNA binding properties of three polyamides, ImPyPyPy-beta-PyPyPyPy-beta-Dp, ImImPyPy-beta-PyPyPyPy-beta-Dp, and ImImImPy-beta-PyPyPyPy-beta-Dp, were analyzed by footprinting experiments on DNA fragments containing the respective match sites 5'-AGTAATTTACT-3', 5'-AGGTATTACCT-3' (Dp = dimethylaminopropylamide). Quantitative footprint titrations reveal that each polyamide binds its respective target site with subnanomolar affinity and 7-fold to over 30-fold specificity over double-base-pair mismatch sites. A 20-fold decrease in binding affinity is observed for placement of a side-by-side beta-beta pairing opposite G•C/C•G relative to placement opposite a A•T/T•A base pair. The use of side-by-side antiparallel beta-alanine residues as an A•T/T•A-specific DNA binding element provides a new pairing rule for polyamide design. Expanding the DNA binding site size targeted by pyrrole-imidazole polyamides represents an important step in the development of cell-permeable synthetic ligands for the control of gene-specific regulation

Discrimination of 5'-GGGG-3',5'-GCGC-3', and 5'-GGCC-3' sequences in the minor groove of DNA by eight-ring hairpin polyamides

Eight-ring hairpin polyamides which differ only by the linear arrangement of pyrrole (Py) and imidazole (Im) amino acids were designed for recognition of six base pair DNA sequences containing four contiguous G,C base pairs. The respective DNA binding properties of three polyamides, ImImPyPy-γ-ImImPyPy-β-Dp, ImPyImPy-γ-ImPyImPy-β-Dp, and ImImImIm-γ-PyPyPyPy-β-Dp, were analyzed by footprinting and affinity cleavage on a DNA fragment containing the respective match sites 5‘-TGGCCA-3‘, 5‘-TGCGCA-3‘, and 5‘-TGGGGA-3‘. Quantitative footprint titrations demonstrate that ImImPyPy-γ-ImImPyPy-β-Dp binds the designed match site 5‘-TGGCCA-3‘ with an equilibrium association constant of Ka = 1 × 1010 M-1 and >250-fold specificity versus the mismatch sequences, 5‘-TGCGCA-3‘ and 5‘-TGGGGA-3‘. The polyamides ImPyImPy-γ-ImPyImPy-β-Dp and ImImImIm-γ-PyPyPyPy-β-Dp recognize their respective 5‘-TGCGCA-3‘ and 5‘-TGGGGA-3‘ match sites with reduced affinity relative to ImImPyPy-γ-ImImPyPy-β-Dp, but again with high specificity with regard to mismatch sites. These results expand the DNA sequence repertoire targeted by pyrrole-imidazole polyamides and identify sequence composition effects which will guide further second-generation polyamide design for DNA recognition

Effects of γ-Turn and β-Tail Amino Acids on Sequence-Specific Recognition of DNA by Hairpin Polyamides

Three-ring polyamides containing pyrrole (Py) and imidazole (Im) amino acids covalently coupled by a turn-specific γ-aminobutyric acid linker (γ-turn) form six-ring hairpins that recognize predetermined 5-base pair (bp) sequences in the minor groove of DNA. To determine the sequence specificity of the γ-turn and C-terminal β-alanine (β-tail) amino acids, the DNA-binding properties of the hairpin polyamide ImImPy-γ-ImPyPy-β-Dp were analyzed by footprinting and affinity cleavage on DNA-restriction fragments containing the eight possible 5‘-ATGGCNA-3‘ and 5‘-ANGGCTA-3‘ sites (N = A, T, G or C; 5-bp hairpin site is in italics). Quantitative footprint titrations demonstrate that both the γ-turn and β-tail amino acids have a >200−400-fold preference for A·T/T·A relative to G·C base pairs at these positions. Effects of the base pairs adjacent to the 5-bp hairpin-binding site were analyzed by footprinting experiments on a DNA-restriction fragment containing the eight possible 5‘-ATGGCTN-3‘ and 5‘-NTGGCTA-3‘ sites. Quantitative footprint titrations demonstrate that the turn and tail amino acids have reduced specificity (3−20-fold preference) for A·T/T·A relative to G·C base pairs at these positions. These results indicate that the turn and tail amino acids do not simply act as neutral linker residues but, in fact, are sequence-specific recognition elements with predictable DNA-binding specificity

Recognition of a 5‘-(A,T)GGG(A,T)_2-3‘ Sequence in the Minor Groove of DNA by an Eight-Ring Hairpin Polyamide

The use of pyrrole−imidazole polyamides for the recognition of core 5‘-GGG-3‘ sequences in the minor groove of double stranded DNA is described. Two hairpin pyrrole−imidazole polyamides, ImImIm-γ-PyPyPy-β-Dp and ImImImPy-γ-PyPyPyPy-β-Dp (Im = N-methylimidazole-2-carboxamide, Py = N-methylpyrrole-2-carboxamide, β = β-alanine, γ = γ-aminobutyric acid, and Dp = ((dimethylamino)propyl)amide), as well as the corresponding EDTA affinity cleaving derivatives, were synthesized and their DNA binding properties analyzed. Quantitative DNase I footprint titrations demonstrate that ImImIm-γ-PyPyPy-β-Dp binds the formal match sequence 5‘-AGGGA-3‘ with an equilibrium association constant of K_a = 5 × 10^6 M^(-1) (10 mM Tris·HCl, 10 mM KCl, 10 mM MgCl_2, and 5 mM CaCl_2, pH 7.0 and 22 °C). ImImImPy-γ-PyPyPyPy-β-Dp binds the same site, 5‘-AGGGAA-3‘, approximately two orders of magnitude more tightly than the six ring polyamide, with an equilibrium association constant of K_a = 4 × 10^8 M^(-1). The eight-ring hairpin polyamide demonstrates greater specifity for single base pair mismatches than does the six ring hairpin. Polyamides with an EDTA·Fe(II) moiety at the carboxy terminus confirm that each hairpin binds in a single orientation. The high affinity recognition of a 5‘-GGG-3‘ core sequence by an eight ring polyamide containing three contiguous imidazole amino acids demonstrates the versatility of pyrrole−imidazole polyamides and broadens the sequence repertoire for DNA recognition

Discrimination of 5'-GGGG-3',5'-GCGC-3', and 5'-GGCC-3' sequences in the minor groove of DNA by eight-ring hairpin polyamides

Eight-ring hairpin polyamides which differ only by the linear arrangement of pyrrole (Py) and imidazole (Im) amino acids were designed for recognition of six base pair DNA sequences containing four contiguous G,C base pairs. The respective DNA binding properties of three polyamides, ImImPyPy-γ-ImImPyPy-β-Dp, ImPyImPy-γ-ImPyImPy-β-Dp, and ImImImIm-γ-PyPyPyPy-β-Dp, were analyzed by footprinting and affinity cleavage on a DNA fragment containing the respective match sites 5‘-TGGCCA-3‘, 5‘-TGCGCA-3‘, and 5‘-TGGGGA-3‘. Quantitative footprint titrations demonstrate that ImImPyPy-γ-ImImPyPy-β-Dp binds the designed match site 5‘-TGGCCA-3‘ with an equilibrium association constant of Ka = 1 × 1010 M-1 and >250-fold specificity versus the mismatch sequences, 5‘-TGCGCA-3‘ and 5‘-TGGGGA-3‘. The polyamides ImPyImPy-γ-ImPyImPy-β-Dp and ImImImIm-γ-PyPyPyPy-β-Dp recognize their respective 5‘-TGCGCA-3‘ and 5‘-TGGGGA-3‘ match sites with reduced affinity relative to ImImPyPy-γ-ImImPyPy-β-Dp, but again with high specificity with regard to mismatch sites. These results expand the DNA sequence repertoire targeted by pyrrole-imidazole polyamides and identify sequence composition effects which will guide further second-generation polyamide design for DNA recognition