Recognition of Seven Base Pair Sequences in the Minor Groove of DNA by Ten-Ring Pyrrole-Imidazole Polyamide Hairpins

A new upper limit of binding site size is defined for the hairpin polyamide-DNA motif. Ten-ring hairpin polyamides containing pyrrole (Py) and imidazole (Im) amino acids were designed for recognition of seven base pair (bp) sequences in the minor groove of DNA. The DNA binding properties of two polyamides, ImPyPyPyPyγ-ImPyPyPyPy-â-Dp, and ImImPyPyPy-γ-ImPyPyPyPy-â-Dp were analyzed by footprinting and affinity cleavage on a DNA fragment containing the respective match sites 5′-TGTAACA-3′ and 5′-TGGAACA-3′. Quantitative footprint titrations demonstrate that ImPyPyPyPy-γ-ImPyPyPyPy-â-Dp binds the 7-bp match sequence 5′-TGTAACA3′ with an equilibrium association constant (Ka) of Ka ) 1.2 × 1010 M-1 and 18-fold specificity versus the single base pair mismatch sequence 5′-TGGAACA-3′. ImImPyPyPy-γ-ImPyPyPyPy-â-Dp differs from ImPyPyPyPy-γImPyPyPyPy-â-Dp by a single amino acid substitution and binds its match 5′-TGGAACA-3′ site with Ka ) 3.6 × 109 M-1 and 300-fold specificity versus its corresponding single base pair mismatch sequence 5′-TGTAACA-3′. Ten-ring hairpin polyamides have binding affinities similar to those of eight-ring hairpin polyamides. These results indicate that the affinity of hairpin binding ceases to increase as the length of the polyamide subunits increases beyond four rings, analogous to the behavior of unlinked subunits. Therefore, recognition of seven base pairs by a ten-ring hairpin polyamide most likely represents an upper limit to the effective targetable site size of the hairpin polyamide-DNA motif. Small molecules that target specific DNA sequences have the potential to control gene expression. Polyamides containing N-methylpyrrole and N-methylimidazole amino acids are synthetic ligands that have an affinity and specificity for DNA comparable to naturally occurring DNA binding proteins.1 DNA recognition depends on side-by-side amino acid pairings in the minor groove. Antiparallel pairing of imidazole (Im) opposite pyrrole (Py) recognizes a G‚C base pair, while a Py-Im combination recognizes C‚G.2 A Py-Py pair is degenerate and recognizes either an A‚T or T‚A base pair.2,3 Four-ring polyamides covalently coupled to form eight-ring hairpin structures bind specifically to 6-bp target sequences and, importantly, have been shown to be cell permeable and to inhibit transcription of specific genes in cell culture.4 The optimal binding site size for effective biological regulation has yet to be determined. This provides impetus to explore the binding site size limitations of the hairpin motif for recognition in the minor groove of DNA.5 Given the failure of g five contiguous rings to maintain precise register with the DNA helix5 combined with the constraint of covalent coupling, it remained to be determined if ten-ring hairpin polyamides could be successfully designed to recognize 7-bp sequences without compromising DNA-binding affinity or sequence specificity. Hairpin Polyamide. In parallel with elucidation of the scope and limitations of the polyamide pairing rules, efforts have been X Abstract published in AdVance ACS Abstracts, August 1, 1997. (1) Trauger, J. W.; Baird, E. E.; Dervan, P. B. Nature 1996, 382, 559. (2) (a) Wade, W. S.; Mrksich, M.; Dervan, P. B. J. Am. Chem. Soc. 1992, 114, 8783. (b) Mrksich, M.; Wade, W. S.; Dwyer, T. J.; Geierstanger, B. H.; Wemmer, D. E.; Dervan, P. B. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 7586. (c) Wade, W. S.; Mrksich, M.; Dervan, P. B. 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Soc. 1997, 119, 7636-7644 S0002-7863(97)01208-0 CCC: $14.00 © 1997 American Chemical Society made to increase DNA-binding affinity and sequence specificity by covalently linking polyamide subunits.6-8 A hairpin polyamide motif with γ-aminobutyric acid (γ) serving as a turnspecific internal-guide-residue provides a synthetically accessible method for C-N linkage of polyamide subunits.8 Head-to-tail linked polyamides bind specifically to designated target sites (6) (a) Mrksich, M; Dervan, P. B. J. Am. Chem. Soc. 1993, 115, 2572. (b) Geierstanger, B. H.; Dwyer, T. J.; Bathini, Y.; Lown, J. W.; Wemmer, D. E. J. Am. Chem. Soc. 1993, 115, 4474. (c) Geierstanger, B. H.; Jacobsen, J. P.; Mrksich, M.; Dervan, P. B.; Wemmer, D. E. Biochemistry 1994, 33, 3055. (7) (a) Mrksich, M.; Dervan, P. B. J. Am. Chem. Soc. 1994, 116, 3663. (b) Chen, Y. H.; Lown, J. W. J. Am. Chem. Soc. 1994, 116, 6995. (8) (a) Mrksich, M.; Parks, M. E.; Dervan, P. B. J. Am. Chem. Soc. 1994, 116, 7983. (b) Parks, M. E.; Baird, E. E.; Dervan, P. B. J. Am. Chem. Soc. 1996, 118, 6147. (c) Parks, M. E.; Baird, E. E.; Dervan, P. B. J. Am. Chem. Soc. 1996, 118, 6153. (d) Trauger, J. W.; Baird, E. E.; Dervan, P. B. Chem. Biol. 1996, 3, 369. (e) Swalley, S. E.; Baird, E. E.; Dervan, P. B. J. Am. Chem. Soc. 1996, 118, 8198. (f) Pilch, D. S.; Pokar, N. A.; Gelfand, C. A.; Law, S. M.; Breslauer, K. J.; Baird, E. E.; Dervan, P. B. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 8306. (g) de Claire, R. P. L.; Geierstanger B. H.; Mrksich, M.; Dervan, P. B.; Wemmer, D. E. J. Am. Chem. Soc. In press. Figure 2. Binding model for the complexes formed between the DNA and either ImImPyPyPy-γ-ImPyPyPyPy-â-Dp (top) or ImPyPyPyPy-γImPyPyPyPy-â-Dp (bottom). Circles with dots represent lone pairs of N3 of purines and O2 of pyrimidines. Circles containing an H represent the N2 hydrogen of guanine. Putative hydrogen bonds are illustrated by dotted lines. Ball and stick models are also shown. Shaded and nonshaded circles denote imidazole and pyrrole carboxamides, respectively. Nonshaded diamonds represent the â-alanine residue. Ten-Ring Pyrrole-Imidazole Polyamide Hairpins J. Am. Chem. Soc., Vol. 119, No. 33, 1997 7637