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

Selective placement of an aliphatic â-alanine (â) 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 â-alanine “spring” aliphatic amino acid residues. The binding affinities and specificities of these polyamides are regulated by the placement of paired â/â, Py/â, and Im/â residues. Quantitative footprint titrations demonstrate that replacing two Py/Py pairings in a 12-ring hairpin (6-γ-6) with two Py/â pairings affords 10-fold enhanced affinity and similar sequence specificity for an 8-bp target sequence. The 6-γ-6 hairpin ImPyImPyPyPy-γ-ImPyPyPyPyPy-â-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-â-ImPyPyPy-γ-ImPyPyPy-â-Py-â-Dp binds to the 8-bp match sequence 5′-TGTGAACA3′ with an equilibrium association constant of Ka ) 2.4 × 1010 M-1 and g48-fold specificity versus the 5′TGTTAACA-3′ single-base-pair mismatch site. Modeling indicates that the â-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-â-Im polyamide subunit. This observation provided the basis for design of a hairpin polyamide, Im-â-ImPy-γ-Im-â-ImPy-â-Dp, which incorporates Im/â pairings to recognize a “problematic” 5′-GCGC-3′ sequence at subnanomolar concentrations. These results identify Im/â and â/Im pairings that respectively discriminate G‚C and C‚G from A‚T/T‚A as well as Py/â and â/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. Polyamides containing N-methylpyrrole and N-methylimidazole amino acids are synthetic ligands that have an affinity and specificity for DNA comparable to those of naturally occurring DNA binding proteins.1 DNA recognition depends on sideby-side aromatic 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 Eight-ring pyrrole-imidazole polyamides have been shown to be cell permeable and to inhibit transcription of designated genes in cell culture.4 This provides impetus to develop an ensemble of motifs which recognize a broad binding site size and sequence repertoire. Given the sequence-dependent microstructure of the DNA helix,5 it is surprising that a simple recognition code can be developed at all.6 In both published and unpublished work, over 100 pyrrole-imidazole polyamides have been synthesized which recognize predetermined sequences. However, within this group, certain difficult Py-Im/DNA base-pair sequences have emerged. Sequence-dependent DNA structure features such as intrinsic minor groove width, minor groove flexibility, and inherent DNA curvature may reduce polyamide binding at certain sites.5 However, it may also be possible to identify polyamide structural elements which will restore affinity at difficult sequences by providing an optimal fit between the hydrogen bond donors and acceptors displayed on the edges of both the Watson-Crick base pairs and the crescent-shaped polyamide dimers. Hairpin Polyamide. Efforts have been made to increase DNA-binding affinity and sequence specificity by covalently linking polyamide heterodimers and homodimers.1,7 A head(1) (a) Trauger, J. W.; Baird, E. E.; Dervan, P. B. Nature 1996, 382, 559. (b) Swalley, S. E.; Baird, E. E.; Dervan, P. B. J. Am. Chem. Soc. 1997, 119, 6953. (c) Turner, J. M.; Baird, E. E.; Dervan, P. B. J. Am. Chem. Soc. 1997, 119, 7636. (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. Biochemistry 1993, 32, 11385. (d) Mrksich, M.; Dervan, P. B. J. Am. Chem. Soc. 1993, 115, 2572. (e) Geierstanger, B. H.; Dwyer, T. J.; Bathini, Y.; Lown, J. W.; Wemmer, D. E. J. Am. Chem. Soc. 1993, 115, 4474. (3) (a) Pelton, J. G.; Wemmer, D. E. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 5723. (b) Pelton, J. G.; Wemmer, D. E. J. Am. Chem. Soc. 1990, 112, 1393. (c) Chen, X.; Ramakrishnan, B.; Rao, S. T.; Sundaralingham, M. Nat. Struct. Biol. 1994, 1, 169. (d) White, S.; Baird, E. E.; Dervan, P. B. Biochemistry 1996, 35, 12532. (4) Gottesfield, J. M.; Nealy, L.; Trauger, J. W.; Baird, E. E.; Dervan, P. B. Nature 1997, 387, 202. (5) (a) Wu, H.; Crothers, D. M. Nature 1984, 308, 509. (b) Steitz, T. A. Annu. ReV. Biophys. 1990, 23, 205. (c) Goodsell, D. S.; Kopka, M. L.; Cascio, D.; Dickerson, R. E. Proc. Natl. Sci. U.S.A. 1993, 90, 2930. (d) Paolella, D. N.; Palmer, R.; Schepartz, A. Science 1994, 264, 1130. (e) Kahn, J. D.; Yun, E.; Crothers, D. M. Nature 1994, 368 163. (f) Geierstanger, B. H.; Wemmer, D. E. Annu. ReV. Biochem. 1995, 24, 463. (g) Hansen, M. R.; Hurley, L. H. Acc. Chem. Res. 1996, 29, 249. (6) (a) Dervan, P. B. Science 1986, 232, 464. (b) Dervan, P. B. In Nucleic Acids and Molecular Biology; Springer-Verlag: Heidelberg, 1988; Vol. 2, 49-64. (c) Thuong, N. T.; Helene, C. Angew. Chem. 1993, 32, 666. (d) Nielsen, P. E. Chem. Eur. J. 1997, 3, 505. 6219 J. Am. Chem. Soc. 1998, 120, 6219-6226 S0002-7863(98)00147-4 CCC: $15.00 © 1998 American Chemical Society Published on Web 06/12/1998 to-tail linked polyamide with γ-aminobutyric acid (γ) serving as a turn-specific internal guide residue binds to predetermined target sites with 100-fold enhanced affinity relative to unlinked subunits.1,7 In contrast to unlinked dimers which can adopt a variety of “slipped” binding modes, the hairpin structure locks the relatiVe positions of the indiVidual subunits and allows greater control of amino acid ring pairings. Three-, four-, and five-ring polyamides covalently coupled to form six-, eight-, and 10-ring hairpin structures bind specifically to 5-, 6-, and 7-bp target sequences, respectively.1,7 Recognition of 7 bps by a 10-ring hairpin represents an upper limit of five contiguous ring pairings which will match the curvature of the DNA helix without energetic penalty.1c,8,9 For complexes of fully overlapped 2:1 polyamide dimers in the minor groove, a flexible â-alanine (â) spring was found to form A,T-specific â/â pairings which were necessary for recognition of longer binding sites.10 It remained to be determined (i) if paired â-alanine residues could be accommodated in the hairpin structure to restore register of the hairpin with the DNA helix for recognition of larger binding sites and (ii) if the antiparallel polyamide dimer could accommodate aromatic amino acids paired side-by-side with aliphatic â-amino acids to give â/Py and â/Im ring pairings (Figures 1 and 2). Eight polyamides were synthesized containing either solely ring amino acids, a side-by-side pairing of two â-alanine residues (â/â), or a side-by-side pairing of a pyrrole and a â-alanine residue (Py/â).11 We report here the DNA-binding affinity and sequence selectivity of the eight hairpin polyamides, ImPyPyPyPyPy-γ-ImPyPyPyPyPy-â-Dp (1), ImPyPyPy-â-Pyγ-Im-â-PyPyPyPy-â-Dp (2), ImPyPy-â-PyPy-γ-ImPy-â-PyPyPy-â-Dp (3), ImPy-â-PyPyPy-γ-ImPyPy-â-PyPy-â-Dp (4), Im-â-PyPyPyPy-γ-ImPyPyPy-â-Py-â-Dp (5), Im-â-PyPyPyPyγ-Im-â-PyPyPyPy-â-Dp (6), ImPy-â-PyPyPy-γ-ImPy-â-PyPyPy-â-Dp (7), and ImPy-â-â-PyPy-γ-ImPy-â-â-PyPy-â-Dp (8) for an 8-bp 5′-TGTTAACA-3′ target sequence and a 5′TGTGAACA-3′ single-base-pair mismatch sequence. Polyamides 2-5 vary the position of a single (â/â) pairing, while polyamides 6 and 7 have two (Py/â) pairings. As a control, polyamide 8 was synthesized with two consecutive (â/â) pairings (Figure 4). Additional polyamides ImPyImPyPyPyγ-ImPyPyPyPyPy-â-Dp (9) and Im-â-ImPyPyPy-γ-ImPyPyPyâ-Py-â-Dp (10) were designed to target the 8-bp sequence 5′TGTGAACA-3′. A polyamide, Im-â-ImPy-γ-Im-â-ImPy-â-Dp (12), which incorporates Im/â and â/Im pairings, was also synthesized, and its DNA binding affinity and specificity were determined for a 5′-TGCGCA-3′ site. Two separate techniques were used to characterize the DNA-binding properties of the designed polyamides: methidiumpropyl-EDTA‚Fe(II) (MPE‚ Fe(II)) footprinting12 and DNase I footprinting.13 Information about precise binding site size is gained from MPE‚Fe(II) footprinting, while quantitative DNase I footprint titrations allow determination of equilibrium association constants (Ka) of the polyamides for respective match and mismatch binding sites. Results and Discussion Placement of Py/â and â/â Pairings. A series of eight hairpin polyamides were synthesized using Boc-chemistry machine-assisted protocols (Figure 3).11 MPE‚Fe(II) footprinting on a 32P end-labeled 254-bp DNA restriction fragment (25 mM Tris-acetate, 10 mM NaCl, 100 mM calf thymus DNA, pH 7.0, 22 °C) reveals that each polyamide is binding to the 5′-TGTTAACA-3′ match site (Figure 4 and Supporting Information).12 Footprinting patterns reveal asymmetrically 3′-shifted protection of the 8-bp sites, consistent with formation of a 1:1 hairpin polyamide-DNA complex in the minor groove. Polyamide 1 at 10 μM concentration protects both the 5′-TGTTAACA-3′ match site and the single-base-pair mismatch 5′TGTGAACA-3′ site. Polyamides 4 and 7 each at 10 μM protect their cognate 5′-TGTTAACA-3′ match site; however, no protection is observed at the 5′-TGTGAACA-3′ single-basepair mismatch site. Quantitative DNase I footprint titration experiments13 (10 mM Tris-HCl, 10 mM KCl, 10 mM MgCl2, and 5 mM CaCl2, pH 7.0, 22 °C) were performed to determine the equilibrium association constants of the polyamides for the two bound sites (Table 1).13 The 5′-TGTTAACA-3′ match site was bound by the polyamides with decreasing affinity: ImPyâ-PyPyPy-γ-ImPyPy-â-PyPy-â-Dp (4, Ka ) 1.2 ((0.1) × 1011 M-1) > Im-â-PyPyPyPy-γ-Im-â-PyPyPyPy-â-Dp (6, Ka ) 4.5 ((2.7) × 1010 M-1) > ImPy-â-PyPyPy-γ-ImPy-â-PyPyPy-â(7) (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. 1997, 119, 7909. (h) White, S.; Baird, E. E.; Dervan, P. B. J. Am. Chem. Soc. 1997, 119, 8756. (i) White, S.; Baird, E. E.; Dervan, P. B. Chem. Biol. 1997, 4, 569. (8) Kelly, J. J.; Baird, E. E.; Dervan, P. B. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 6981. (9) Kielkopf, C. L.; Baird, E. E.; Dervan, P. B.; Rees, D. C. Nat. Struct. Biol. 1988, 5, 104. (10) (a) Trauger, J. W.; Baird, E. E.; Mrksich, M.; Dervan. P. B. J. Am. Chem. Soc. 1996, 118, 6160. (b) Swalley, S. E.; Baird, E. E.; Dervan, P. B. Chem. Eur. J. 1997, 3, 1600. (c) Trauger, J. W.; Baird, E. E.; Dervan. P. B. J. Am. Chem. Soc. 1998, 120, 3534. (11) Baird, E. E.; Dervan, P. B. J. Am. Chem. Soc. 1996, 118, 6141. (12) (a) Van Dyke, M. W.; Dervan, P. B. Nucleic Acids Res. 1983, 11, 5555. (b) Van Dyke, M. W.; Dervan, P. B. Science 1984, 225, 1122. (13) (a) Brenowitz, M.; Senear, D. F.; Shea, M. A.; Ackers, G. K. Methods Enzymol. 1986, 130, 132. (b) Brenowitz, M.; Senear, D. F.; Shea, M. A.; Ackers, G. K. Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 8462. (c) Senear, D. F.; Brenowitz, M.; Shea, M. A.; Ackers, G. K. Biochemistry 1986, 25, 7344. Figure 1. Ribbon model for the use of aliphatic amino acids in hairpin imidazole-pyrrole polyamides. Arrows represent aromatic imidazole and pyrrole amino acids, while springs represent â-alanine residues. (a) Six consecutive aromatic amino acid pairings. (b) One â/â pairing. (c) Two aromatic/â pairings. 6220 J. Am. Chem. Soc., Vol. 120, No. 25, 1998 Turner et al.