Orientation Preferences of Pyrrole-Imidazole Polyamides in the Minor Groove of DNA

نویسندگان

  • Sarah White
  • Eldon E. Baird
  • Peter B. Dervan
چکیده

In order to determine whether there is an orientation preference of pyrrole-imidazole (Py-Im) polyamide dimers with respect to the 5′-3′ direction of the backbone in the DNA helix, equilibrium association constants (Ka) were determined for a series of six-ring hairpin polyamides which differ with respect to substitution at the N and C termini. Affinity cleaving experiments using hairpin polyamides of core sequence composition ImPyPy-γ-PyPyPy with an EDTA‚Fe(II) moiety at the C-terminus reveal a single binding orientation at each formal match site, 5′(A,T)G(A,T)3-3′ and 5′-(A,T)C(A,T)3-3′. A positive charge at the C-terminus and no substitution at the N-terminus imidazole affords the maximum binding orientation preference, calculated from Ka(5′-TGTTA-3′)/Ka(5′-TCTTA3′), with the N-terminal end of each three-ring subunit located toward the 5′ side of the target DNA strand. Removal of the positive charge, rearrangement of the positive charge to the N-terminus or substitution at the N-terminal imidazole decreases the orientation preference. These results suggest that second generation design principles superimposed on the simple pairing rules can further optimize the sequence-specificity of Py-Im polyamides for double helical DNA. Polyamides containing pyrrole (Py) and imidazole (Im) amino acids bind cooperatively as antiparallel dimers in the minor groove of the DNA helix.1,2 Sequence-specificity depends on the side-by-side pairings of N-methylpyrrole and N-methylimidazole amino acids.1 A pairing of Im opposite Py targets a G‚C base-pair, while Py opposite Im targets a C‚G base-pair.1 A pyrrole/pyrrole combination is degenerate and targets both T‚A and A‚T base-pairs.2 Py-Im polyamides have been shown to be cell permeable and to inhibit the transcription of genes in cell culture.3 This provides impetus to develop second generation polyamide design rules that provide for enhanced sequencespecificity and perhaps optimal biological regulation. Although the polyamides bind DNA antiparallel to each other, the “pairing rules” do not distinguish whether there should be any energetic preference for alignment of each polyamide (NC) with respect to the backbone (5′-3′) of the DNA double helix (Figure 1). In a formal sense the homodimer (ImPyPy)2 could bind 5′-WGWCW-3′ or 5′-WCWGW-3′ and still not violate the binary code. Remarkably, even in the first report on the binding specificity of the three ring polyamide ImPyPyDp there were qualitative data to suggest that there was indeed a binding preference 5′-WGWCW-3′ > 5′-WCWGW-3′.1a,4 This suggested that pyrrole-imidazole polyamide dimers align N-C with the 5′-3′ direction of the DNA strand. This orientation preference superimposed on the pairing rules confers added specificity by breaking a potential degeneracy for recognition. It would be useful to find out whether this preference is general and which aspects of the ligand design control the energetics of orientation preference. Therefore we describe here a study to address the influence on orientation of (1) positive charge or lack of, (2) position of the positive charge at the Nor C-terminus, and (3) substitution of the terminal imidazole. Three-ring polyamide subunits covalently coupled by a γ-aminobutyric acid linker form six-ring hairpin structures that bind to 5-bp target sequences with enhanced affinity and specificity relative to the unlinked polyamide pair.5 In principle, a hairpin polyamide:DNA complex can form at two different DNA sequences depending on the N-C alignment of the polyamide with the walls of the minor groove of DNA (5′-3′). A six-ring hairpin polyamide of core sequence composition ImPyPy-γ-PyPyPy which places the N-terminus of each threering polyamide subunit at the 5′-side of each recognized DNA strand would bind 5′-TGTTA-3′. Placement of the polyamide N-terminus at the 3′ side of each recognized strand would result in targeting of a 5′-TCTTA-3′ sequence (Figure 2). Four six-ring hairpin polyamides, ImPyPy-γ-PyPyPy-â-Dp 1, ImPyPy-γ-PyPyPy-â-EtOH 2, Ac-ImPyPy-γ-PyPyPy-â-Dp 3, and Dp-ImPyPy-γ-PyPyPy-â-Me 4, were synthesized by solid phase methods (Figure 3).6 The corresponding EDTA analogs ImPyPy-γ-PyPyPy-â-Dp-EDTA 1-E, ImPyPy-γ-PyPyPy-â-C7X Abstract published in AdVance ACS Abstracts, September 15, 1997. (1) (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) Trauger, J. W.; Baird, E. E.; Dervan, P. B. Nature 1996, 382, 559. (2) (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. Nature Struct. Biol. 1994, 1, 169. (d) White, S.; Baird, E. E.; Dervan, P. B. Biochemistry 1996, 35, 12532. (3) Gottesfield, J. M.; Nealy, L.; Trauger, J. W.; Baird, E. E.; Dervan, P. B. Nature 1997, 387, 202. (4) See Table 1, ref 1a. (5) (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) de Claire, R. P. L.; Geierstanger B. H.; Mrksich, M.; Dervan, P. B.; Wemmer, D. E. J. Am. Chem. Soc. 1997, 119, 7906. (6) Baird, E. E.; Dervan, P. B. J. Am. Chem. Soc. 1996, 118, 6141. Figure 1. Antiparallel polyamide subunits are depicted as filled arrows. Arrowheads correspond to C-terminal end of the polyamide: (a) polyamide binding with N-terminal end located toward 5′-side of the targeted DNA strand and (b) binding with the C-terminal end of the polyamide located toward 5′-side of binding site. 8756 J. Am. Chem. Soc. 1997, 119, 8756-8765 S0002-7863(97)01569-2 CCC: $14.00 © 1997 American Chemical Society EDTA 2-E, Ac-ImPyPy-γ-PyPyPy-â-Dp-EDTA 3-E, and DpImPyPy-γ-PyPyPy-â-C7-EDTA 4-E were also constructed in order to confirm a single orientation of each hairpin:DNA complex. We report here the DNA-binding affinity, orientation, and sequence-selectivity of the four polyamides for the two match five base-pair binding sites, 5′-TGTTA-3′ and 5′-TCTTA3′. Three separate techniques are used to characterize the DNAbinding properties of the polyamides: affinity cleaving7 and MPE‚Fe(II)8 and DNase I9 footprinting. Affinity cleavage studies determine the specific binding orientation and stoichiometry of each hairpin:DNA complex. Binding site size is accurately determined by MPE‚Fe(II) footprinting, while quantitative DNase I footprint titration is more suitable for measurement of equilibrium association constants (Ka) for the polyamide binding to designated sequences.

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تاریخ انتشار 1997