Orientation Preferences of Pyrrole-Imidazole Polyamides in theMinor Groove of DNASarah White, Eldon E. Baird, and Peter B. Dervan*Contribution from the DiVision of Chemistry and Chemical Engineering,California Institute of Technology, Pasadena, California 91125ReceiVed May 14, 1997XAbstract: In order to determine whether there is an orientation preference of pyrrole-imidazole (Py-Im) polyamidedimers 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 Ctermini. Affinity cleaving experiments using hairpin polyamides of core sequence composition ImPyPy-γ-PyPyPywith 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-terminusimidazole affords the maximum binding orientation preference, calculated from Ka(5′-TGTTA-3′)/Ka(5′-TCTTA-3′), with the N-terminal end of each three-ring subunit located toward the 5′ side of the target DNA strand. Removalof the positive charge, rearrangement of the positive charge to the N-terminus or substitution at the N-terminalimidazole decreases the orientation preference. These results suggest that second generation design principlessuperimposed on the simple pairing rules can further optimize the sequence-specificity of Py-Im polyamides fordouble helical DNA.Polyamides containing pyrrole (Py) and imidazole (Im) aminoacids bind cooperatively as antiparallel dimers in the minorgroove of the DNA helix.1,2Sequence-specificity depends onthe side-by-side pairings of N-methylpyrrole and N-methylimi-dazole amino acids.1A pairing of Im opposite Py targets aG‚C base-pair, while Py opposite Im targets a C‚G base-pair.1A pyrrole/pyrrole combination is degenerate and targets bothT‚A and A‚T base-pairs.2Py-Im polyamides have been shownto be cell permeable and to inhibit the transcription of genes incell culture.3This provides impetus to develop second genera-tion polyamide design rules that provide for enhanced sequence-specificity and perhaps optimal biological regulation.Although the polyamides bind DNA antiparallel to each other,the “pairing rules” do not distinguish whether there should beany energetic preference for alignment of each polyamide (N-C) with respect to the backbone (5′-3′) of the DNA doublehelix (Figure 1). In a formal sense the homodimer (ImPyPy)2could bind 5′-WGWCW-3′ or 5′-WCWGW-3′ and still notviolate the binary code. Remarkably, even in the first reporton the binding specificity of the three ring polyamide ImPyPy-Dp there were qualitative data to suggest that there was indeeda binding preference 5′-WGWCW-3′ > 5′-WCWGW-3′.1a,4Thissuggested that pyrrole-imidazole polyamide dimers align N-Cwith the 5′-3′ direction of the DNA strand. This orientationpreference superimposed on the pairing rules confers addedspecificity by breaking a potential degeneracy for recognition.It would be useful to find out whether this preference is generaland which aspects of the ligand design control the energeticsof orientation preference. Therefore we describe here a studyto address the influence on orientation of (1) positive charge orlack of, (2) position of the positive charge at the N- orC-terminus, and (3) substitution of the terminal imidazole.Three-ring polyamide subunits covalently coupled by aγ-aminobutyric acid linker form six-ring hairpin structures thatbind to 5-bp target sequences with enhanced affinity andspecificity relative to the unlinked polyamide pair.5In principle,a hairpin polyamide:DNA complex can form at two differentDNA sequences depending on the N-C alignment of thepolyamide with the walls of the minor groove of DNA (5′-3′).A six-ring hairpin polyamide of core sequence compositionImPyPy-γ-PyPyPy which places the N-terminus of each three-ring polyamide subunit at the 5′-side of each recognized DNAstrand would bind 5′-TGTTA-3′. Placement of the polyamideN-terminus at the 3′ side of each recognized strand would resultin targeting of a 5′-TCTTA-3′ sequence (Figure 2).Four six-ring hairpin polyamides, ImPyPy-γ-PyPyPy-β-Dp1, ImPyPy-γ-PyPyPy-β-EtOH 2, Ac-ImPyPy-γ-PyPyPy-β-Dp3, and Dp-ImPyPy-γ-PyPyPy-β-Me 4, were synthesized by solidphase methods (Figure 3).6The corresponding EDTA analogsImPyPy-γ-PyPyPy-β-Dp-EDTA 1-E, ImPyPy-γ-PyPyPy-β-C7-XAbstract 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 thetargeted DNA strand and (b) binding with the C-terminal end of thepolyamide located toward 5′-side of binding site.8756 J. Am. Chem. Soc. 1997, 119, 8756-8765S0002-7863(97)01569-2 CCC: $14.00 © 1997 American Chemical SocietyEDTA 2-E, Ac-ImPyPy-γ-PyPyPy-β-Dp-EDTA 3-E, and Dp-ImPyPy-γ-PyPyPy-β-C7-EDTA 4-E were also constructed inorder to confirm a single orientation of each hairpin:DNAcomplex. We report here the DNA-binding affinity,