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8756 J Am Chem Soc 1997 119 8756 8765 Orientation Preferences of Pyrrole Imidazole Polyamides in the Minor Groove of DNA Sarah White Eldon E Baird and Peter B Dervan Contribution from the DiVision of Chemistry and Chemical Engineering California Institute of Technology Pasadena California 91125 ReceiVed May 14 1997X Abstract 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 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 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 X S0002 7863 97 01569 2 CCC 14 00 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 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 N or C terminus and 3 substitution of the terminal imidazole Three ring polyamide subunits covalently coupled by a 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 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 PyPyPy Dp 1 ImPyPy PyPyPy EtOH 2 Ac ImPyPy PyPyPy Dp 3 and Dp ImPyPy PyPyPy Me 4 were synthesized by solid phase methods Figure 3 6 The corresponding EDTA analogs ImPyPy PyPyPy Dp EDTA 1 E ImPyPy PyPyPy C7 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 1997 American Chemical Society Pyrrole Imidazole Polyamides in the Minor GrooVe of DNA J Am Chem Soc Vol 119 No 38 1997 8757 Figure 2 Binding models for ImPyPy PyPyPy Dp in complex with 5 WGWWW 3 top and 5 WCWWW 3 bottom W A or T 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 For schematic binding models the imidazole and pyrrole rings are represented as shaded and unshaded spheres respectively the curved line represents aminobutyric acid and the alanine residue is represented as an unshaded diamond Shaded arrows represent the orientation of individual

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