Enhanced Sequence Specific Recognition in the Minor Groove of DNA by Covalent Peptide Dimers: Bis(

The designed peptide pyridine-2-carboxamidonetropsin (2-PyN) binds to the minor groove of double-helical DNA at two very different sequences, 5’-TTTTT-3’ and 5’-TGTCA-3’, with comparable energetics but quite different structures. 2-PyN likely binds the 5’-TTTTT-3’ site as a 1:l complex, whereas 2-PyN binds 5’-TGTCA-3’ sites as a 2:l complex. In order to enhance the binding affinity of 2-PyN for the 5’-TGTCA-3’ site, covalently linked dimers of 2-PyN have been synthesized wherein the nitrogens of the central pyrroles are connected with propyl, butyl, pentyl, and hexyl linkers. DNase I footprint t i tration experiments reveal that these bis(pyridine-2-carboxamidenetropsin)(CH2)34 peptides bind to a 5’-TGTCA-3’ site with binding affinities 10-fold greater than that of 2-PyN. By taking advantage of the different structures of peptides bound in the minor groove, the ratio of binding affinities of 2-PyN for 5’-TGTCA-3’ and 5’-TTTTT-3’ sites have been altered from 1:l to 25:l. 1:l and 2:l PeptideDNA Complexes. The natural products netropsin and distamycin A are crescent shaped diand tripeptides, respectively, that bind in the minor groove of DNA at sites of four or five successive A,T base pairs ( b ~ ) . l ~ The structures of a number of peptideDNA complexes have been determined by X-ray diffraction4 and NMR spectro~copy,~ and the thermodynamic profiles have been studied for these complexes.6 This work suggests that favorable electrostatic interactions and extensive van der Waals contacts between the peptide and the floor and walls of the minor groove contribute to complex stability. The carboxamide NH’s of the peptides participate in bifurcated hydrogen bonds with adenine N 3 and thymide 0 2 atoms on the floor of the minor groove. The aromatic hydrogens of the N-methylpyrrole rings are set too deeply in the minor groove to allow room for the guanine 2-amino group of a G,C base pair, affording binding specificity for A,T-rich sequences. Although this model has aided in thedesign of oligopeptides for recognition a Abstract published in Advance ACS Abstracts, October 1, 1993. (1) (a) Krylov, A. S.; Grokhovsky, S. L.; Zasedatelev, A. S.; Zhuze, A. L.; Gursky, G. V.; Gottikh, B. P. Nucl. Acids. Res. 1979, 6, 289-304. (b) Zasedatelev, A. S.; Gursky, G. V.; Zimmer, Ch.; Thrum, H. Mol. Biol. Rep. 1974, 1, 337-342. (c) Zasedatelev, A. S.; Zhuze, A. L.; Zimmer, Ch.; Grokhovsky, S. L.; Tumanyan, V. G.; Gursky, G. V.; Gottikh, B. P. Dokl. Acad. Nauk SSSR 1976, 231, 1006-1009. For a review, see: (d) Zimmer, C.; Wihnert, U. Prog. Biophys. Molec. Biol. 1986, 47, 31-112. (2) (a) Van Dyke, M. W.; Hertzberg, R. P.; Dervan, P. B. Proc. Natl. Acad. Sci. U.S.A. 1982, 79,5470-5474. (b) Van Dyke, M. W.; Dervan, P. B. Cold Spring Harbor Symposium on Quantitative Biology 1982,47,347353. (c) Van Dyke, M. W.; Dervan, P. B. Biochemistry 1983,22,2373-2377. (d) Harshman, K. D.; Dervan, P. B. Nucl. Acids Res. 1985, 13,48254335. (e ) Fox, K. R.; Waring, M. J. Nucl. Acids Res. 1984, 12, 9271-9285. (f) Lane, M. J.; Dobrowiak, J. C.; Vournakis, J. Proc. Natl. Sci. U.S.A. 1983, 80, 3260-3264. (3) (a) Schultz,P. G.; Taylor, J. S.; Dervan, P. B. J. Am. Chem. SOC. 1982, 104,6861-6863. (b) Taylor, J. S.; Schultz, P. G.; Dervan, P. B. Teirahedron 1984,40,457-465. (c) Schultz, P. G.; Dervan, P. B. J . Biomol. Struct. Dun. 1984, 1, 1133-1147. (d) Dervan, P. B. Science 1986, 232, 464471. (4) (a) Kopka, M. L.; Yoon, C.; Goodsell, D.; Pjura, P.; Dickerson, R. E. Proc. Natl. Acad.Sci. U.S.A. 1985,82, 1376-1380. (b) Kopka, M. L.;Yoon, C.; Goodsell, D.; Pjura, P.; Dickerson, R. E. J . Mol. Biol. 1985,183,553-563. (c) Coll, M.; Frederick, C. A.; Wang, A. H.-J.; Rich, A. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 8385-8389. (5) (a) Patel, D. J.; Shapiro, L. J . Bioi. Chem. 1986,261, 1230-1240. (b) Klevitt, R. E.; Wemmer, D. E.; Reid, B. R. Biochemistry 1986, 25, 32963303. (c) Pelton, J. G.; Wemmer, D. E. Biochemistry 1988,27,8088-8096. (6) (a) Markey, L. A.; Breslauer, K. J. Proc. Narl. Acad. Sci. U.S.A. 1987, 84,4359-4363. (b) Breslauer, K. J.; Remeta, D. P.; Chou, W.-Y.; Ferrante, R.; Curry, J.; Zaunczkowski, D.; Snyder, J. G.; Marky, L. A. Proc. Narl. Acad. Sci. U.S.A. 1987,84, 8922-8926. 0002-786319311515-9892$04.00/0 of longer tracts of A,T-rich DNA? efforts to design peptides capable of binding mixed A,T and G,C sequences have proven inconsistent with a 1:l peptide-DNA model.8.9 There have been several recent reports of peptides which bind in the minor groove of DNA as antiparallel side-by-side dimers.1G15 Pelton and Wemmer found that distamycin at high concentrations ( 2 4 mM) is capable of binding in theminor groove of 5’-AAATT-3‘ as a dimer.10 Shortly thereafter, the designed peptide 1 -methylimidazole-2-carboxamidonetropsin (2-1”) was shown to bind exclusively to the mixed sequence 5’-TGTCA-3’ as a 2: 1 complex.11J2 Another synthetic peptide analog pyridine2-carboxamidonetropsin (2-PyN) was found to bind to the minor groove of double-helical DNA at two very different sequences, 5’-TTTTT-3‘ and 5’-TGTCA-3’.9J1 From quantitative footprint titration experiments, the apparent first-order binding affinities for 2-PyN in complex with 5’-TTTTT-3’ and 5’-TGTCA-3’ are comparable, K, = 2.3 X 105 and 2.7 X 10s M-1, respectively (20 mM Tris.HC1 and 100 mM NaCl a t pH 7.0 and 37 OC).16 2-PyN likely binds 5’-TTTTT-3’ as a 1 : 1 complex and 5’-TGTCA-3’ as a 2:l complex (Figure 1). Experimental Design. One strategy for increasing the affinity and hence the sequence specificity of peptides that bind DNA sites as side-by-side dimers in the minor groove is to covalently tether the two peptides.17 The overall free energy of complex formation is expected to be more favorable for a covalent dimer since one bis-peptide should bind with more favorable entropy than do two peptides. Examination of 2:l peptide-DNA models (7) Dervan, P. B. Science 1986, 232, 464-471. (8) (a) Lown, J. W.; Krowicki, K.; Bhat, U. G.; Ward, B.; Dabrowiak, J. C. Biochemistry 1986, 25, 7408-7416. (b) Kissinger, K.; Krowicki, K.; Dabrowiak, J. C.; Lown, J . W. Biochemistry 1987, 26, 559&5595. (9) Wade, W. S.; Dervan, P. B. J . Am. Chem. SOC. 1987,109,1574-1575. (IO) (a) Pelton, J. G.; Wemmer, D. E. Proc. Nail. Acad.Sci. U.S.A. 1989, 86,5723-5727. (b) Pelton, J. G.; Wemmer, D. E. J . Am. Chem. Soc. 1990, ( 1 1) Wade, W. S.; Mrksich, M.; Dervan, P. B. J . Am. Chem. SOC. 1992, (12) Mrksich, M.; Wade, W. S.; Dwyer, T. J.;Geierstanger,B. H.; Wemmer, (13) Dwyer,T. J.;Geierstanger, B. H.; Bathini, Y.;Lown, J. W.; Wemmer, (14) Mrksich,M.;Dervan,P.B. J.Am.Chem.Soc. 1993,115,2572-2576. (15) Geierstanger, B. H.; Jacobsen, J.-P.; Mrksich, M.; Dervan, P. B.; (16) Wade, W. S.; Mrksich, M.; Dervan, P. B. Biochemistry In press. 112, 1393-1399.