ZUSCHRIFTEN Angew. Chem. 2001, 113, Nr. 24 ¹ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2001 0044-8249/01/11324-4859 $ 17.50+.50/0 4859 Design and Synthesis of a Peptide That Binds Specific DNA Sequences through Simultaneous Interaction in the Major and in the Minor Groove** M. EugenioVa ¬zquez,Ana M.Caaman ƒo,Jose ¬ MartÌnez- Costas, Luis Castedo, and Jose ¬ L. Mascaren ƒas* Most transcription factors (TFs) bind specific DNA se- quences using dimeric or multimer motifs, with more or less equal binding energy contributions from each domain and usually strong cooperativity between them. [1] One of the most remarkable examples of bivalent DNA recognition is provid- ed by the bZIP family of TFs, which bind DNA as leucine zipper mediated homo- or heterodimers, with the N-terminal basic region (BR) of each monomer inserting into adjacent DNA major grooves. [2] It has been shown that the leucine zipper unit can be replaced by other noncovalent dimerizing units, or by artificial covalent linkers, without significantly compromising the recognition properties of the system. [3, 4] However, monovalent bZIP BRs exhibit very low DNA binding affinities. [3a, 5] It was recently demonstrated that covalent attachment of one basic region of a bZIP protein (GCN4) to DNA at an appropriate position allows sequence- specific binding. [6] This results suggests that monomeric bZIP- basic region peptides might be able to fold and bind DNA specifically, provided they could be properly delivered to their cognate recognition site. On these grounds we envisaged that suitable tethering of a bZIP-BR domain to a molecule that binds with moderate-to-good affinity in the minor groove of an adjacent sequence might provide for specific binding of the peptide to its cognate major groove site (Figure 1). Most importantly, the resulting conjugate should exhibit higher affinity for its target hybrid DNA site than its individual components have for their target sites. [7] Herein we demon- strate the validity of this approach by reporting the first designed peptide derivative capable of binding to DNA with nanomolar affinity through a bivalent, major± minor groove recognition motif. [8] Figure 1. Strategy for DNA recognition. For minor-groove recognition we used distamycin A, [9] a well known tripyrrole antibiotic that binds DNA preferen- tially at A±T rich sites. For the major-groove counterpart we chose the basic region of GCN4, a bZIP protein which binds specifically to the cAMP response element site (CRE: 5'- ATGAcgTCAT-3'). From the X-ray structures of the DNA complexes of GCN4 [10] and distamycin A, [11] we built an hypothetical model for simultaneous interaction of the GCN4 BR and distamycin with adjacent DNA sites (Figure 2). [12] On Figure 2. Model for the simultaneous interaction of the GCN4 basic region and distamycin at adjacent sites with DNA the basis of this model we designed the hybrid 1 (Scheme 1), in which the tether connects the nitrogen atom of the N-terminal pyrrole of a distamycin analogue with the side chain of a glutamic acid. The latter replaces the Arg245 residue of the natural protein. The tether was chosen to span the required distance between the attachment points while allowing for crossover of the phosphate backbone, a process that may be facilitated by the presence of the secondary amine in the chain. The key step for the synthesis of this hybrid consisted of the coupling of the tripyrrole derivative 2 with the peptide while it was still attached to the resin and fully protected except at the Glu245 residue (Scheme 1). [13] The DNA binding properties of hybrid 1 were first assessed by circular dichroism, a spectroscopic technique particularly useful for studying DNA ± b-ZIP interactions because of the well-established folding of the b-ZIP BRs from random coil to an a-helix upon specific DNA binding. [4] The CD spectrum of compound 1 in the absence of DNA is similar to that of peptide 3 (Figure 3a), which lacks the tripyrrole tether and hence can be used as a control in the binding studies. Unfortunately, addition of an 18 base pair (bp) duplex containing the designated hybrid DNA sequence (T/ CRE hs ) [14] to 1 at 4 8C, did not produce a significant variation of the CD signal at 222 nm, which indicates that the basic region is not inserting into the groove. The considerable [*] Prof. Dr. J. L. Mascaren ƒas, M. E. Va ¬zquez, A. M. Caaman ƒo, Prof. Dr. L. Castedo Departamento de QuÌmica Orga ¬nica y Unidad Asociada al CSIC Universidad de Santiago de Compostela 15782 Santiago de Compostela (Spain) Fax: ( 34)981-595-012 E-mail: qojoselm@usc.es Dr. J. MartÌnez-Costas Departamento de BioquÌmica y BiologÌa Molecular Universidad de Santiago de Compostela 15782 Santiago de Compostela (Spain) [**] This work was supported by the Spanish M.E.C. (PB97-0524) and the Xunta de Galicia (PGIDT00PXI20912PR). M.E.V. and A.M.C. thank the Xunta de Galicia and the University of Santiago for their predoctoral fellowships. We are very grateful to Prof. G. L. Verdinefor his input and support in the early phases of this work. We also thank Prof. J. Benavente for allowing us to use the radioactivity facilities and Prof. C. Abell for critical reading of the manuscript. Supporting information for this article is available on the WWW under http://www.angewandte.com or from the author.