Antiparallel Triple Helices. Structural Characteristics and Stabilization by 8-Amino Derivatives Anna Avin ˜o ´ , ² Elena Cubero, ‡ Carlos Gonza ´ lez, § Ramon Eritja,* ,² and Modesto Orozco* ,‡ Contribution from the Institut de Biologia Molecular de Barcelona, CSIC, C/Jordi Girona 18-26, E-08034 Barcelona, Institut de Recerca Biome ´ dica de Barcelona, Parc Cientı ´fic de Barcelona, C/Josep Samitier 1-5, E-08028 Barcelona, Departament de Bioquı ´mica i Biologı ´a Molecular, Facultat de Quı ´mica, UniVersitat de Barcelona, Martı ´ i Franque ` s 1, E-08028 Barcelona, and Instituto de Quı ´mica-Fı ´sica Rocasolano, CSIC, C/Serrano 119, E-28006 Madrid, Spain Received March 7, 2003; E-mail: recgma@cid.csic.es; modesto@mmb.pcb.ub.es Abstract: The structural, dynamical, and recognition properties of antiparallel DNA triplexes formed by the antiparallel d(G#G‚C), d(A#A‚T), and d(T#A‚T) motifs (the pound sign and dot mean reverse-Hoogsteen and Watson-Crick hydrogen bonds, respectively) are studied by means of “state of the art” molecular dynamics simulations. Once the characteristics of the helix are defined, molecular dynamics and thermodynamic integration calculations are used to determine the expected stabilization of the antiparallel triplex caused by the introduction of 8-aminopurines. Finally, oligonucleotides containing 8-aminopurine derivatives are synthesized and tested experimentally using several approaches in a variety of systems. A very large stabilization of the triplex is found experimentally, as predicted by simulations. These results open the possibility for the use of oligonucleotides carrying 8-aminopurines to bind single-stranded nucleic acids by formation of antiparallel triplexes. Introduction DNA is a largely polymorphic molecule, which in near- physiological conditions can adopt a variety of structures. 1-3 Triple helices are one of these minor conformations that appear when a DNA duplex containing a polypurine track interacts with a third strand by means of specific H-bonds in the major groove of the duplex. DNA triple helices were theoretically proposed in 1953 by Pauling and Corey, 4 and demonstrated experimentally by Rich and co-workers in 1957. 5 Triplexes have been since then the subject of intense research effort owing not only to their role in the cell cycle but also to their possible biomedical (the antigene strategy) and biotechnological applications. 6-12 Depending on the orientation of the third strand with respect to the central polypurine Watson-Crick (WC) strand, triplexes are classified into two main categories: (i) parallel and (ii) antiparallel. The former (also named pyrimidine triplexes) are defined by three types of Hoogsteen triads (Figure 1): d(T-A‚ T), d(C-G‚C), and d(G-G‚C), where the first base refers to the Hoogsteen strand and the symbols dot and dash refer to Watson-Crick and Hoogsteen pairings, respectively. The anti- parallel triplexes (also named purine triplexes) are based on three reverse-Hoogsteen triads (Figure 1): d(G#G‚C), d(A#A‚T), and d(T#A‚T), where the pound sign refers to reverse-Hoogsteen hydrogen bonds. Most structural studies on DNA triplexes have focused on parallel helices, which, under normal laboratory conditions, are more stable than the corresponding antiparallel conformations. 15-18 Accurate structural models of parallel triplexes have been derived from IR and NMR experiments 19 and molecular dynamics (MD) simulations. 20 This large amount of information about the structure, reactive properties, and flexibility of these triplexes has allowed the design and synthesis of new molecules for the stabilization of the structure in physiological conditions (for a review, see ref 21). Especially powerful are the 8-ami- nopurine derivatives developed by our groups, which are able to dramatically stabilize parallel triple helices built on the d(T-A‚T) or d(C-G‚C) triads. 22 * To whom correspondence should be addressed. ² Institut de Biologia Molecular de Barcelona, CSIC. ‡ Parc Cientı ´fic de Barcelona and Universitat de Barcelona. § Instituto de Quı ´mica-Fı ´sica Rocasolano, CSIC. (1) Saenger, W. Principles of Nucleic Acid Structure; Springer-Verlag: New York, 1984. (2) Bloomfield, V. A., Crothers, D. M., Tinoco, I., Eds. Nucleic Acids: Structures, Properties and Functions; University Science Books: Sausalito, CA, 2000. (3) Blackburn, G. M., Gait, M. J., Eds. Nucleic Acids in Chemistry and Biology; IRL Press: Oxford, 1990. (4) Pauling, L.; Corey, R. B. Proc. Natl. Acad. Sci. U.S.A. 1953, 39, 84. (5) Felsenfeld, G.; Davis, D. R.; Rich, A. J. Am. Chem. Soc. 1957, 79, 2023. (6) Soyfer, V. N.; Potaman, V. N. Triple-Helical Nucleic Acids; Springer- Verlag: New York, 1996. (7) Malvy, C.; Harel-Bellan, A.; Pritchard, L. L. Triple helix forming oligonucleotides; Kluwer Academic: Dordrecht, The Netherlands, 1999. (8) Fox, K. R. Curr. Med. Chem. 2000, 7, 17. (9) Micklefield, J. Curr. Med. Chem. 2001, 8, 1157. (10) Sun, J. S.; Garestier, T.; He ´le `ne, C. Curr. Opin. Struct. Biol. 1996, 6, 327. (11) Giovannangeli, C.; He ´le `ne, C. Nat. Biotechnol. 2000, 18, 1245. (12) Vasquez, K. M.; Narayanan, L.; Glazer, P. M. Science 2000, 290, 530. (13) Cooney, M.; Czernuszewicz, G.; Postel, E. H.; Flint, S. J.; Hogan, M. E. Science 1988, 241, 456. (14) Lyamichew, V. I.; Frank-Kamenetskii, M. D.; Soyfer, V. Nature 1990, 344, 568. (15) Scaria, P. V.; Shafer, R. H. Biochemistry 1996, 35, 10985. (16) Chandler, S. P.; Fox, K. R. Biochemistry 1996, 35, 15038. (17) Washbrook, E.; Fox, K. R. Biochem. J. 1994, 301, 569. (18) Cheng, Y. K.; Pettitt, B. M. J. Am. Chem. Soc. 1992, 114, 4465. Published on Web 11/27/2003 10.1021/ja035039t CCC: $25.00 © 2003 American Chemical Society J. AM. CHEM. SOC. 2003, 125, 16127-16138 9 16127