Observation of Spontaneous Base Pair Breathing Events in the Molecular Dynamics Simulation of a Difluorotoluene-Containing DNA Oligonucleotide Elena Cubero, ² Edward C. Sherer, ‡ F. Javier Luque, § Modesto Orozco, ² and Charles A. Laughton* ,‡ Departament de Bioquı ´mica i Biologı ´a Molecular Facultat de Quı ´mica, UniVersitat de Barcelona Martı ´ i Franque ` s 1, Barcelona 08028, Spain Cancer Research Laboratories School of Pharmaceutical Sciences UniVersity of Nottingham, NG7 2RD U.K. Departament de Fisicoquı ´mica Facultat de Farmacia, UniVersitat de Barcelona AVgda. Diagonal sn, Barcelona 08028, Spain ReceiVed April 5, 1999 We report the results of extended molecular dynamics simula- tions of a DNA oligonucleotide containing an adenine-difluoro- toluene (A‚F) base pair, and of the corresponding “parent” oligonucleotide containing an adenine-thymine base pair. The observation in the former case of spontaneous breathing events involving the A‚F base pair gives further insight into the controversial subject of the thymine-mimicking characteristics of difluorotoluene. Difluorotoluene (F, Figure 1) has been designed as a nonpolar homologue of thymine and investigated extensively in an attempt to understand the origins of fidelity in DNA replication. 1-4 Studies in chloroform show no evidence that F forms hydrogen-bonding interactions with adenine, 5 but DNA polymerase I will incorporate F across from A, and A across from F, in a precise fashion. 5,6 Despite this specificity, thermal denaturation studies 7 show that replacing T by F destabilizes DNA duplexes by 3.0-3.6 kcal mol -1 . The significance of these results has been debated in quantum mechanical (QM) calculations. 8,9 Recently, the structure of an A‚F-containing DNA dodecamer has been determined by NMR. 10 The structure refinement involved numerous short (∼25 ps) molecular dynamics (MD) simulations with NMR-derived distance restraints. No unusual behavior of the dodecamer was observed during the MD simulations, and the refined structure showed standard B-type characteristics. Extended MD simulations of oligonucleotides and related molecules, including solvent and considering long-range electro- static effects, can give reliable structural and dynamic informa- tion. 11,12 Using these methods, 13 we have performed a total of 10 ns of MD simulations on an A‚F-containing dodecamer and 1.5 ns on its “parent”, A‚T-containing sequence (Figure 1). The first 1.5-ns MD trajectories for both dodecamers were monitored by measuring the RMS deviation of the snapshots from reference canonical A- and B-form structures. Within 0.5 ns, both simulations reached a plateau about 4 Å RMS deviation from the B-form reference, but about 5 Å away from the A-form refer- ence (not shown). An essentially B-form structure was maintained in both cases, the RMS deviation resulting largely from a reduction in helical twist. 12d The last 1 ns of each simulation was used to generate time-averaged structures. These showed an RMS deviation of only 1.4 Å, indicating that both sequences were adopting similar conformations, in agreement with the NMR data 10 However, plotting the lengths of the “hydrogen bonds” in the A‚F base pair over the simulation (Figure 2) showed that the conventional orientation between these bases was lost for about 200 ps, beginning at about 600 ps. This event was found to involve the swinging out into the major groove of the A and F basessa base pair breathing motion. Cieplak et al. 12f have previously reported the breathing of a terminal base pair, but “end effects” inevitably limit the generality of conclusions that can be drawn from that study. To our knowledge, this is the first time that a spontaneous breathing event of an “internal” base pair has been observed in the MD simulation of a DNA duplex, at least with the current generation of force fields and simulation protocols. This is not unexpected; for a natural Watson-Crick base pair, the breathing frequency is estimated to be in the microsecond range. The weaker nature of the A-F interaction would appear to have moved this process into a time scale accessible by atomistic MD. Solvent exchange data from the NMR studies support this conclusion. 10 ² Facultat de Quı ´mica, Universitat de Barcelona. ‡ University of Nottingham. § Facultat de Farmacia, Universitat de Barcelona. (1) Petruska, J.; Goodman, M. F.; Boosalis, M. S.; Sowers, L. C.; Cheong, C. Proc. Natl. Acad. Sci. 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Biol. 1998, 281, 675. (b) Weerasinghe, S.; Smith, P. E.; Mohan, V.; Cheng, Y.-K.; Pettit, B. M. J. Am. Chem. Soc. 1995, 117, 2147. (c)York, D. M.; Yang, W.; Lee, H.; Darden T.; Pedersen, L. G. J. Am. Chem. Soc. 1995, 117, 5001. (d) Feig, M.; Pettitt, B. M.; Biophys. J. 1998, 75, 134. (e) Spackova, N.; Berger, I.; Egli, M.; Sponer, J. J. Am. Chem. Soc. 1998, 120, 6147. (f) Cieplak, P.; Cheatham, T. E.; Kollman, P. A. J. Am. Chem. Soc. 1997, 119, 6722. (g) Foloppe, N.; MacKerell, A. D. J. Phys. Chem. B. 1998, 102, 6669. (h) Duan, Y.; Wilkosz, P.; Crowley, M.; Rosenberg, J. M. J. Mol. Biol. 1997, 272, 553. (13) All MD simulations were performed using the AMBER suite of programs. 14 Full details of the simulation protocols are included in the Supporting Information. (14) Case, D. A.; Pearlman, D. A.; Caldwell, J. W.; Cheatham, T. E.; Ross, W. S.; Simmerling, C. L.; Darden, T. A.; Merz, K. M.; Stanton, R. V.; Cheng, A. L.; Vincent, J. J.; Crowley, M.; Ferguson, D. M.; Radmer, R. J.; Seibel, G. L.; Singh, U. C.; Weiner, P. K.; Kollman, P. A. AMBER 5; University of California, San Francisco, 1997. Figure 1. Structures of (left) A‚T and (right) A‚F base pairs and dodecamer sequences simulated, showing numbering scheme. Figure 2. Variation in selected A‚F nonbonded distances (in Å) through the trajectory. 8653 J. Am. Chem. Soc. 1999, 121, 8653-8654 10.1021/ja991067t CCC: $18.00 © 1999 American Chemical Society Published on Web 09/01/1999