Extended molecular dynamics (MD) and thermodynamic integration (MD-TI) calculations have been used to determine the structural and energetic changes in DNA that accompany the replacement of thymine (T) by the nonnatural isostere difluorotoluene (F). In a duplex DNA oligonucleotide, it is found that the TfF mutation leads to only small changes in the average structure, but to important alterations in flexibility, hydration, and recognition properties. The TfF mutation in the Watson-Crick or Hoogsteen position of a pyrimidine‚ purine‚pyrimidine type DNA triplex does not lead to dramatic changes in the general structure of the triplex, but again, detailed analysis shows some alterations in flexibility, hydration, and recognition properties. MDTI calculations on the TfF mutation in duplex DNA reproduce the experimentally determined free energy differences with good accuracy, and detailed analyses of the trajectories have enabled us to rationalize these. Finally, MD-TI simulations have been used to predict the changes in stability of a triplex due to a TfF mutation in either the Watson-Crick or Hoogsteen-binding pyrimidine strands. We predict that in either case the mutation will reduce stability, being most unfavorable in the Watson-Crick strand. Introduction DNA replication by DNA polymerase is a high-fidelity process with an error rate of the order of one mismatch per 104 to 105 bases.1,2 It might be thought that this fidelity has its origins in the specificity of Watson-Crick hydrogen bonding, but it has been argued that this is not the case. The free energy difference between matched and mismatched terminal base pairs in solution is estimated3 to range between 0.2 and 0.4 kcal mol-1. This difference is too low to account for the observed error rate, suggesting that shape-complementarity must also play an important role in ensuring the fidelity of replication.4 However, other workers5 have argued that within the environment of the polymerase, the free energy difference between matched and mismatched base pairs may be much greater due to reduced solvation and so the hydrogen-bonding argument is sufficient. These competing hypotheses have been tested using the thymine mimic difluorotoluene (F, Figure 1). F was designed as a nonpolar homologue of thymine,6 which lacks H-bonding capability, even in apolar solvents such as chloroform.7 Despite its nonpolar nature, it was found that DNA polymerase I would incorporate F across from A, and A across from F, in a precise fashion.7,8 Despite this specificity, thermal denaturation studies9 show that replacing T by F destabilized DNA duplexes by 3.03.6 kcal mol-1. The significance of these results was challenged by Evans and Seddon,10 who argued on the basis of quantum mechanical calculations that F was a significantly polar molecule and could form nonclassical hydrogen bonds. Recently, this conclusion has been disputed by Wang and Houk,11 whose quantum mechanical and molecular mechanical calculations support the view that F does not hydrogen bond. The structure of an AF-containing DNA dodecamer has been recently determined by NMR.12 The structure refinement involved numerous short (approximate 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. On the theoretical side, very recent MD simulations13 have confirmed that the TfF mutation does not induce major changes in the average structure of a duplex, but revealed important changes in its flexibility. It was possible to detect, within a 10 ns trajectory, several “breathing” movements of the AF base pair, whereas normal AT and GC base pairs only breath on the microsecond time scale. In contrast to the wealth of information on the effect of TfF mutations on the structure and stability of duplex DNA, there † Facultat de Quimica, Universitat de Barcelona. ‡ University of Nottingham. § Facultat de Farmacia, Universitat de Barcelona. (1) Kornberg, A.; Baker, T. A. DNA Replication, 2nd ed.; W. H. Freeman: New York. (2) Goodman, M. F.; Creighton, S.; Bloom, L. B.; Petruska, J. Crit. ReV. Biochem. Mol. Biol. 1993, 28, 83. (3) Petruska, J.; Goodman, M. F.; Boosalis, M. S.; Sowers, L. C.; Cheong, C. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 6252. (4) Echols, H.; Goodman, M. F. Annu. ReV. Biochem. 1991, 60, 477. (5) Johnson, K. A. Annu. ReV. Biochem. 1993, 62, 685. (6) Guckian, K. M.; Kool, E. T. Angew. Chem., Int. Ed. 1997, 36, 2825. (7) Moran, S.; Ren, R. X.-F.; Kool, E. T. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 10506. (8) Liu, D.Y.; Moran, S.; Kool, E. T. Chem. Biol. 1997, 4, 919. (9) Moran, S.; Ren, R. X.-F.; Rumney, S.; Kool, E. T. J. Am. Chem. Soc. 1997, 119, 2056. (10) Evans, T. A.; Seddon, K. R. J. Chem. Soc., Chem. Commun. 1997, 2023. (11) Wang, X.; Houk, K. N. J. Chem. Soc., Chem. Commun. 1998, 2631. (12) Guckian, K. M.; Krugh, T. R.; Kool, E. T. Nature Struct. Biol. 1998, 5, 954. (13) Cubero, E.; Sherer, E. C.; Luque, F. J.; Orozco, M.; Laughton, C. A. J. Am. Chem. Soc. 1999, 121, 8653. 6891 J. Am. Chem. Soc. 2000, 122, 6891-6899 10.1021/ja000117n CCC: $19.00 © 2000 American Chemical Society Published on Web 07/08/2000 have been, to our knowledge, no similar studies looking at triplex DNA. In this paper we present an extension of our previous MD simulations of F-containing oligonucleotides. In addition to a 10 ns trajectory of a duplex containing a TfF mutation (and the corresponding 1.5 ns of trajectory for a control duplex containing no mutation), we have produced 3 ns trajectories for DNA triplexes containing the TfF mutation in both the Watson-Crick (d(T-A‚F) motif) and the Hoogsteen (d(F-A‚T) motif) positions, and a 1.5 ns control trajectory for the parent unmutated triplex. In addition, a series of MD-TI simulations have been performed to study in more detail the influence of the TfF mutation on the stability of both duplex and triplex DNA. MD-TI simulations are expected to be very useful to determine the stability of nucleic acid structures containing unnatural bases pairs,14,15 but due to technical problems MD-TI calculations on these systems are scarce. Calculations provide a detailed view on the changes in structure, flexibility, reactivity, and stability induced in duplex and triplex DNA by the TfF mutation. The implications of the results in the design of new antigene and antisense strategies are discussed. Methods MD Simulations. All molecular dynamics simulations were performed using the AMBER 5.0 suite of programs16 in association with the AMBER 95 force field. Missing AMBER parameters for difluorotoluene were those previously determined in our previous studies.13 All simulations were performed at constant temperature (300 K) and pressure (1 atm). Long-range electrostatic interactions were handled using the particle mesh Ewald (PME)17 method. SHAKE18 was used to constrain all bonds, allowing the use of a 2 fs time step. The DNA sequences studied are shown in Scheme 1. The duplex structures 1 and 2 were constructed in standard B-type conformation.19 Triplexes 3, 4, and 5 were generated using our equilibrated structure (14) Soliva, R.; Luque, F. J.; Orozco, M. Nucleic Acid. Res. 1999, 27, 2248. (15) Hernandez, B.; Soliva, R.; Luque, F. J.; Orozco, M. J. Am. Chem. Soc. 2000. Submitted for publication. (16) 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. (17) Cheetham. T. E.; Miller, J. L.; Fox, T.; Darden, T. A.; Kollman, P. A. J. Am. Chem. Soc. 1995, 117, 4193. (18) Ryckaert, J. P.; Ciccotti, G.; Berendsen, H. J. C. J. Comput. Phys. 1977, 23, 327. (19) Arnott, S.; Hukins, W. L. Biochem. Biophys. Res. Commun. 1972, 47, 1504. Figure 1. Chemical structure of 1,3-difluorotoluene (F) and thymine bound to adenine in the Watson-Crick and Hoogsteen orientations. Scheme 1. DNA Duplex and Triplex Sequences Used in This Study 6892 J. Am. Chem. Soc., Vol. 122, No. 29, 2000 Cubero et al.