Thermodynamic Insights of Base Flipping in TNA Duplex: Force Fields, Salt Concentrations, and Free Energy Simulation Methods

Threofuranosyl nucleic acid (TNA) is an analogue of DNA. Its inter-nucleotide linkages are shifted from the wild-type 5'-to-3' one to the 3'-to-2' one. As a result, the number of covalent bonds between consecutive phosphates is reduced from 6 to 5. This leads to higher chemical stability, less reactive groups, and lower conformational flexibility. Experimental observations indicate that the interaction network is perturbed at the minimal level and the thermodynamic stability of the duplex is unaltered upon the TNA mutation. Whether computational modelling could reproduce this result will be studied in the base flipping of the middle T (DNA) residue or its T-to-TFT mutation (TNA). We applied the equilibrium free energy simulation and the nonequilibrium stratification method proposed previously in the base flipping case, proving the applicability of alternative free energy simulation protocols. As the force field is the main accuracy-limiting factor when converged phase space sampling is obtained, we benchmarked three popular AMBER force fields for nucleotides. The last-generation force fields include bsc1 and OL15, both of which perform similarly in reproducing the structures near the crystal conformation in previous benchmark studies. Our results indicate that all these three force fields provide similar descriptions of the base-paired state. However, with free energy simulation constructing the free energy profiles along the conformational change pathway, high-energy regions are explored and these three force fields behave differently. The bsc1 force field is found to perform best in reproducing the similarity of stabilities of DNA and TNA duplexes. The free energy barrier of base flipping under the OL15 force field is lowered modestly in TNA, and thus this force field is also usable. However, the bsc0 force field provides wrong results. The TNA duplex is significantly less stable than the DNA duplex. Therefore, the bsc0 force field is not recommended in any application in modern nucleotide simulations. The salt concentration in nucleotide simulations is another factor influencing the thermodynamics of the system. Previous reports conclude that the net-neutral and excess-salt simulations provide similar results. However, the simulation method limits the phase space region explored in previous computational modelling. Our free energy simulation explores high-energy regions, where the excess salt does affect the thermodynamic stability. The free energy barrier along the base flipping pathway is generally elevated upon the addition of excess salts, but the relative height of the free energy barriers in DNA and TNA duplexes is not significantly changed. This phenomenon emphasizes the importance of adding sufficient salts to reproduce the experimental condition.