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 with a shift in the internucleotide linkages from the wild-type 5'-to-3' direction to 3'-to-2.' This alteration leads to higher chemical stability, less reactive groups, and lower conformational flexibility. Experimental observations indicate that these characteristic changes are attributable to a minimal perturbation of the interaction network, but the thermodynamic stability of the duplex remains unaltered in the TNA mutation. We applied the equilibrium and nonequilibrium free-energy simulations employing three popular assisted model building with energy refinement (AMBER) force fields for nucleotides to investigate this mutation-dependent behavior in the base flipping from T (DNA) residue to the T-to-TFT mutation (TNA) computationally. The force fields were performed similarly, as described in the base-paired state. However, after exploring the high-energy regions with free-energy simulations, we observed that these three force fields behaved differently. Previous reports conclude that the net-neutral and excess-salt simulations provided similar results. Nonetheless, our free-energy simulation indicated that the presence of excess salt affected the thermodynamic stability. The free-energy barrier along the base-flipping pathway was generally elevated upon the addition of excess salts, but the relative height of the free-energy barriers in DNA and TNA duplexes did not change significantly. This phenomenon emphasizes the importance of adding sufficient salts in the simulation scheme to reproduce the experimental condition.

Automated summary

TNA, an analogue of DNA, exhibits higher chemical stability and less reactive groups, while maintaining the thermodynamic stability in the duplex. Simulation studies investigated the mutation-dependent behavior of TNA in base flipping, highlighting the importance of simulating experimental conditions with sufficient salt concentrations.