Nearest-Neighbor dsDNA Stability Analysis Using Alchemical Free-Energy Simulations

Abstract

The thermodynamic stability of double-stranded (ds)­DNA depends on its sequence. It is influenced by the base pairing and stacking with neighboring bases along DNA molecules. Semiempirical schemes are available that allow us to predict the thermodynamic stability of DNA sequences based on empirically derived nearest-neighbor contributions of base pairs formed in the context of all possible nearest-neighbor base pairs. Current molecular dynamics (MD) simulations allow one to simulate the dynamics of DNA molecules in good agreement with experimentally obtained structures and available data on conformational flexibility. However, the suitability of current force field methods to reproduce dsDNA stability and its sequence dependence has been much less well tested. We have employed alchemical free-energy simulations of whole base pair transversions in dsDNA and in unbound single-stranded partner molecules. Such transversions change the sequence context but not the nucleotide content or base pairing in dsDNA and allow a direct comparison with the empirical nearest-neighbor dsDNA stability model. For the alchemical free-energy changes in the unbound single-stranded (ss)­DNA partner molecules, we tested different setups assuming either complete unstacking or unrestrained simulations with partial stacking in the unbound ssDNA. The free-energy simulations predicted nearest-neighbor effects of similar magnitude, as observed experimentally but showed overall limited correlation with experimental data. An inaccurate description of stacking interactions and other possible reasons such as the neglect of electronic polarization effects are discussed. The results indicate the need to improve the realistic description of stacking interactions in current molecular mechanic force fields.

Isotope-Edited Variable Temperature Infrared Spectroscopy for Measuring Transition Temperatures of Single A-T Watson–Crick Base Pairs in DNA Duplexes

“…Interactions such as hydrogen bonding and pi-pi stacking in DNA and protein complexes play important roles in determining which of the large number of possible conformations are populated by the molecules under a given set of conditions, as their energetic landscape is sensitive to the physicochemical conditions they experience. − Therefore, detecting and measuring the strengths of these noncovalent interactions to determine their contributions to the stability of the whole nucleic acid assembly is of substantial interest for optimizing duplex hybridization reactions. − Prediction of the melting temperature for even short duplexes from primary sequence information alone remains an active area of research due to dependencies of the total melting temperature on nearest neighbor interactions exhibited by these biopolymers. − While substantial work was performed to measure the contribution of sequence order to duplex structural stability, few “thermometers” exist that are small enough to measure transitions at individual base pairs located at specified duplex positions, as this would require a minimum molecular-level spatial resolution of the probe. Fluorophores are a well-known probe for sensing local environments in duplexes, and their strong signal can simplify the time and effort required to collect a complete set of thermodynamic increments.…”

Isotope-edited variable temperature infrared spectroscopy for measuring transition temperatures of single A-T Watson-Crick base pairs in DNA duplexes

“…[31][32][33][34] Prediction of the melting temperature for even short duplexes from primary sequence information alone remains an active area of research due to dependencies of the total melting temperature on nearest neighbor interactions exhibited by these biopolymers. [35][36][37][38][39][40] While substantial work was performed to measure the contribution of sequence order to duplex structural stability, few "thermometers" exist that are small enough to measure transitions of individual base pairs located at specified duplex positions, as this would require a minimum molecular-level spatial resolution of the probe. Fluorophores are a well-known probe for sensing local environments in duplexes, and their high signal can simplify the time and effort required to collect a complete set of thermodynamic increments.…”