Exploring Nucleic Acid Structure and Function with Molecular Dynamics and Fragment Molecular Orbital Methods

In recent years, nucleic acid therapeutics have attracted increasing attention as a new modality for drug discovery and as a novel therapeutic approach for rare and intractable diseases. The G-quadruplex structure of nucleic acids is of interest as both a target in drug development and as a candidate for nucleic acid therapeutics, as it can inhibit replication, transcription, reverse transcription, translation, and elongation reactions. The G-quadruplex structures are known to undergo significant changes in both conformation and stability in response to environmental factors such as ionic concentration, pH, temperature, and molecular crowding. To develop nucleic acid therapeutics, including G-quadruplexes, effectively, it is necessary to consider not only interactions within the target nucleic acid but also those with binding nucleic acids, proteins, and various crowding molecules present in the cellular environment. In this study, in order to elucidate the interactions and behaviors of crowding molecules that affect nucleic acid stability, we performed molecular dynamics simulations and fragment molecular orbital (FMO) calculations of hairpin DNA and three types of G-quadruplexes, which are involved in transcription and translation in the absence and presence of tetraethylene glycol (EG4) as crowding molecules. The results of FMO calculations on multiple snapshots from MD simulations of DNA under dilute conditions showed that backbone interactions were similar across nucleic acid topologies, while base-base interactions showed distinct fluctuations in energy. Molecular dynamics simulations in the presence of EG4 molecules as crowding agents demonstrated that EG4 molecules, which are generally considered not to directly interact with target biomacromolecules, localized around DNAs for extended periods. From the FMO calculation, it was found that EG4 interacts with the backbone fragment through hydrogen bonding, whereas it interacts with the base fragment through CH-π interactions. These CH-π interactions were mainly observed with the G-quartets and with the bases stacked on the G-quartets in parallel- and hybrid-type G-quadruplexes that exhibited low fluctuation. On the other hand, in the anti-parallel-type G-quadruplex whose G-quartet was covered by highly fluctuating loop bases and in the hairpin DNA without a G-quartet, a few stable interactions were observed. These findings provide insight into how crowding molecules shape the intracellular environment surrounding nucleic acids.