Molecular Dynamics Simulations Reveal a Broadly Open Solution Ensemble for the DNA Holliday Junction: Mg2+ Crossover Bridging and Weak Force-Field Sensitivity

Holliday junctions, four-way DNA crossovers central to DNA origami and key intermediates of homologous recombination, must be accurately modeled for rational DNA-nanodevice design, yet their solution conformational ensemble remains incompletely characterized. We characterize this ensemble for the experimental junction d(TCGGTACCGA)4 by all-atom molecular dynamics, comparing the AMBER force fields parmbsc1, OL21, and OL24 in OPC water with matched ionic conditions. In every force field, the junction explores a broad, nearly flat inter-arm-angle landscape, with <1 kcal/mol variation across inter-arm angles of 2–90°: the open state is favored by 0.45–0.63 kcal/mol, the crystallographic-like inter-arm angle occurs in 2–5% of frames, and the crossover remains coaxially stacked whether open or compact. A Mg2+ ion occupies the crossover pocket in 92–94% of frames, specifically bridging the A- and C-strand phosphates (Li–Merz CM Mg2+ set). Stacking-isomer exchange between the A/C-continuous and crystallographic B/D isomers proceeds at ~0.5 ns−1 in this minimal short-arm construct, independent of force field, with A/C-continuous favored ~2:1. parmbsc1 yields a more open junction than OL21 and OL24, which reproduce the crystallographic inter-arm angle of 39–43°; the sugar-pucker shift targeted in OL24 is also visible at the junction. These results establish a force-field-robust baseline for simulation-guided design of crossover-containing nanostructures.