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.

The kinetics and energetics of load-driven conformational rearrangements underlie DNA replication, repair, and DNA nanodevice function. DNA tension-gate tethers exploit these dynamics, yet their rupture thresholds are set almost exclusively by single-molecule calibration; here, an atomistic steered-molecular-dynamics pipeline predicts relative thresholds and characterizes the underlying rearrangements. Seven constructs—duplexes of varying length and GC content, single-stranded poly(dT)₃₀, a GC-clamped hairpin, and G-quadruplex—were pulled with 50 trajectories per construct per rate (0.01/0.001 nm/ps). At MD loading rates, the ordering G-quadruplex (911 pN) > GC-rich duplex (786 pN) > hairpin (643 pN) > mixed duplexes (559/558 pN) > ssDNA (388 pN) holds at both rates, and the design rules deconfound, +68 pN for GC content (47% → 100%), −160 pN for doubled length, and 208 pN for pull geometry—larger than GC itself. Dissipated work falls 31–58% at the slower rate; slow-rate Jarzynski estimators converge for low-dissipation elements, while fast-rate profiles of high-dissipation motifs do not. Absolute thresholds at experimental rates remain out of reach, but the ordering—the actionable output for TGT design—comes with transferable structure-gating and convergence-diagnostic protocols, positioning atomistic simulation as a quantitative complement to single-molecule spectroscopy.