BPS2026 – Multi-axial DNA origami force spectroscopy reveals quasi-ergodic dynamics of Holliday junction

Biomolecules experience complex multi-directional mechanical forces that govern their structure, dynamics, and function. However, most single-molecule techniques primarily exert force along a single axis, thereby failing to simulate the complex mechanical cellular environment. Here, we present Multi-axial entropic spring tweezer along rigid DNA origami (MAESTRO), a molecular platform that applies up to 9 pN forces from up to four directions simultaneously using programmable ssDNA entropic springs anchored to a rigid circular DNA origami scaffold. Combining MAESTRO, single-molecule Förster resonance energy transfer (smFRET), and Bayesian non-parametric FRET (BNP-FRET) enables a high-throughput study of biomolecules under different complexities of multi-axial tension forces. We applied MAESTRO to Holliday junctions (HJs), four-way DNA intermediates that experience multi-directional tension during homologous recombination. Counterintuitively, we discovered ≥ 5 × slower kinetics of the HJ conformations under multi-axial tension than under tension-free conditions, for the first time enabling direct single-molecule observations of HJ open conformation. Most remarkably, we discovered that multi-axial tension reveals quasi-ergodic dynamics by overcoming the rugged energy landscape of HJs, enabling direct observation of kinetic class interconversion within individual molecules—a phenomenon previously thought impossible—that reveals that the energy landscape is far more interconnected than understood and fundamentally challenges existing models. Furthermore, we demonstrated that this conformational control regulates T7 endonuclease I cleavage site selection on HJs, directly linking mechanical environments and molecular mechanics to enzymatic function. By achieving multi-axes capability, MAESTRO enables exciting frontiers in molecular mechanobiology, revealing how physiologically relevant multi-directional forces enables dynamic exploration of rugged energy landscape and can serve as master regulators for biomolecular function through mechanisms inaccessible to conventional single-axis approaches.