Multi-axial DNA origami force spectroscopy unlocks conformational dynamics hidden under single-axial tension

Biomolecules in living cells experience complex multi-directional mechanical forces that regulate their structure, dynamics, and function. However, most single-molecule techniques primarily exert force along a single axis, thereby failing to emulate the mechanical environment of cells. Here, we demonstrate that single-axis force application fundamentally restricts conformational dynamics by kinetically trapping molecules within distinct dynamic classes, preventing interconversion and exploration of the full conformational landscape. We developed MAESTRO (Multi-Axial Entropic Spring Tweezer along a Rigid Origami), a molecular platform that applies up to 9 pN forces from up to 4 directions simultaneously using programmable ssDNA entropic springs anchored to a DNA origami scaffold. We applied MAESTRO to Holliday junctions (HJs), 4-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, enabling direct observations of previously hidden HJ conformational intermediates. Most remarkably, we discovered that multi-axial forces unlock conformational dynamics, enabling interconversion between 5 distinct kinetic classes that remain kinetically inaccessible under zero force or single-axial tension. Furthermore, we demonstrated that tension regulates T7 endonuclease I cleavage site selection, directly linking mechanical environments and molecular mechanics to enzymatic function. By overcoming single-axis limitations, MAESTRO opens new frontiers in molecular mechanobiology, revealing how multi-directional cellular force environments are essential for unlocking the full conformational landscape of biomolecules, and that these complex force patterns serve as master regulators of biological function through mechanisms hidden from conventional approaches.