Free-energy landscapes of unlocking transitions in DNA origami nanocarriers.

Abstract

Unlocking transitions in DNA origami nanocarriers regulate structural stability and trigger payload release, yet quantifying the associated free-energy changes is challenging because duplex rupture occurs stochastically and is coupled to large-scale structural deformation. Here, we combine coarse-grained oxDNA simulations with ensemble-averaged multistate Bennett acceptance ratio (MBAR) analysis to reconstruct effective free-energy profiles along mechanically driven opening pathways. Using a simplified single-stranded DNA construct with terminal locking duplexes, we show that the energetic cost of unlocking increases with duplex length but represents only a fraction of the available base-pairing stabilization, indicating increasing energetic redundancy in longer locks. We then apply the same approach to a rectangular DNA origami nanocarrier stabilized by six locking duplexes and operationally separate the energetic contributions associated with duplex rupture and global structural deformation. Trajectory-level analysis shows that individual locks rupture asynchronously and heterogeneously while preserving reproducible ensemble-level energetic trends. Direction-resolved analysis reveals a pronounced asymmetry between alternative bending pathways: the unlocked nanocarrier intrinsically favors bottom-bent deformation, whereas sufficiently long locking strands selectively stabilize the opposing top-bent configuration. These results clarify how lock-mediated hybridization and mechanical deformation jointly shape the effective pathway-dependent energetic response of DNA origami nanocarriers and provide quantitative guidance for tuning structural stability and directional response in programmable DNA devices.