Thermomechanical Response of Protein-DNA Hybrid Tetrahedrons Tuned by Linker Length

Protein-DNA hybrid assemblies provide a versatile platform for constructing functional bionanomaterials, yet the molecular determinants of their thermal and mechanical stability remain incompletely understood. Here, we characterize the thermomechanical response of a hybrid tetrahedral cage in which a homotrimeric KDPG aldolase is covalently linked to a DNA vertex through three duplex handles of variable hybridization lengths (21, 15, 10, and 5 bp). Using the combined ANM-oxDNA coarse-grained framework, we quantify how duplex length modulates junction energetics, equilibrium stability, and nonequilibrium rupture behavior. Equilibrium simulations reveal that short linkers (5 bp) destabilize rapidly upon thermal perturbation, while longer duplexes preserve the structural integrity of the protein-DNA vertex across comparable conditions. Under mechanical loading, rupture forces, extension at break, and mechanical work increase systematically with linker length, while elevated temperature induces softening and broadens the distribution of failure pathways. Together, these results establish a direct molecular connection between hybridization length, thermal robustness, and mechanical resilience. They provide quantitative insight into the physical chemistry governing protein-DNA interfaces and offer design guidelines for thermoresponsive biohybrid nanostructures. Importantly, comparison with DNA-only reference systems shows that the protein vertex redistributes forces among the linkers, leading to more gradual and cooperative rupture behavior without significantly altering the intrinsic mechanical strength of the junction.