Cosolvents induce sharp softening and RNA-like twist-stretch coupling of RNA-DNA hybrid.

Cellular environments generally contain abundant organic cosolvents, yet how in-cell cosolvents modulate the mechanics of RNA-DNA hybrid (RDH) duplex remains unclear. Here, we combined high-precision magnetic tweezer experiments with all-atom molecular dynamics simulations to investigate the structural elasticity of RDH duplexes using PEG200, PEG1000, and ethanol as model cosolvents. We found that high-concentration cosolvents sharply soften RDH duplexes in bending and induce an apparent RNA-like positive twist-stretch coupling. Specifically, for RDH duplex, the bending persistence length P decreases from ∼54 nm to ∼27-35 nm (∼35-50% decrease) by 40% cosolvents. Meanwhile, the twist-stretch coupling parameter dL/dN shifts from ∼0 to -0.56--0.45 nm/turn, approaching that of RNA duplex (∼-0.81 nm/turn). Microscopic analyses revealed that sharp bend softening mainly arises from local hydrogen-bond disruption associated with structural mismatch between RNA and DNA strands and enhanced strand fluctuation driven by increased ion neutralization. Moreover, the RNA-like twist-stretch coupling originates from cosolvent-induced shifts of structure and deformation pathway of RDH duplex toward RNA-like ones from comprehensive analyses at the helix, base-pair, and strand levels. Our findings revealed that cosolvents can substantially reshape the structure and mechanics of RDH duplexes, potentially providing a physical basis for understanding their behavior in cells.