Drying parameters and aging modulate protective properties of vitrified trehalose

• Nucleic acids are generally stable under various drying conditions/vitrified systems • Protein preservation benefits from trehalose coincubation during drying and the vitrified properties that are achieved • Drying method and parameters tune vitrified properties and protection • Vitrified systems age over time, changing properties and protectiveness • Initially high Tg is protective, increasing Tg and fragility over time indicate aging Storage of biological materials underpins medical, research, and biotechnological applications. Although cold-chain preservation is effective, it is costly, infrastructure-dependent, and vulnerable to disruption. Room-temperature dry storage, inspired by desiccation-tolerant organisms, offers an alternative by stabilizing biomolecules in vitrified matrices that limit molecular motion and degradation. Trehalose is widely used as a vitrifying agent, but its protective capacity depends on glassy properties shaped by drying conditions, environment, storage duration, and biomolecule type. However, systematic links between these factors and stability remain poorly defined. Here, we examine how drying conditions and storage duration influence the stability of DNA, RNA, and enzymes in trehalose-based vitrified systems. DNA remained stable across all conditions, independent of trehalose or drying parameters, reflecting intrinsic resistance to desiccation damage. RNA exhibited moderate sensitivity to drying without trehalose but was stabilized in its presence, although RNA integrity did not consistently correlate with measured glassy properties. In contrast, enzymes were highly sensitive to drying in the absence of trehalose and strongly protected under conditions that promoted favorable vitrified properties. Short-term enzyme protection (30 min) positively correlated with higher glass transition temperature (Tg). However, during prolonged dry storage, higher Tg was inversely correlated with enzyme stability and instead tracked detrimental physical aging of the vitrified matrix. These findings demonstrate that optimal glass properties depend on both biomolecule class and timescale, providing a framework for rationally designing room-temperature preservation strategies.