Application of protective polymer coatings to enhance the biostability of DNA-based nanomaterials (DONs) has become common practice in in vitro and in vivo experiments. While the functional effect of these coatings is obvious, a detailed molecular picture of what is protected and for how long remains unclear. Additionally, the use of the oligolysine-1kPEG protective polymer has been limited due to aggregation issues. In this study, we evaluated the colloidal stability, structural integrity, and functional preservation of DONs coated with oligolysine (K)-1k/5kPEG block copolymers. Dynamic light scattering and transmission electron microscopy were employed to assess colloidal stability before and after degradation. A FRET-based assay was developed to monitor the directionality of degradation, while quantitative PCR measured the protection of functional DNA handles, crucial for the design of ligand-functionalized DONs. Our results show that K10-1kPEG, while prone to aggregation, can offer similar protection against nucleases as K10-5kPEG, provided buffer conditions are carefully chosen. Maintaining the colloidal, structural, and functional stability before and after nuclease exposure supports DON applications, particularly at the biointerface. These insights provide valuable guidelines for selecting the most effective protection strategy and enhancing DON functionality across diverse biological environments.