The intrinsic properties of RNA and DNA biopolymers emphasized by engineered nucleic acid nanoparticles (NANPs) offer accelerated development of next-generation therapies. The rational design of NANPs facilitates programmable architectures intended for regulated molecular and cellular interactions. The conventional bottom-up assembly of NANPs relies on the thermal annealing of individual strands. Here, we introduce a concept of nuclease-driven production of NANPs where selective digestion of functionally inert structures leads to isothermal self-assembly of liberated constituents. The working principles, morphological changes, assembly kinetics, and the retention of structural integrity for system components subjected to anhydrous processing and storage are assessed. We show that the assembly of precursors into a single structure improves stoichiometry and enhances the functionality of nuclease-driven products. Furthermore, the experiments with immune reporting cell lines show that the developed protocols retain the immunostimulatory functionality of tested NANPs. The presented approach enables exploitation of the advantages of conditionally produced NANPs and demonstrates that NANPs' stability, immunorecognition, and assembly can be regulated to allow for a more robust functional system.