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.