Nucleic acid biopolymers are essential to all living organisms. The chemical makeup of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) consist of sequences of only four monomers. However, they transmit and express genetic information for various biological functions based on stored blueprints. Along with communicating the flow of genetic information (DNA → RNA→ protein), nucleic acids have also become a preferred material for fabrication of objects and scaffolds at the nanoscale. Nucleic acid-based assemblies that interact with each other and communicate with the cellular machinery represent a new class of reconfigurable nanostructures that enable precise control over their formulation and physicochemical properties and activation of multiple biological functionalities. Therefore, the use of nucleic acids offers a unique platform for development of nanoparticles which consist of nanoscale-size oligonucleotides designed to fold into predicted three-dimentional structures. They can serve as scaffolds capable of carrying numerous functional moieties such as: RNA interference activators for gene silencing, aptamers for specific targeting of selected molecules, immunostimulatory sequences for modulating immune responses, and fluorescent entities for bioimaging. Programmable, controllable, biocompatible, rationally designed, and self-assembled nucleic acid nanoparticles have become attractive candidates for diverse therapeutic options. Despite profound advances in the field of therapeutic nucleic acids, their negative charges decrease membrane permeation capacity, thus hindering their translation from experimental research to clinical application. This dissertation focuses on the development of nucleic acid nanoparticles as therapeutics with defined immunostimulatory properties accompanied by rationally designed systems able to regulate the duration of therapeutic activity. Furthermore, a safe, efficient and stable intracellular delivery system for multi-functional nucleic acid nanoparticle platforms is investigated.